Effects of N and P fertilizers on the growth, nodulation and N2-fixation of fababean (Vicia faba L.),green pea (Pisum sativum L.) and dry bean (Phaseoulus vulgaris L.)by Saidou Koala
Abstract:The most common grain legumes in temperate and sub tropical regions are Pisum, Phaseolus and Viciabeans. Their yields are often lower than the potential yield due to deficiencies in both P and N. Theobjectives of this research were to evaluate the relative effectiveness of different sources of phosphorusfertilizers, levels and methods of application on dry beans (Phaseolus vulgaris L.) and fababean (Viciafaba L.) and also to evaluate the effects of N and P fertilizers and their interaction on nodulation, N2-fixation and growth of fababean, dry bean and green pea (Pisum sativum L.) grown in the field.
In 1980, a split plot, randomized complete block design with four replications was used. Main plotswere 0 and 100 Kg ha-1 N applied as ammonium nitrate (NH4 NO3). Sub plots were a no P control,two P sources, orthophosphoric acid (H3 PO4) as liquid P fertilizer and triple superphosphate. In 1981,the orthophosphoric acid was replaced by monoammonium phosphate. In 1982, 1983 and 1984,factorials in randomized complete block designs with four replications were used with varying levels ofN and P fertilizers.
There were differential responses of fababean and dry bean grain yields to P sources and methods ofapplication. Nodulation and N2-fixation in fababean reached a maximum at pod filling and remainedconstant until pod filling was complete and then showed a decline. In dry bean, however, maximumnodulation and N2Tixation reached a maximum during pod set and declined rapidly during the finalweeks of growth. Application of 100 Kg ha-1 of fertilizer N reduced nitrogenase activity by 75, 72, 82and 75 percent in dry bean at the four harvests but only 47,60,62 and 57 in fababean. Excellent positivelinear correlations between acetylene reduction rates and nodule number and mass were found withboth fababean and dry bean in 1980.
Increasing P supply increased nodule number and nodule dry weight but these increases paralleledincreases in shoot and root dry weight and suggested that increasing P supply increases nodulation andN2fixation in the three different species of host plants by stimulating the plant growth rather than byaffecting nodule initiation and function. A model is proposed to explain the inhibitory effects ofammonia on nitrogenase activity. It suggests that ammonia acts as an uncoupler or ion ionophore anddissipates the electrochemical proton gradient created by the bacteriod respiratory chain. Moreimportantly, the destruction of the membrane potential suppresses the low potential electrons thatmight be necessary in reduction reactions within the bacteroids.
EFFECTSOF N AND P FERTILIZERS ON THE GROWTH, NODULATION AND
N2-F i XATION OF Fa b a b e a n (Vicia faba L.), GREEN PEA (Pisum sativum L-)
AND DRY BEAN {Phaseolus vulgaris L.)
by
Saidou Koala
A thesis submitted in partial fulfillment of the requirements for the degree
9f
Doctor of Philosophy
in
Crop and Soil Science
MONTANA STATE UNIVERSITY Bozeman, Montana
June 1985
Ds??
dop’^
ii
APPROVAL
of a thesis submitted by
Saidou Koala
This thesis has been read by each member of the thesis committee and has oeen found to be satisfactory regarding content, English usage, format, citation, bibliographic style, and consistency, and is ready for submission to the College of Graduate Studies.
a 6 . / ?rrhairperson, Graduate Committee
Approved for the Major Department
Date Head, Major Department
£
Approved for the College of Graduate Studies
Date Graduate Dean
iii
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the requirements for a doctoral degree
at Montana State University, I agree that the Library shall make it available to borrowers
under rules of the Library. I further agree that copying of this thesis is allowable only for
scholarly purposes, consistent with "fair use" as prescribed in the U.S. Copyright Law.
Requests for extensive copying or reproduction of this thesis should be referred to Uni
versity Microfilms International, 300 North Zeeb Road, Ann Arbor, Michigan 48106, to
whom I have granted "the exclusive right to reproduce and distribute copies of the disser
tation in and from microfilm and the right to reproduce and distribute by abstract in any
format.
Signature
Dedicated to my wife Bernadette and our children Koutou, Kotima and Maimouna
V
V ITA
Saidou Koala was born in 1951 to Rasmata and Issaka Koala, inThyou, Burkina Faso. He attended elementary school at Thyou and secondary school at "Lyc6e Philippe Zinda Kabor6", OUA GADOUGOU, where he graduated in 1970. He received a B.Sc (Ag) degree from MacDonald College of McGill University, Montreal, Canada in 1976.
He worked as an agricultural engineer in the Burkina Faso Ministry of Agriculture from 1976 to 1980.
In June, 1980, he entered graduate school at Montana State University and earned a M.S. degree in Soils in 1982. He continued to pursue a Ph.D. program and intends to finish in June 1985. Mr. Koala is married to Bernadette Laurent and they have three children.
vi
ACKNOWLEDGMENTS ,i
The author would like to thank the following people who directly or indirectly con
tributed in making my Ph.D. program successful: Dr. J. R. Sims, my major professor, for |
his guidance, inspiration and friendship during my graduate training; Drs. Ron Lockerman, !
Ray Ditterline, Hayden Ferguson, R. E. Lund and R. D. Dahl, members of my graduate .
committee, for sharing their time, efforts and enthusiasms; Dr. C. F. McGuire for his assis
tance in the lab; Drs. Hatim El-Attar and Mohammed El-Alfawi, postdoctoral fellows, for
their invaluable help in the fifeld as well as in the lab; the staff members of Plant and Soil
Science Department for their teaching.
The Burkina Faso government and the SAFGRAD project for sponsoring my gradu
ate training.
The USDA-SEA/USAID small BNF special grant No. 59-2301-0-5-001-0 and the
Montana Wheat Research and Marketing Committee for partially funding the study.
My wife, Bernadette, and our children, Koutou, Kotima and Maimouna for their love,
sacrifice and understanding during this entire program.
Mrs. Jean Julian for typing this manuscript.
