GROWTH AND YIELD RESPONSES OF Glycine max and Phaseolus
vulgaris TO MODE OF NITROGEN NUTRITION AND TEMPERATURE
CHANGES WITH ELEVATION
A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF
THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN AGRONOMY AND SOIL SCIENCE
Duane P. Bartholomew, Chairman
B. Ben Bohlool
Paul W. Singleton
James A. Silva
Francoise M. Robert
My sincere gratitude goes to Dr. Duane P. Bartholomew, Dr. B. Ben Bohlool and Dr. Paul W. Singleton for their constant guidance, constructive criticisms and spirited support during this research. I am especially appreciative of the help given by Dr. Singleton and Dr. Bartholomew in the preparation of the manuscript. I am grateful to Dr. James A. Silva and Dr. Francoise M. Robert for their helpful suggestions and advice.
I would like to acknowledge Kevin Keane, Rick Koglin, Goeff Haines, Brian Duffy and Thomas Walker for their assistance in the field, the NifTAL Project staff for their untiring help, Robert Abaidoo for his participation in a part of this research.
Lastly, my heartfelt thanks goes to Maria Luz Caces, my best friend.
This research was supported in part by the U.S. Agency for International Development grants DAN-1406-G-SS-4081-00 (Indo/US Initiative on Science and Technology) and DAN-0613-C-00-2064-00 (NifTAL Project), National Science Foundation/Ecology Program, and the International Atomic Energy Agency.
Efficient exploitation of the legume-Rhizobium symbiosis in varied environments requires an understanding of the responses of both the host and the bacteria to management and environmental variables. The objective of this research was to study the growth, development and yield responses of soybean (Glycine max L. Merrill) and common bean (Phaseolus vulgaris L.) to mode of N nutrition and temperature at sites in an elevational transect differing in mean temperatures. In the first experiment five soybean varieties and their non-nodulating isolines from four maturity groups were grown after inoculating with three Bradyrhizobium japonicum strains. The proportion of nodules formed by each B. japonicum strain was not affected significantly by temperature, soil type, or soybean genotype. Soybean grain yield and total plant N decreased with increasing elevation. At a given site, the proportion of total plant N derived from symbiotic N2 fixation was approximately constant. Between sites, the proportion of fixed N was inversely related to soil N availability. Vegetative and total crop durations were prolonged by cool temperature. Maturity of soybean was hastened by N insufficiency. In a second experiment, inoculated and uninoculated soybean and common bean were grown with either 9, 120, or 900 kg N ha-1. As a result of extended crop durations at the higher elevation, total biomass production was similar between elevations in both legumes. Grain yield of common bean was similar at the cool and warm sites; in contrast the grain yield of soybean was greatly reduced at the cool site. Yield and total plant N of both legumes increased at mineral N levels of 120 and 900 kg ha-1 compared to 9 kg N ha-1. Significant symbiotic N2 fixation limited yield and total N assimilation in soybean. Common bean utilized proportionately more mineral N than soybean throughout growth and had a higher fertilizer N use efficiency than soybean during the vegetative phase. Common bean also had a lower N requirement and N assimilation was more uniform throughout growth when compared to soybean. Maximizing N derived from fixation in common bean may require altering its N assimilation characteristics.