vii
TABLE OF CONTENTS
Page
APPROVAI........ .................................................................................................................................... ii
STATEMENT OF PERMISSION TO USE.............. : ............................................................ iii
DEDICATION.................................: ............................................................................................ iv
V IT A .......................................................... .................................................................................. v
ACKNOWLEDGMENTS........................................................................................................... vi
TABLE OF CONTENTS........................................................................................................... vii
LIST OF TABLES...................................................................................................................... ix
LISTO F F IG U R E S .................................................................................................................. xxi
A B S TR A C T................................................................................................................................ xxiii
Chapter
1. IN TR O D U C TIO N .............................................................. I
2. LITERATURE REVIEW......... ................................................................................ 3
Effect of Placement on Nodulation, N2-Fixation and G ro w th .............. 3Effect of P Sources on Nodulation, N2 -Fixation and Growth................ 7Effect of Combined N on Nodulation, N2-Fixation and Growth........... 10Effect of N Fertilizer on Fababean Nodulation, N2-Fixation
and Growth. . . .......................................................................................... 12Effect of N Fertilization on Green Pea Nodulation, N2-Fixation
and Growth................................................................................................. 13Effect of N Fertilization on Dry Bean Nodulation, N2-Fixation
and Growth....................... <............................ .. . . ................................... 14Mode of Action of Combined Nitrogen........................................................ 16Effect of P on Nodulation, N2 -Fixation and Growth of Legumes. . . . . 19N and P Interaction on Nodulation, N2-Fixation and Growth
of Legumes................................................................................................. 21Methodology for Assessing Phosphorus Involvement in
3 MATERIALS AND METHODS..................... ...................................................... 24
Field Experiment 1980.................................................................................... 24Field Experiment 1981.................................................................................... 27Field Experiment 1982............................................................................... 30Field Experiments 1983 and 1984 ............................................................... 31
4 RESULTS AND DISCUSSION...................................................................... 33
Effects of Placement and Source of P Fertilizer on NodulationN2 Fixation and Growth of Fababean, 1980....................................... 33
Effects of Placement and Source of P Fertilizer on Nodulation,N2-Fixation and Growth of Fababean, 1981........................................ 40
Discussion on Fababean 1980 and 1981 Field Experiments................... 45Effects of Placement and Source of P Fertilizer oh Nodulation,
N2-Fixation and Growth of Dry Bean, 1980........................................ 46Effects of Placement and Source of P Fertilizer on Nodulation,
N2-Fixation and Growth of Dry Bean, 1981........................................ 53Effect of Inoculation of Fababean and Dry Bean Seed on
Growth and Nodulation.............................................................................. 61Effect of N and P on Growth, Nodulation and Nitrogen
Fixation in Fababean.............................................. 65Assessment of the Role of P in Fababean Nodulation and
N2 Fixation.................................................................................................. 87Effects of N and P on Growth, Nodulation and Nitrogen Fixation
on Green Pea................................................................................. i ........... 90Assessment of the Role of P in Green Pea Nodulation and
N2 Fixation.................................................................................................... 108Effects of N and P on Growth, Nodulation and Nitrogen
Fixation in Dry Bean.............................................. .................................. 114Assessment of the Role of P in Dry Bean Nodulation and
N2 Fixation....................................................................................... 127Mechanism of Inhibition by NH4+.................................................................... 128
5 SUMMARY AND CONCLUSIONS........................................................................ 134
LITERATURE C IT E D ................................................................................. 138
viii
Page
APPENDIX 149
ix
LIST OF TABLES
Tables Page
1. Summary of Fertilizer Treatments used in Subplots at Bozeman,Montana, 1980 ................................................................................... 24
2. Summary of Soil Properties at Experimental Site, 1 980 ........................ 27
3. Summary of Contrast Comparisons used in Analysis, 1980................................. 28
4. Summary of Contrast Comparison Coefficients, 1980..................................................29
5. . Summary of Soil Properties at Experimental Site, 1 981 ...................................... 30
6. Effects of N Fertilizer, Phosphorus Sources and Methods ofApplication on Fababean Shoot Dry Weight at Bozeman, Montana,1 9 8 0 ................ 34
7. Fababean Nodule Number as Affected by N and P Applications atBozeman, Montana, 1 9 8 0 ..................................................................... 34
8. Fababean Nodule Dry Weight as Influenced by N, P Source andMethod Application at Bozeman, Montana, 1980 ........... .................................... 34
9. Effect of Sources of P on Fababean Nitrogenase Activity at Bozeman,Montana, 1980 ................................ 36
10. Correlation Coefficients of Nitrogenase Activity with Nodule Numberand Weight in Fababean at Bozeman, Montana, 1980.......................................... 36
11. Shoot Nitrogen Concentrations as a Function of N, P Sources andMethods of Application in Fababean at Bozeman, Montana, 1 9 8 0 ................... 37
12. Root Nitrogen Concentrations as a Function of N, P Source andMethod of Application in Fababean at Bozeman, Montana, 1980....................... 38
13. Effects of Phosphorus and Nitrogen Application on Fababean Final Forage and Grain Yields, and Grain Percent Total Nitrogen at Bozeman,Montana, 1980 ......................................... 39
14. Contrast Comparison Mean Squares for Fababean Forage and GrainYields and Grain N Concentrations at Bozeman, Montana, 1980 ..................... 39
X
15. Analysis of Variance for Fababean Shoot Dry Weight at Bozeman,Montana, 1 9 8 1 ................................................................................. 41
16. Analysis of Variance for Fababean Root Dry Weight at Bozeman,Montana, 1 9 8 1 .................................i ................................................ ............... .. 41
17. Analysis of Variance for Fababean Nodule Number at Bozeman,Montana, 1 9 8 1 ................................................ 42
18. Analysis of Variance for Fababean Nodule Dry Weight at Bozeman,Montana, 1 9 8 1 ............................................................................................................. 43
19. Analysis of Variance for Fababean Shoot %N at Bozeman, Montana,1 98 1 ................................................................................................................................... 43
20. Analysis of Variance for Fababean Root %N at Bozeman,Montana, 1 9 8 1 ................................................................................................................. 44
21. Analysis of Variance for Fababean Shoot %P at Bozeman,Montana, 1 9 8 1 ................................................................................................................. 44
22. Analysis of Variance for Fababean Root %P at Bozeman,Montana, 1 9 8 1 .......................................................................... 45
23. Effect of P on Dry Bean Nodule Number at Bozeman,Montana, 1980 ............................................................................................................. 48
24. Effect of Source of P on Dry Bean Nodule Weight at Bozeman,Montana, 1980 ..................................... 48
25. Effect of Source of P on Dry Bean Nitrogenase Activity (C2 H4 )at Bozeman, Montana, 1 9 8 0 ...................................................................................... 48
26. Effect of Source of P on Dry Bean Specific Activity at Bozeman,Montana, 1980 ............................................................................................................. 49
27. Effect of Banded (S) and Broadcast (B) Applications on Dry Bean Nodule Number, Dry Weight, N2-ase and Specific Activities atBozeman, Montana, 1 98 0 ........................................................................................... 49
28. Correlation Coefficients of Dry Bean Nitrogenase Activity with Nodule Number and Weight, Shoot and Root %N and Specificat Bozeman, Montana, 1 98 0 ...................................................................................... 50
29. Effects of P Source, Method of Application and N Treatments onDry Bean Shoot N Concentration at Bozeman, Montana, 1980.......................... 51
Tables Page
30. Analysis of Variance for Dry Bean Shoot Dry Weight at Bozeman,Montana, 1 9 8 1 ............................................................................................ 53
31. Analysis of Variance for Dry Bean Root Dry Weight at Bozeman,Montana, 1 9 8 1 ............................................................................................................. 54
32. Analysis of Variance for Dry Bean Nodule Number at Bozeman,Montana, 1 9 8 1 ............................................................................................................. 55
33. Analysis of Variance for Dry Bean Shoot %N at Bozeman,Montana, 1 9 8 1 ............................................................................................................. 55
34. Analysis of Variance for Dry Bean Root %N at Bozeman, Montana,1 9 8 1 ..................................................... , ........................................................................ 56
35. Analysis of Variance for Dry Bean Shoot %P at Bozeman, Montana,1 98 1 .......................................................................... 57
36. Analysis of Variance for Dry Bean Root %P at Bozeman, Montana,1 9 8 1 ........................................................................................................................ 57
37. Analysis of Variance for Dry Bean Grain Yield at Bozeman, Montana,1 9 8 1 ................ 58
38. Effect of Inoculation of Fababean Seed on Growth and Nodulationat Bozeman, Montana, 1 9 8 1 ..................................................... 62
39. Effect of Inoculation of Dry Bean Seed on Growth and Nodulationat Bozeman, Montana, 1 98 1 ..................................................... 63
40. Effect of Inoculation (C|) of Dry Bean Seed on Pods Number andDry Weight at Bozeman, Montana, 1981............................................ 64
41 Effect of Inoculation (C|) of Dry Bean Seed on Final Straw and GrainYields at Bozeman, Montana, 1981.................. 64
42. Analysis of Variance for Fababean Shoot Dry Weight as Affected byP Supply and Mode of N Nutrition at Bozeman, Montana, 1982....................... 65
43. Analysis of Variance for Fababean Root Dry Weight as Affected by PSupply and Mode of N Nutrition at Bozeman, Montana, 1 98 2 .......................... 67
44. Analysis of Variance for Fababean Nodule Number as Affected byP Supply and Mode of N Nutrition at Bozeman, Montana, 1982....................... 68
45. Analysis of Variance for Fababean Nodule Dry Weight as Affected byP Supply and Mode of N Nutrition at Bozeman, Montana, 1982....................... 69
xi
Tables Page
46. Analysis of Variance for Fababean Shoot N Concentrations as Affectedby P Supply and Mode of N Nutrition at Bozeman, Montana, 1 9 8 2 ................ 70
47. Analysis of Variance and Orthogonal Polynomials for Fababean ShootDry Weight at Bozeman, Montana, 1983................................................................. 74
48. Mean Values of Fababean Shoot Dry Weight g/2.Plants at Bozeman,Montana, 1983 ..................................... .. ................................................................... 74
49. Analysis of Variance and Orthogonal Polynomials for Fababean NoduleNumber at Bozeman, Montana, 1983 ............................................................. ........ 76
50. Mean Values for Fababean Nodule Number N°/2 Plants at Bozeman,Montana, 1983 ........................................................................................................... 76
51. Analysis of Variance and Orthogonal Polynomials for Fababean NoduleDry Weight at Bozeman, Montana, 1983.....................................................................