TABLE OF CONTENTS
ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . iii
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . iv
LIST OF TABLES . . . . . . . . . . . . . . . . . . . vii
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . xii
CHAPTER I. INTRODUCTION . . . . . . . . . . . . . . 1
CHAPTER II. NODULATION AND INTERSTRAIN COMPETITION BY
Bradyrhizobium japonicum IN SOILS ALONG AN
ELEVATIONAL TRANSECT . . . . . . . . . . 6
CHAPTER III. EFFECT OF TEMPERATURE AND MATURITY GROUP
ON PHENOLOGY OF FIELD GROWN SOYBEAN . . 28
CHAPTER IV. YIELD, SOIL N UPTAKE AND N2 FIXATION BY
SOYBEANS FROM FOUR MATURITY GROUPS GROWN
AT THREE ELEVATIONS . . . . . . . . . . 48
CHAPTER V. GROWTH RESPONSES OF FIELD GROWN SOYBEAN
AND COMMON BEAN TO TEMPERATURE AT VARYING LEVELS OF N NUTRITION . . . . . . . . . 67
CHAPTER VI. NITROGEN ASSIMILATION AND N2 FIXATION IN
SOYBEAN AND COMMON BEAN AS AFFECTED BY
ELEVATION AND MINERAL N AVAILABILITY . . 89
SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . 109
APPENDIX . . . . . . . . . . . . . . . . . . . . . . 114
LITERATURE CITED . . . . . . . . . . . . . . . . . . 123
LIST OF TABLES
II-1 Soil and site characteristics of three
locations in an elevational transect on
the island of Maui, Hawaii . . . . . . . . . . . 21
II-2 Proportion of nodules formed by B. japonicum
strains from five soybean varieties grown in
three soils in the field and greenhouse . . . . . 22
II-3 Proportion of nodules formed by B. japonicum
strains from five soybean varieties grown in
the field and greenhouse . . . . . . . . . . . . 23
II-4 Number and dry weight of nodules from five
soybean varieties at 45 days after planting
at three different elevations . . . . . . . . . . 24
II-5 Number and dry weight of nodules from five
soybean varieties at 45 days after planting
in three different soils in the greenhouse . . . 25
II-6 Shoot dry weight of five soybean varieties
at 45 days after planting at three different
elevations . . . . . . . . . . . . . . . . . . . 26
II-7 Shoot nitrogen content of five soybean varieties
grown at three different elevations at 45 days
after planting . . . . . . . . . . . . . . . . . 27
III-1 Mean maximum and minimum temperatures during
growth of five soybean varieties at three
elevations on the island of Maui, Hawaii . . . . 40
III-2 Days to first flower and physiological maturity
of nodulating isolines of five soybean varieties
grown at three elevations . . . . . . . . . . . . 42
III-3 Growing degree days accumulated during
vegetative growth and up to physiological
maturity for nodulating isolines of five
soybean varieties grown at three elevations . . . 43
III-4 Duration of seed-filling of nodulating isolines
of five soybean varieties at three elevations . . 44
III-5 Rate of seed dry matter accumulation of
nodulating isolines of five soybean varieties
grown at three elevations . . . . . . . . . . . . 45
III-6 Mean days to first flower and physiological
maturity for nodulating and non-nodulating
isolines of five soybean varieties grown at
two elevations . . . . . . . . . . . . . . . . . 46
III-7 Mean grain and shoot nitrogen concentrations of
nodulating and non-nodulating isolines and grain
yield of non-nodulating isolines of five soybean
varieties grown at three elevations . . . . . . . 47
IV-1 Characteristics of three sites in an elevational
transect on the island of Maui, Hawaii . . . . . 59
IV-2 Grain and stover yields of five nodulating
soybean varieties grown at three different
elevations . . . . . . . . . . . . . . . . . . . 60
IV-3 Total nitrogen assimilation by five nodulating
soybean varieties grown at three different
elevations . . . . . . . . . . . . . . . . . . . 61
IV-4 Symbiotically fixed nitrogen by five soybean
varieties grown at three different elevations . . 62
IV-5 Average rate of nitrogen assimilation by five
soybean varieties grown at three different
elevations . . . . . . . . . . . . . . . . . . . 63
IV-6 Percent of total nitrogen derived from symbiotic
fixation by five soybean varieties grown at
three elevations . . . . . . . . . . . . . . . . 64
IV-7 Comparison of estimated amounts of mineralized N
and actual soil nitrogen uptake by five soybean
varieties at three elevations . . . . . . . . . . 65
IV-8 Nitrogen uptake by Clark non nodulating plants
grown in three soils in the greenhouse at a mean
soil temperature of 27 C . . . . . . . . . . . . 66
V-1 Environmental characteristics at two sites in an
elevational transect on the island of Maui,
Hawaii . . . . . . . . . . . . . . . . . . . . . 81
V-2 Days to first flower and physiological maturity
of inoculated soybean and common bean grown at
two elevations with three levels of N . . . . . . 82
V-3 Grain yield of soybean and common bean as
affected by elevation and N application . . . . . 83
V-4 Effect of elevation by legume species interaction
on harvest index and whole plant tissue N
concentration at physiological maturity . . . . . 84