52. Mean Values for Fababean Nodule Dry Weight g/2 Plants at Bozeman,Montana, 1983 ................................................................................................... 79
53. Analysis of Variance and Orthogonal Polynomials for Fababean RootDry Weight (root + nodules) at Bozeman, Montana, 1983................................... 80
54. Mean Values for Fababean Root Dry Weight g/2 Plants at Bozeman,Montana, 1983 ............................................................................................................... 80
55. Analysis of Variance and Orthogonal Polynomials for Fababean ShootPercent N at Bozeman, Montana, 1983................................................................... 81
56. Mean Values for Fababdan Shoot %N at Bozeman, Montana, 1983................... 81
57. Analysis of Variance and Orthogonal Polynomials for Fababean Forageand Grain Yields Kg/ha at Bozeman, Montana, 1983 .......................................... 83
58. Mean Values for Fababean Forage and Grain Yields Kg/ha at Bozeman,Montana, 1983 ...................................................................................................... • ■ ■ 83
59. Analysis of Variance and Orthogonal Polynomials for Fababean ShootDry Weight at Bozeman, Montana, 1984.................................................... 85
60. Mean Values of Fababean Shoot Dry Weight g/4 Plants at Bozeman,Montana, 1984 ............................................................................................................. 85
61. Analysis of Variance and Orthogonal Polynomials for Fababean NoduleDry Weight at Bozeman, Montana, 1984................................................................ 86
62. Mean Values of Fababean Nodule Dry Weight g/4 Plants at Bozeman,Montana, 1984 .................................. 86
63. Analysis of Variance and Orthogonal Polynomials for Fababean RootDry Weight at Bozeman, Montana, 1984................................................................. 88
64. Mean Values of Fababean Root Dry Weight g/4 Plants at Bozeman,Montana, 1984 ....................................... ........................... .... ................................... 88
65. Analysis of Variance and Orthogonal Polynomials for Fababean Nodule+ Root Dry Weight at Bozeman, Montana, 1984............................................ .. 89
66. Mean Values of Fababean Nodule + Root Dry Weight g/4 Plants atBozeman, Montana, 1984 ............................ ,............................................................. 89
67. Analysis of Variance for Green Pea Shoot Dry Weight as Affected byP Supply and Mode of N Nutrition at Bozeman, Montana, 1982.............. 91
68. Analysis of Variance for Green Pea Root Dry Weight as Affected byP Supply and Mode of N Nutrition at Bozeman, Montana, 1982....................... 92
69. Analysis of Variance for Green Pea Nodule Number as Affected by PSupply and Mode of N Nutrition at Bozeman, Montana, 1 9 8 2 .......................... 93
70. Analysis of Variance for Green Pea Nodule Dry Weight as Affectedby P Supply and Mode of N Nutrition at Bozeman, Montana, 1 9 8 2 ................ 94
71. Analysis of Variance and Orthogonal Polynomials for Green PeaShoot Dry Weight g/2 Plants at Bozeman, Montana, 1983 ................ ............... 97
72. Mean Values of Green Pea Shoot Dry Weight Averaged over N andAverage over P Levels, Respectively, at Bozeman, Montana, 1983 ................... 97
73. Analysis of Variance and Orthogonal Polynomials for Green PeaRoot Dry Weight g/2 Plants at Bozeman, Montana, 1983 ..................... ............. 100
74. Mean Values of Green Pea Root Dry Weight Averaged over N andP Levels, Respectively, at Bozeman, Montana, 1983 ............................................ 100
75. Analysis of Variance and Orthogonal Polynomials for Green PeaNodule Number at Bozeman, Montana, 1 9 8 3 ....................................................... 102
76. Mean Values of Green Pea Nodules Number Averaged over N andP Levels, Respectively, at Bozeman, Montana, 1983 ............................................ 102
77. Analysis of Variance and Orhogonal Polynomials for Green PeaNodule Dry Weight g/2 Plants at Bozeman, Montana, 1983 .............................. 105
78. Mean Values of Green Pea Nodules Dry Weight Averaged over N and Averaged over P Levels, Respectively, at Bozeman, Montana,1 98 3 ...................................................................................................... ......................... 105
xiii
Tables Page
79. Analysis of Variance and Orthogonal Polynomials for Green PeaShoot %IN, at Bozeman, Montana, 1983 .......................................................... 107
80. Mean Values of Green Pea Shoot %N Averaged over N and Averagedover P Levels, Respectively, at Bozeman, Montana, 1 98 3 ................... ............ 107
81 Mean Values of Green Pea Seed Yield Averaged over N and Averaged over P Levels, Respectively, Kg/ha at Bozeman,Montana, 1983 ......................................... 108
82. Analysis of Variance and Orthogonal Polynomials for Green PeaShoot Dry Weight at Bozeman, Montana, 1984..................................... ............... 109
83. Mean Values of Green Pea Shoot Dry Weight Averaged over INand P Levels, Respectively, at Bozeman, Montana, 1984..................................... 109
84. Analysis of Variance and Orthogonal Polynomials for Green PeaRoot Dry Weight at Bozeman, Montana, 1984 ..................................................... 110
85. Mean Values of Green Pea Root Dry Weight Averaged over Narid P Levels, Respectively, at Bozeman, Montana, 1984.................................... 110
86. Analysis of Variance and Orthogonal Polynomials for Green PeaNodule + Root Dry Weight at Bozeman, Montana, 1984..................................... Tl I
87. Mean Values of Green Pea Nodule + Root Dry Weight Averagedover N and P Levels, Respectively, at Bozeman, Montana, 1984 ....................... I l l
88. Analysis of Variance and Orthogonal Polynomials for Green PeaNodule Dry Weight at Bozeman, Montana, 1984................................................... 112
89. Mean Values of Green Pea Nodule Dry Weight Averaged over N andP Levels, Respectively, at Bozeman, Montana, 1984 ............................................ 112
90. Analysis of Variance and Orthogonal Polynomials for Green PeaShoot Nitrogen Concentrations at Bozeman, Montana, 1984 ............................ 113
91. Mean Values of Green Pea Shoot Nitrogen Concentrations Averagedover N and P Levels, Respectively, at Bozeman, Montana, 1984 ....................... 113
92. Analysis of Variance for Dry Bean Shoot Dry Weight as Affectedby P Supply and Mode of N Nutrition at Bozeman, Montana, 1 9 8 2 ................ 115
93. Analysis of Variance for Dry Bean Root Dry Weight as Affected by PSupply and Mode of N Nutrition at Bozeman, Montana, 1 98 2 .......................... 115
94. Analysis of Variance for Dry Bean Shoot N Concentrations as Affectedby P Supply and Mode of N Nutrition at Bozeman, Montana, 1 9 8 2 ................ 116
xiv
Tables Page
95. Dry Bean Grain Yield as Affected by P Supply and Mode of NNutrition at Bozeman, Montana, 1982 ................................................................... 117
96. Analysis of Variance and Orthogonal Polynomials for Dry Bean ShootDry Weight at Bozeman, Montana, 1983................................................................ 119
97. Mean Values of Dry Bean Shoot Dry Weight Averaged over N andAveraged over P Levels, Respectively, at Bozeman, Montana, 1983 ................ 119