V-5 Main effects of elevation, legume species and N
level on dry weights at different growth stages . 85
V-6 Main effects of elevation, legume species and
N levels on mean crop growth rate during
different growth phases . . . . . . . . . . . . . 86
V-7 Effect of elevation by legume species
interaction on leaf characteristics at different
growth stages . . . . . . . . . . . . . . . . . . 87
V-8 Effect of elevation and legume species on unit
leaf rate during different growth phases . . . . 88
VI-1 Elevation by legume by N level interaction on
N assimilation at physiological maturity . . . . 103
VI-2 Effect of elevation, N level and growth stage
on N2 fixation by soybean at different growth
stages . . . . . . . . . . . . . . . . . . . . . 104
VI-3 Effect of elevation, N application and legume
species on the rate of N assimilation during
crop cycle . . . . . . . . . . . . . . . . . . . 105
VI-4 Relative N assimilation during different growth
phases of soybean and common bean grown at two
elevations . . . . . . . . . . . . . . . . . . . 106
VI-5 Effect of legume and N levels on fertilizer N
use efficiency during the crop cycle . . . . . . 107
VI-6 Effect of legume and N levels on KCl-extractable
soil N during the crop cycle . . . . . . . . . . 108
A-1 Effect of elevation and N level on nodule dry
weight at end bloom . . . . . . . . . . . . . . 114
A-2 Days to flowering and physiological maturity
of the non-nodulating isoline of Clark soybean
grown at two elevations with three levels
of mineral N . . . . . . . . . . . . . . . . . . 115
A-3 Effect of N level on harvest index of legumes
grown at two elevations. . . . . . . . . . . . . 116
A-4 Grain yield of the non-nodulating isoline of
Clark soybean grown at two elevations with three
levels of mineral N . . . . . . . . . . . . . . 117
A-5 Total N assimilation by the non-nodulating
isoline of Clark soybean grown at two elevations
with three levels of mineral N . . . . . . . . . 118
A-6 Effect of N level on the leaf area and leaf weight
of legumes at different growth stages . . . . . 119
A-7 Effect of N level on the unit leaf rate of
legumes at different growth stages . . . . . . . 120
A-8 Fertilizer use efficiency of legumes grown at two
elevations with three N levels . . . . . . . . . 121
A-9 KCl-extractable soil N after harvest of legumes
grown at two elevations with three N levels . . 122
LIST OF FIGURES
1 Weekly mean temperatures and daylengths during the
growth of five soybean varieties at three elevations
on the island of Maui, Hawaii . . . . . . . . . . . 41
Legumes respond to the environment in the same way other plants do, but what makes them physiologically distinct from other plants is their ability to harbor nitrogen-fixing rhizobia in a symbiotic relationship. The establishment of the legume-Rhizobium symbiosis starts with the infection of the legume host by compatible rhizobia, resulting in the formation of nodules.
Both symbiotic N2 fixation and mineral N assimilation contributes to the N requirement of legumes. The symbiosis is functional when the legume is stressed for nitrogen and, to some degree, compensates for decreased mineral N availability. However, mineral nitrogen suppresses symbiotic N2 fixation (Allos and Bartholomew, 1959; Chen and Phillips, 1977; Dart and Wildon, 1970; Oghoghorie and Pate, 1971). While N2 fixation is readily replaced by mineral N, evidence presented by Gibson and Pagan (1977), Manhart and Wong (1980), and Streeter (1985) do not show any direct inhibitory effect of mineral N on N2 fixation.
The general consensus regarding the N nutrition of legumes is that legumes require N from both symbiotic and mineral N sources for maximum yield (Bhangoo and Albritton, 1976; Harper, 1974). However, there are indications that legumes relying on mineral N produce yields similar to or greater than symbiotic plants (Kato et al. 1984; Ryle et al. 1978; Silsbury, 1977). Schweitzer and Harper (1985) found that increasing light interception by a soybean canopy using reflectors increased nitrate reductase activity in nitrate-supplied plants more than it increased nodule activity in inoculated plants, and the nitrate-supplied plants consequently produced greater dry weight. Also, Tanaka (1986) showed that if photosynthetic activity decreased, N2 fixation decreases more than mineral N absorption. Accordingly, Mahon and Child (1979) argued that it is difficult to increase N2 fixation and the productivity of legumes simultaneously. Salsac et al., (1984) also concluded that plants entirely or partly dependent on mineral N had yields higher than symbiotic plants. The assimilation of mineral nitrogen does not involve the maintenance of specialized structures like root nodules as in the symbiotic plant, so it is a more energy efficient source of N. Finke et al. (1982) concluded that dependence of the plant solely on N2 fixation is an energy expensive process.