98. Analysis of Variance and Orthogonal Polynomials for Dry Bean RootDry Weight at Bozeman, Montana, 1983................................................................. 121
99. Mean Values of Dry Bean Root Dry Weight Averaged over N andAveraged over P Levels, Respectively, at Bozeman, Montana, 1983 ................ 121
100. Analysis of Variance and Orthogonal Polynomials for Dry BeanNodule Number at Bozeman, Montana, 1 98 3 ........................................................ 123
101. Mean Values of Dry Bean Nodule Number Averaged over N andAveraged over P Levels, Respectively, at Bozeman, Montana, 1983 ................ 123
102. Analysis of Variance and Orthogonal Polynomials for Dry BeanShoot %N at Bozeman, Montana, 1983................................................................... 125
103. Mean Values for Dry Bean Shoot %N Averaged over N and Averagedover P Levels, Respectively, at Bozeman, Montana, 1 983 ................................... 125
104. Mean Values of Dry Bean Seed Yield Averaged over N and Averagedover P Levels, Respectively, at Bozeman, Montana, 1983 ................................... 126
Appendix Tables
105. Average Monthly Temperatures Recorded at Experimental S ite ....................... 150
106. Total Rainfall, Evaporation and Number of Days with Precipitationat Experimental S ite ............................................. ..................................................... 151
107. Calibration Equations Relating Total Percent N Determined by the Nitrogen Autoanalyzer and the Infra-Red Analyzer Test Values forFababean, Green Pea and Dry Bean.......................................................................... 152
108. Effects of P and Applications on Fababean Shoot Dry Weight, g perPlant, as a Function of Time at Bozeman, Montana, 1980 ................................. 153
109. Contrast Comparisons for Fababean Shoot Dry Weight as a Functionof Time at Bozeman, Montana, 1980 ...................................................................... 153
XV
Tables Page
110. Effects of P and N Applications on Fababean Root Dry Weight,g Plant-1 , as a Function of Time at Bozeman, Montana, 1980 ......................... 154
111. Contrast Comparisons for Fababean Root Dry Weight as a Functionof Time at Bozeman, Montana, 1980 ...................................................................... 154
112. Effects of P and N Applications on Fababean Nodule Number as aFunction of Time at Bozeman, Montana, 1980..................................................... 155
113. Contrast Comparisons for Fababean Nodule Number as a Functionof Time at Bozeman, Montana, 1980 ....................................... ............................. 155
114. Effects of P and N Applications on Fababean Nodule Dry Weight as aFunction of Time at Bozeman, Montana, 1980..................................................... 156
115. Contrast Comparisons for Fababean Nodule Dry Weight as a Functionof Time at Bozeman, Montana, 1980 ..................................................... ................ 156
116. Effects of P and N Applications on Nitrogenase Activity (C2 H4 ) ofFababean as a Function of Time at Bozeman, Montana, 1980 .......................... 157
117. Contrast Comparisons for Fababean Nitrogenase Activity as a Functionof Time at Bozeman, Montana, 1980 ..................................................................... 157
118. Effects of P and N Applications on Specific Nitrogenase Activity forFababean as a Function of Time at Bozeman, Montana, 1980 .......................... 158
119. Contrast Comparisons for Fababean Specific Activity as a Functionof Time at Bozeman, Montana, 1980 ...................................................................... 158
120. Effects of P and N Applications on Fababean Shoot Total PercentNitrogen as a Function of Time at Bozeman, Montana, 1 98 0 ............................ 159
121. Contrast Comparisons for Fababean Shoot Tltal Percent N as aFunction of Time at Bozeman, Montana, 1980.............................. ...................... 159
122. Effects of P and N Applications on Fababean Root Total PercentNitrogen as a Function of Time at Bozeman, Montana, 1980 ............................ 160
123. Contrast Comparisons for Fababean Root Total Percent N as aFunction of Time at Bozeman, Montana, 1980..................................................... 160
124. Effects of P Rates, Sources and Application Methods and N on Fababean Shoot Weight as a Function of Time at Bozeman, Montana,1 9 8 1 ............................................................................................................................... 161
125. Effects of P Rates, Sources and Application Methods and N on Fababean Root Weight as a Function of Time at Bozeman, Montana,1 9 8 1 ................................................................................................................................ 161
xvi
Tables Page
xvii
126. Effects of P Rates, Sources and Application Methods and N on Fababean Nodule Number as a Function of Time at Bozeman,Montana, 1981............................................................................................................... 162
127. Effects of P Rates, Sources and Application Methods and N on Fababean Nodule Dry Weight as a Function of Time at Bozeman,Montana, 1 9 8 1 ............................................................................................................. 162
128. Effects of P Rates, Sources and Application Methods and N on Fababean Shoot Total Percent Nitrogen as a Function of Timeat Bozeman, Montana, 1 98 1 ...................................................................................... 163
129. Effects of P Rates, Sources and Application Methods and N on Fababean Root Total Percent Nitrogen as a Function of Time
' at Bozeman, Montana, 1 9 8 1 ................................................................................... 163
130 Effects of P Rates, Sources and Application Methods and N onFababean Shoot P% as a Function of Time at Bozeman, Montana,1 9 8 1 ........................................................................................................ ...................... 164
131. Effects of P Rates, Sources and Application Methods on FababeanRoot %P as a Function of Time at Bozeman, Montana, 1 98 1 ............................ 164
132. Effects of P and N Applications on Dry Bean Shoot Dry Weight,g per plant, as a Function of Time at Bozeman, Montana, 1 98 0 ....................... 165
133. Contrast Comparisons for Dry Bean Shoot Dry Weight as a Functionof Time at Bozeman, Montana, 1980 ..................... ................................................ 165
134. Effects of P and N Applications on Dry Bean Root Dry Weight,g plant-1 , as a Function of Time at Bozeman, Montana, 1980 ................ .. 166
135. Contrast Comparisons for Dry Bean Root Dry Weight as a Function ofTime at Bozeman, Montana, 1980.............. .............................................................. 166
136. Effects of P and N Applications on Dry Bean Nodule Number as aFunction of Time at Bozeman, Montana, 1980..................................................... 167
137. Contrast Comparisons for Dry Bean Nodule Number as a Function ofTime at Bozeman, Montana, 1980................................................... .. ■ ■ ................ 167
138. Effects of P and N Applications on Dry Bean Nodule Dry Weight as aFunction of Time at Bozeman, Montana, 1980...................................................: 168
139. Contrast Comparisons for Dry Bean Nodule Dry Weight as a Functionof Time at Bozeman, Montana, 1980 ...................................................................... 168
140. Effects of P and N Applications on Nitrogenase Activity (C2H4 ) of DryBean as a Function of Time at Bozeman, Montana, 1 98 0 ................................... 169
Tables Page
Tables Page
xviii
141. Contrast Comparisons for Dry Bean Nitrogenase Activity as a Functionof Time at Bozeman, Montana, 1980 ..................................................................... 169
142. Effects of P and N Applications on Specific Nitrogenase Activity forDry Bean as a Function of Time at Bozeman, Montana, 1980............................ 170
143. Contrast Comparisons for Dry Bean Specific Activity as a Functionof Time at Bozeman, Montana, 1980 ..................................................................... 170
144. Effects of P and N Applications on Dry Bean Shoot Total PercentNitrogen as a Function of Time at Bozeman, Montana, 1 98 0 ............................ 171
145. Contrast Comparisons for Dry Bean Shoot Total Percent Nitrogenas a Function of Time at Bozeman, Montana, 1980 ............................................ 171
146. Effects of P and N Applications on Dry Bean Root Total PercentNitrogen as a Function of Time at Bozeman, Montana, 1980 ..................... .. 172
147. Contrast Comparisons for Dry Bean Root Total Percent Nitrogenas a Function of Time at Bozeman, Montana, 1980 ............................................ 172
148. Effects of P and N Applications on Dry Bean Final Forage and Grain Yield and Grain Percent Total Nitrogen at Bozeman, Montana,1 9 8 0 ......................... 173
149. Contrast Comparisons for Dry Bean Forage and Grain Yields atBozeman, Montana, 1 9 8 0 .......................................................................................... 173
150. Effects of P Rates, Sources and Application Methods and N on DryBean Shoot Weight g Plant-1 at Bozeman, Montana, 1 9 8 1 ................................ 174
151. Effects of P Rates, Sources and Application Methods and N on DryBean Root Weight g Plant-1 at Bozeman, Montana, 1981 ................................... 174
152. Effects of P Rates, Sources and Application Methods and N on DryBean Nodule Number at Bozeman, Montana, 1981............................................... 175
153. Effects of P Rates, Sources and Application Methods and N on DryBean Shoot N% at Bozeman, Montana, 1 9 8 1 .................................. 175
154. Effects of P Rates, Sources and Application Methods and N on DryBean Root N % at Bozeman, Montana, 1 98 1 ............................... ......................... 176
155. Effects of P Rates, Sources and Application Methods and N on DryBean Shoot P % at Bozeman, Montana, 1 9 8 1 ........................................................ 176
156. Effects of P Rates, Sources and Application Methods and N on DryBean Root % P at Bozeman, Montana, 1981 .......................................................... 177
157. Effects of P Rates, Sources and Application Methods and N on DryBean Pods Numbers and Weight at Bozeman, Montana, 1981............................ 177
158. Effects of P Rates, Sources and Application Methods and N on DryBean Straw and Grain Yields at Bozeman, Montana, 1 9 8 1 ................................ 178
159. Fababean Shoot Dry Weight as Affected by P Supply and Mode ofN Nutrition at Bozeman, Montana, 1982 .............................................................. 179
160. Fababean Root Dry Weight as Affected by P Supply and Mode ofN Nutrition g/2 Plants at Bozeman, Montana, 1982 ............................................ 179
161. Fababean Nodule Number per 2 Plants as Affected by P Supplyand Mode of N Nutrition at Bozeman, Montana, 1 982 ..................... .................. 180
162. Fababean Nodule Dry Weight as Affected by P Supply and Modeof N Nutrition at Bozeman, Montana, 1982 .......................................................... 180
163. Fababean Shoot N Concentrations as Affected by P Supply andMode of N Nutrition at Bozeman, Montana, 1982 .............................................. 181
164. Fababean Pods Number and Weight as Affected by P Supply andMode of N Nutrition at Bozeman, Montana, 1982 .............................................. 181
165. Green Pea Shoot Dry Weight as Affected by P Supply and Modeof N Nutrition at Bozeman, Montana, 1982 .......................................................... 182
166. Green Pea Root Dry Weight as Affected by P Supply and Mode ofN Nutrition at Bozeman, Montana, 1982 .............................................................. 182
167. Green Pea Nodule Number as Affected by P Supply and Mode ofN Nutrition at Bozeman, Montana, 1982 ............................................................... 183
168. Green Pea Nodule Dry Weight as Affected by P Supply and Modeof N Nutrition at Bozeman, Montana, 1982 .......................................................... 183
169. Green Pea Shoot N Concentrations as Affected by P Supply andMode of N Nutrition at Bozeman, Montana, 1982 .............................................. 184
170. Green Pea Grain Yield as Affected by P Supply and Mode of NNutrition at Bozeman, Montana, 1982 ..................... .............................................. 184
171. Dry Bean Shoot Dry Weight as Affected by P Supply and Modeof N Nutrition at Bozeman, Montana, 1982 ............................................ ............. 185
172. Dry Bean Root Dry Weight as Affected by P Supply and Modeof N Nutrition at Bozeman, Montana, 1982 .......................................................... 185
173. Dry Bean Nodule Number as Affected by P Supply and Modeof N Nutrition at Bozeman, Montana, 1982 .......................................................... 186
xix
XX
Tables Page
174. Dry Bean Shoot N Concentrations as Affected by P Supply andMode of N Nutrition at Bozeman, Montana, 1982 ............................................... 186
175. Analysis of Variance and Orthogonal Polynomials for Dry BeanShoot Dry Weight at Bozeman, Montana, 1984..................................................... 187
176. Mean Values of Dry Bean Shoot Dry Weight Averaged over N and Averaged over P Levels, Respectively, at Bozeman, Montana,1 9 8 4 ............................................................................................................................... 187
177. Analysis of Variance and Orthogonal Polynomials for Dry BeanRoot Dry Weight at Bozeman, Montana, 1984 ...................... ............................... 188
178. Mean Values of Dry Bean Root Dry Weight Averaged over N and Averaged over P Levels, Respectively, at Bozeman, Montana,1 98 4 ................................................................................................................................ 188
179. Analysis of Variance and Orthogonal Polynomials for Dry BeanNodule Dry Weight at Bozeman, Montana, 1984.............................................. . 189
180. Mean Values of Dry Bean Nodule Dry Weight Averaged over N andAveraged over P Levels, Respectively, at Bozeman, Montana, 1984 ................ 189
181. Analysis of Variance and Orthogonal Polynomials for Dry Bean Root+ Nodule Dry Weight at Bozeman, Montana, 1 98 4 ............................................. 190
182. Mean Values of Dry Bean Root + Nodule Dry Weight Averaged over N and Averaged over P Levels, Respectively, at Bozeman, Montana,1 9 8 4 ................................................................................................................ ; ............ 190