If there is sufficient N in the soil to meet the plant's requirement, and if assimilation from the soil is not limited by other factor(s), then symbiotically fixed N will not be a major source of N for the plant. Any factor which limits the availability, uptake or assimilation of soil N should favor N2 fixation. Low temperature is one factor which reduces the rate of soil N mineralization (Stanford et al., 1973; Cassmann and Munns, 1980), and uptake of available soil nitrogen (McDuff and Hopper, 1986; Tolley and Raper, 1985). Low temperature, therefore, may favor N2 fixation over mineral N assimilation unless it decreases nitrogenase activity to the same degree that soil N assimilation is decreased.
Nitrogen nutrition presumably can range from complete dependence on mineral N to complete dependence on symbiotic N2 fixation. Plants dependent on N2 fixation or mineral N may respond differently to the environment because of the differences in their root morphology, N assimilation process, and N nutritional level. In the case of the symbiotic plant, the response may be further complicated by specific interactions between the legume host and the rhizobial microsymbiont. The interaction between the legume host and the rhizobial microsymbiont is specific, and the compatibility of the host with any one of the competing rhizobial strains may determine whether or not a productive symbiosis will be established. Thus, any attempt to correlate legume response to environment requires careful consideration of the mode of N nutrition and the N requirement of the plant.
Developing a holistic approach to legume productivity research requires the evaluation of the legume rhizobium system from soil, plant, and microbial perspectives in the field. Such an approach would render the results more relevant to productivity research and improve the degree of predictability of outcomes in varied environments.
Considering the complexity of environmental factors in agricultural ecosystems, the delineation of effects of single factors is often difficult. Although management variables may be made optimum and thus, eliminated as sources of variation, the effects of environmental variables such as temperature, photoperiod and irradiance are often confounded.
Temperature has direct and indirect effects on biological or biochemical processes and in legumes, these responses are readily modified by photoperiod (Salisbury, 1981). The field evaluation of temperature effects is simplified when the confounding effects of irradiance and photoperiod are minimal. Sites in an elevational transect may permit this kind of evaluation if changes in irradiance with elevation are small.
Two experiments were conducted to study the effects of mode of N nutrition, temperature and other elevation-associated variables on the ecophysiological response of soybean and common bean. The approach taken was to study host-Rhizobium-environment interactions and plant genotype-temperature-mode of nitrogen nutrition interactions in soybeans adapted to a wide range of latitudes, and use the data thus obtained as the basis for a second experiment to evaluate the growth and yield responses of soybean and common bean to temperature under a range of mineral N availability conditions.
The Objectives of the experiments were:
1. Determine the effects of elevation-associated site characteristics (temperature, soil type) and soybean genotype on the competition pattern of Bradyrhizobium japonicum strains.
2. Determine the effects of temperature and mode of N nutrition on the phenology of a range of soybean maturity groups under similar photoperiodic regimes.
3. Assess the effects of site characteristics and soybean genotype on the contribution of N2 fixation to plant N.
4. Evaluate the growth responses of soybean and common bean to cool and warm environments at varying levels of soil N availability.
5. Evaluate the responses of soybean and common bean to mineral N application and contrast the N assimilation patterns of the two legumes.