183. Analysis of Variance and Orthogonal Polynomials for Dry BeanShoot Nitrogen Concentrations at Bozeman, Montana, 1984 ............................ 191
184. Mean Values of Dry Bean Shoot Nitrogen Concentrations Averaged over N and Averaged over P Levels, Respectively, at Bozeman,Montana, 1984 191
xxi
LIST OF FIGURES
Figures Page
1. Shoot N concentrations of 4 week-old fababean plants in responseto P applications with and without supplied N, 1982 ................................... .. 71
2. Shoot N concentrations of 3 week-old fababean plants in responseto P applications with and without supplied N, 1982 ............................ ............. 71
3. Shoot N concentrations If 12 week-old fababean plants in responseto P applications with and without supplied N, 1982 .......................................... 72
4. Shoot weight (g/2 plants) of 5 week-old fababean plants in responseto P and N applications at Bozeman, Montana, 1983 ......................................... 75
5. Shoot weight (g/2 plants) of 9 week-old fababean plants in responseto P and N applications at Bozeman, Montana, 1983 .......................................... 75
6. Number of. nodules per 2 plants on 5 week-old fababean plants inresponse to P and N applications at Bozeman, Montana, 1983 .......................... 78
7. Nodule dry weight (g/2 plants) of 11 week-old fababean plants inresponse to P and N applications at Bozeman, Montana, 1983 .......................... 78
8. Fababean grain yield, kg/ha, as affected by P and N applications atBozeman, Montana, 1 98 3 ................................................................................... .. 84
9. Shoot weight (g/2 plants) of 5 week-old green pea plants in responseto P and N applications at Bozeman, Montana, 1983 ..................................... .. . 98
10. Shoot weight (g/2 plants) of 7 week-old green pea plants in responseto P and N applications at Bozeman, Montana, 1983 .......................................... 98
11. Shoot weight (g/2 plants) of 9 week-old green pea plants in responseto P and N applications at Bozeman, Montana, 1983 .......................................... 99
12. Root weight (g/2 plants) of 7 week-old green pea plants in responseto P and N applications at Bozeman, Montana, 1983 .......................................... 99
13. Root weight (g/2 plants) of 9 week-old green pea plants in responseto P and N applications at Bozeman, Montana, 1983 .......................................... 101
14. Nodule number per 2 plants of 5 week-old green pea plants inresponse to P and N applications at Bozeman, Montana, 1983 ....................... 101
Figures Page
15. Number of nodules per 2 plants of 7 week-old green pea plants inreponse to P and N applications at Bozeman, Montana, 1983............................ 103
16. Number of nodules per 2 plants of 9 week-old green pea plants inresponse to P and N applications at Bozeman, Montana, 1983 .............. 103
17. Shoot weight (g/2 plants) of 9 week-old dry bean plants in responseto P and N applications at Bozeman, Montana, 1983 .......................... ................ 120
18. Nodule number per 2 plants of 5 week-old dry bean plants inresponse to P and N applications at Bozeman, Montana, 1983 ................ .. 122
19. Number of nodules per 2 plants of 5 week-old dry bean plants inresponse to P and N applications at Bozeman, Montana, 1983 .......................... 122
20. A model for symbiotic N2 -fixation by bacteria..................................................... 129
The most common grain legumes in temperate and sub tropical regions are Pisum, Phaseolus and Vicia beans. Their yields are often lower than the potential yield due to deficiencies in both P and N. The objectives of this research were to evaluate the relative effectiveness of different sources of phosphorus fertilizers, levels and methods of application on dry beans (Phaseolus vulgaris L.) and fababean [Vicia faba L.) and also to evaluate the effects of N and P fertilizers and their interaction on nodulation, N2 -fixation and growth of fababean, dry bean and green pea (Pisum sativum L.) grown in the field.
In 1980, a split plot, randomized complete block design with four replications was used. Main plots were 0 and 100 Kg ha-1 N applied as ammonium nitrate (NH4 NO3). Sub plots were a no P control, two P sources, orthophosphoric acid (H3 PO4 ) as liquid P fertilizer and triple superphosphate. In 1981, the orthophosphoric acid was replaced by monoammonium phosphate. In 1982, 1983 and 1984, factorials in randomized complete block designs with four replications were used with varying levels of N and P fertilizers.
There were differential responses of fababean and dry bean grain yields to P sources and methods of application. Nodulation and N2-fixation in fababean reached a maximum at pod filling and remained constant until pod filling was complete and then showed a decline. In dry bean, however, maximum nodulation and N2Tixation reached a maximum during pod set and declined rapidly during the final weeks of growth. Application of 100 Kg ha-1 of fertilizer N reduced nitrogenase activity by 75, 72, 82 and 75 percent in dry bean at the four harvests but only 4 7 ,6 0 ,6 2 and 57 in fababean. Excellent positive linear correlations between acetylene reduction rates and nodule number and mass were found with both fababean and dry bean in 1980.
Increasing P supply increased nodule number and nodule dry weight but these increases paralleled increases in shoot and root dry weight and suggested that increasing P supply increases nodulation and N2Tixation in the three different species of host plants by stimulating the plant growth rather than by affecting nodule initiation and function. A model is proposed to explain the inhibitory effects of ammonia on nitrogenase activity. It suggests that ammonia acts as an uncoupler or ion ionophore and dissipates the electrochemical proton gradient created by the bacteriod respiratory chain. More importantly, the destruction of the membrane potential suppresses the low potential electrons that might be necessary in reduction reactions within the bacteroids.
I
CHAPTER I
INTRODUCTION
Grain legumes represent the most economic source of protein for human nutrition
and many compete successfully with animal protein sources in relation to protein and
essential amino-acid content (IVIacgiIIivry and Bosley, 1962). Protein production from I ha
of land is 28.8 times greater from soybeans and 14.5 times, greater from dry beans, than
from beef (Harkness, 1967).
Pisum, Phaseofus and Vicia beans are the most common legume crops in the tempera-
tate and sub-tropical regions. Peas probably originated from the Middle East and are grown
over the world on about 9 million hectares annually. The production is approximately 10
million metric tons per year (Allen and Allen, 1981).
The common bean (Phaseo/us vulgaris L.) is frequently cultivated in most agricultural
areas of the world and is the primary food staple in many developing countries, especially
Latin America (Bazan, 1975; Pinchinat, 1977).
Fababean (Vicia faba) is a major food legume in the Middle East and is used as a for
age in cereal-legume rotations throughout much of the Canadian, Northern Great Plains.
Fababean is capable of achieving high seed yields (7 metric tons/ha) and protein contents
(23 to 32%) (Koala, 1982; Evans et al., 1972). They require large quantities of nitrogen to
attain full yield and protein potential and may be influenced by phosphorus availability
(McEween, 1970; Richards, 1977).
Bean yields are often lower than the potential yield due to deficiencies in both P and
N in the tropics (FAO, 1979; Hernandez-Bravo, 1973; Graham, 1978). An alternative to
I
2
extensive fertilizer application which is too costly for many small farmers is to utilize IM2-
fixation through the \eQume-Rhizobium symbiosis. It is also assumed that P deficiency is
the most important single limiting factor for N2-fixation and legume production.
About 85 percent of the cultivated soils in Montana tested medium, low or very low
in available P (Sims, 1971). Consequently, most cultivated soils in Montana will give an
economic response to P fertilizer additions unless there is a more limiting factor. The
most common phosphorus fertilizer used in Egypt is a low quality ordinary superphos
phate which is broadcast applied (El-Attar, 1981; Sims, 1981, personal communication).
Many current methods in both developed and developing countries do not include banding
P fertilizer near the seed or placement with the seed. Therefore, source, rate and method of
application of P may be negatively influencing symbiotic N2 -fixation in those conditions.
There are many questions regarding the role of phosphorus in nodulation and N2-
fixation. Studies with some of these species do not separate out the effects of phosphorus
from other factors such as host plant effects. It is well documented that high rates of com
bined N inhibit nodulation and N2Tixation (Oghoghorie and Pate, 1971). The inhibition
mechanism is still unclear even though a few studies have considered the PxN interaction
as a means of overcoming some of the ammonia inhibition of nodulation and N2Tixation.
The literature is limited on the effect and mechanism of P and PXN interactions on
food legumes. The objectives of this research were to: ( I) evaluate the relative effective
ness of different sources and levels of phosphorus fertilizers; (2) evaluate methods of appli
cation on dry bean and fababean in soils of low P availability; and (3) to evaluate the
effects of N and P fertilizers and their interaction on nodulation N2Tixation and growth of
fababean, dry bean and green pea.
3
CHAPTER 2
LITERATURE REVIEW
Effect of P Placement on !Modulation, N2-Fixation and Growth
Cummings (1943) stated that the "most efficient and most effective placement of
fertilizer is that which provides for an adequate supply of soluble nutrients in a well aerated
zone of moist soil occupied by actively absorbing plant roots at periods of growth when the
demands of the plant for nutrients are most acute." These factors have been recognized,
but they have not been quantitatively characterized for many legumes, such as fababean
and dry bean. A major problem has been the difficulty in achieving the ideal conditions
which Cummings described.
Band application of fertilizer places the fertilizer in a smaller soil volume than broad
cast application when the fertilizer is added at the same rate. Consequently, roots in
contact with banded fertilizer will be in zones of higher fertilizer concentration than roots
with broadcast application. However, broadcast application usually results in more zones
of root-fertilizer contact.
Diwit (1953) developed an equation that expresses the relation between plant uptake
of both banded and broadcast fertilizer, whereby the smaller volume of fertilized soil with
banded placement is compensated by the higher concentration that results in greater ferti
lizer uptake per unit volume of fertilized soil. Singh and Black (1964) confirmed that
Diwit's compensation function represents the results to a first approximation.
Currently, there is little information available to compare the efficiency of broadcast
versus band placement of P on nodulation, N2-fixation and growth of fababean and dry
4
bean. However, there is considerable information available involving the effect of fertilizer
placement on the corn growth (Zea mays L ). Bates et al. (1965) reported that the growth
and nutrient content of corn was substantially increased by fertilizers placed with the seed
in comparison to the same rate located either 5 cm below or beside the seed. Nelson
(1956) concluded that 224 kg/ha of fertilizer in the row is frequently as effective as 448
kg/ha broadcast for increasing corn yields. Werkhoven et al. (1967) reported that banding
28 was as effective as broadcasting 56 kg P/ha. Nelson and Randall (1968) found a signifi
cant response in early growth and yield when the fertilizer was placed in a band near or in
direct contact with the seed without significant difference between the two treatments.
Radioactive P has permitted determination of the quantity of fertilizer P absorbed
by plants with different placement methods. Nelson et al. (1949) reported that fertilizer P
absorbed by plants was less for broadcast than for placement with the seed or for mixing in
the row. However, broadcast was equal to the other placement methods with respect to
corn yield and total P content.
The effects on plant uptake pf banded versus broadcast P are dependent on such fac
tors as soil structure, temperature, moisture, and chemical form of the P. Olsen et al.
(1967) reported that absorption of P by corn seedlings was inversely related to the soil
moisture tension. The decreased P uptake with increased soil moisture tension may play an
important role when comparing banded and broadcast application. Banded P is near the
plant and the soil moisture develops high tension sooner than an area further removed
from the plant due to water uptake by the plant. Consequently, broadcast and incorpo
rated P might be more readily absorbed than banded P during dry periods.
Robinson et al. (1959) studied the effect of temperature on response of red clover to
banded P in a P-deficient soil. They noted a 22% yield increase to band application at IO0C
and only 34% at 27°C. This was not due to the banded application being less effective at
higher temperatures, but due to the broadcast being more effective at higher temperatures.
5
It was concluded that band placement was apparently the more effective because of an
increased concentration of phosphorus in a small portion of the root zone. Furthermore a
band application would be particularly important on soils low in available phosphate,
especially if they were high in P fixing capacity. Ketcheson (1957) found that fertilizers
distributed in bands compared to seed placement resulted in a greater increase in yield of
dry matter at IS 0C than at 20°C for greenhouse grown corn.
Some investigators have not found band application to be superior to broadcast. Ham
et al. (1973) reported that soybean seed yields increased with increasing rates of applied P
and the yield from seed placed fertilizer was greater per unit P than the yield from band
and broadcast P. Ham et al. (1978) also studied, the effects of fertilizer placement on
soybean seed yield, N2-fixation, and 33P uptake in soybean. Seed yield and total plant P
increased significantly from adding P fertilizer, although no differences were found among
the various placements. It was speculated that the Iackof differences among fertilizer place
ments may have been due to the warm soil temperatures on a well-drained soil with a pH
of approximately 7.0.
Yost et al. (1979) reported that broadcast treatments gave greater yields than band
treatments at the same rates for the first corn crop grown. However, total yields in the
field and P uptake at the end of four seasons were very similar for broadcast and band
treatments in which the same total amount of P had been applied to a high P-fixation
capacity soil.
Duell (1974) reviewed the literature on P fertilization for forage establishment and
found that legume seedlings are usually less capable than grasses to obtain P from the low
soil-P concentrations associated with broadcast fertilizer applications. Seedling growth of
both grasses and legumes is often enhanced by placing P in concentrated bands directly
underneath the seed row (band seeding). Moving the fertilizer band as little as 2 or 3 cm to
the side of the seed row is often sufficient to significantly reduce early growth of legume
6
seedlings. Brown (1959) seeded alfalfa with triple superphosphate banded and broadcast at
rates ranging from 100 to 800 kg/ha. All banded rates except the lowest resulted in a
doubling of alfalfa seedling size. However, only the highest broadcast rate increased the
alfalfa seedling growth rate.
Sleight et al. (1984) reported that the amount of P uptake by oats (Avena sativa L.)
was nearly proportional to the volume of the soil containing the applied P fertilizer.