NODULATION AND INTERSTRAIN COMPETITION BY Bradyrhizobium
japonicum IN SOILS ALONG AN ELEVATIONAL TRANSECT
A better understanding of the ecology of Bradyrhizobium japonicum in relation to its host and its environment may aid in making appropriate management decisions for soybean production in a wide range of conditions. Success of one or more of the competing rhizobial strains in forming nodules and establishing an effective symbiosis is influenced by host genotype, soil type and temperature. The effects of temperature and soil type on interstrain competition, nodulation and nitrogen assimilation in five soybean varieties belonging to maturity groups 00, IV, VI, and VIII were investigated at three sites devoid of soybean rhizobia along an elevational transect in Hawaii. The soils are classified as Humoxic Tropohumult (clayey, ferritic, isohyperthermic), Humoxic Tropohumult (clayey, oxidic, isothermic) and Entic Dystrandept (medial over loamyskeletal, isomesic) at elevations of 320, 660 and 1050 m, respectively. Sites were amended with Ca(OH)2 to a pH between 5.5 and 6.0. Nutrients, except nitrogen, were provided in non limiting amounts. Seeds were inoculated with equal numbers of Bradyrhizobium japonicum strains USDA 110, USDA 136b (CB 1809) and USDA 138. The mean soil/air temperatures during the experiment were 25/24, 24/22 and 22/18 C at the respective elevations. A greenhouse pot experiment was also conducted using the same soils and inoculum under a fixed temperature regime. Plant tops and nodules were sampled at 45 days after seeding. Nodule occupancy was determined by immunofluorescence. Competition patterns of the three strains were unaffected by soil type or soil temperature. Strain USDA 110 was the best competitor, occupying on the average (5 cultivars) 81 and 64% of the nodules in the field and greenhouse experiments, respectively. Strain USDA 138 was the least successful in the field. Its nodule occupancy under greenhouse conditions, however, was significantly greater than in the field. Nodule occupancy was also related to soybean maturity group. Strain USDA 110 formed 61, 71, 88, 88, and 98% of the nodules in the field on Clay (00), Clark (IV), D68 0099 (VI), N77 4262 (VI), and Hardee (VIII), respectively. Strain USDA 136b formed very few nodules on Hardee, an Rj2 soybean variety incompatible with that strain; its nodule occupancy on all other cultivars in the field averaged about 30% (including nodules containing more than one strain). Both nodule number and weight were significantly reduced as elevation increased. Nodule number increased with increasing maturity group within a site, but nodule weight did not. Shoot dry weight and nitrogen assimilation decreased drastically with increased elevation. Nitrogen assimilation decreased from 246 mg N per plant at 320 m elevation to 26 mg N per plant at 1050 m. While soil type and temperature had no effect on strain competition, temperature had a profound influence on nodule parameters and plant growth.
The establishment of an effective and efficient symbiosis between rhizobia and the host legume is basic to viable legume production without mineral N fertilization. The symbiosis is modified by various environmental factors. An understanding of the effects of environmental factors on the symbiosis is therefore, essential for selecting adapted cultivars and management practices that will enhance symbiotic nitrogen fixation and yield.
Bradyrhizobium japonicum strains differ considerably in competitive ability, nodulation and nitrogen fixation (Abel and Erdman, 1964; Caldwell, 1969; Johnson and Means, 1964). The competition and nodulation characteristics of the strains have been shown to be influenced by the host genotype (Caldwell and Vest, 1968; Materon and Vincent, 1980).
Soil type may also influence the competition between strains for nodule sites. Dominance of different B. japonicum strains in soybean nodules from different soils have been reported (Damirgi et al., 1967; Ham et al., 1971; Johnson and Means, 1963; Keyser et al., 1984). Soil type greatly influenced the distribution of strains, with distinctly different populations of rhizobia in soybean nodules from six different soils (Damirgi et al., 1967). According to Ham et al. (1971), the presence of individual strains in soybean nodules was related to two or more of the soil properties, the most important of which was soil pH. Kosslak and Bohlool (1985) have reported that in soils devoid of native B. japonicum, the pattern of competition between two introduced strains remained the same regardless of soil type or soil amendments.
The recovery of specific rhizobial strains in the nodules, and nodulation and N2 fixation are also related to temperature. Caldwell and Weber (1970) found that the dominance of different serogroups in soybean nodules was altered by planting dates, and Weber and Miller (1972) concluded that the effect was due to differences in soil temperature. Low soil temperatures have also been shown to adversely affect both nodulation and nitrogen fixation (Lindeman and Ham, 1979; Waughman, 1977).
To understand the ecology of rhizobia in relation to the host and its environment, experiments were conducted along an elevational transect located on the island of Maui, Hawaii (part of the Maui Soil, Climate and Land Use Network: Mauinet), which provided a field laboratory of varied edaphic and climatic (primarily temperature) conditions. The purpose of the investigation reported here was to study B. japonicum strain competition, nodulation and early nitrogen assimilation on selected genotypes of soybean in field environments that differ in soil type, and soil and air temperatures. Other soil variables were assumed to be controlled by equalizing pH, and applying nutrients other than N at maximum levels.