Apparently, the early beneficial effects of banding are obtained primarily from placing all
of the fertilizer where contact by active roots is more likely, rather than from any increase
in availability that may be obtained from decreased soil-fertilizer contact associated with
banding. This suggests that the most efficient use of P fertilizer in which P is relatively
immobile in soils will be made by the young plants if the fertilizer is mixed thoroughly
with the soil near the seed.
Effects of band and broadcast placements might be affected by N availability. Miller
et al. (1958) reported that placement of N fertilizer caused a relative increase in the
feeding power of the root system on band-placed phosphorus. N had a greater influence
when mixed with P than when placed in a band 3 to 4 inches from the band. The influence
was nearly independent of the soil phosphate level when the N was mixed in the band
phosphorus. However, it was not independent of the soil phosphate level when the nitro
gen was separated from the phosphorus band.
In a split-root experiment with corn, Engelstad and Allen (1971) showed that P
applied to one side of the root system was translocated throughout the entire root system
and was effective in promoting root and top growth. They found that the presence of
ammonium N enhanced the uptake of P from a band, but had no effect on uptake of P
mixed throughout the soil.
7
The management of P placement might be different on the tropics with soils of high P
fixing capacity. The traditional way to cope with the high P fixation is to apply the fertil
izer in bands to minimize the volume of soil with which it will react. The high cost of
superphosphate and other energy-dependent inputs has led to exploring additional ways of
managing high P-fixing soils with limited capital resources, particularly in small farming,
systems in the tropics. The results are completely different in soils with extremely high
fixation capacity and very low levels of available P. Studies by Yost et al. (1979) on a
Brazilian oxisol which requires 750 ppm P at the standard solution concentration indicate
that banded applications are inferior to broadcast application for the first corn crop. The
available P in the soil was so low that root development was limited to the regions where P
was applied. However, the very limited root development around the banded treatments
caused the plants to be less resistant to periods of moisture stress. Similar results were
reported by Hansen (1979). The best method for applying P to these high adsorbing soils
appears to be an initial broadcast application followed by maintenance band applications.
Effect of P Sources on Modulation, N2-Fixation and Growth
Any soil condition may cause some degree of reversion from soluble to insoluble
forms. Divalent and polyvalent cations in the soil cause reversion of water soluble phos
phates to less soluble forms when P fertilizer is applied to soil. The term fixation there
fore refers to the degree of reversion which adversely affects the recovery of applied P by
plants or chemical extractants (McLean and Logan, 1970). Soils differ greatly in P fixing
capacity, plants respond differentially to a given source of P depending on the P fixing
capacity of the soil. This implies that there is a best source of P for a given soil condition.
This has not been evaluated for fababeans and dry beans with respect to nodulation,
N2-fixation and yield in Montana.
8
McLean and Logan (1970) evaluated the sources of P for plants grown in soils with
differing phosphorus fixation tendencies. They found that P content of corn seedlings
increased in direct proportion to water solubility of "available" P in relatively low fix
ation soils. However, P content decreased with increased water solubility of P in high fix
ation soils.
Many other workers have reported similar results. Increased yields or P availability to
crops has been obtained with increased water solubility of P fertilizer in low P fixation
soils (Lawton et al., 1956; Webb and Pesek, 1958; Webb et al., 1961). However, several
reports (McLean and Wheeler, 1964; Webb and Pesek, 1959) indicated that increased water
solubility is of little or no benefit on acid soils. Rock phosphate under acid conditions has
produced crop yields equal to or better than those from superphosphate (McLean et al.,
1952).
The development of superphosphoric acid, containing approximately equal amounts
of ortho and condensed phosphates has also created much interest in the agronomic ,effec
tiveness of condensed phosphates (Gordon and Kamprath, 1971). However, their effec
tiveness as fertilizers is considered to be almost entirely dependent upon their hydrolysis to
orthophosphate (OP) (Sutton and Larsen, 1964). Although several factors affect the
hydrolysis rate. The above authors found that the level of biological activity was the most,
important factor in soils. In soils with low levels of biological activity, P uptake by rye
grass (Lolium mu/tif/orum Lam.) was significantly lower with pyrophosphate than with OP.
Pyrophosphate was a relatively ineffective source of P prior to hydrolysis to the ortho
phosphate form. Differences between P sources in soils with higher levels of biological
activity were detectable only in the first cutting. Uptake of P by barley [Hordeum vulgare
L.) from solutions containing pyrophosphate was lower by a factor of 2.4 than that from
OP solutions.
9
Soil phosphorus levels, formulations, and plant growth stage might also explain
differences obtained with different P sources. Bureau et al. (1953) found that superphos
phate and double superphosphate were equally available as a source of phosphorus on the
high phosphorus soil throughout the growing season. However, superphosphate was slightly
superior on the medium and low phosphorus soils. Calcium metaphosphate was less availa
ble than superphosphate or double superphosphate in the early portion of the season, but
equaled the availability of the superphosphate carriers during the latter part of the season.
However, calcium metaphosphate furnished less phosphorus to plants throughout the
season than either of the above sources on the high phosphorus soil.
Robertson and Hutton (1972) evaluated ten phosphorus sources on the growth of
corn, peanut (Arachis hypogae L ), oat and soybean (Glycine max. L.) and found that
these crops responded differentially, However, the phosphorus sources could be arranged
in the following descending drder of response: superphosphate, dinitra phosphate (17-22-
Statistics were based on contrast comparisons as shown in Table 3. The treatments
were divided into 9 mutually orthogonal contrasts and 8 additional contrasts representing
the interaction between Ci and each of C2, C3, C4 , C5, C6, C7, C8 and C9, respectively.
The contrast comparison coefficients are reported in Table 4.
Means and error mean squares used in the comparisons were previously obtained by
analyzing the experiment as a split plot. All programs used were from 'MSUSTAT' (Lund,
1979).
Field Experiment 1981
Field experiments were established on June 23, 1981 as described for 1980 with the
following modifications. The two sources of phosphorus used were monoammonium phos
phate, 11-48-0; (MP) and triple superphosphate, 0-45-0, (TP) at two levels of application,
60 kg P2O5Zha and 120 kg P2O5Zha applied either broadcast and incorporated prior to
seeding (B) or banded with the seed (S). Treatments were split into main plots containing 0
28
Table 3. Summary of Contrast Comparisons used in Analysis, 1980.
Contrast Designation Contrast Description
I C1 N vs no N2 C2 Control vs P treatments3 C3 Ortho vs TP4 C4 Banded vs Broadcast application5 Cs P1 low level vs P2 at high level6 C6 P1 vs P2 in Ortho7 C7 P1 vs P2 in TP8 C8 Banded vs Broadcast in Ortho9 Cg Banded vs Broadcast in TP
10 C1 X C2 N X (control vs P treatments)11 C1 X C3 N X (Ortho vs TP)12 C1 X C4 N X (Banded vs Broadcast)13 C1 X Cs N X (P1 vs P2 )14 C1 X C6 N X (P1 vs P2 in Ortho)15 C1 X C7 N X (P1 vs P2 in TP)16 C1 X Cs N X (Banded vs Broadcast in Ortho)17 C1 X Cg N X (Banded vs Broadcast in TP)
and 100 kg ha-1 nitrogen applied as ammonium nitrate. The experimental design was a
2X2X2 factorial in a split plot with the nitrogen levels as main plots and P sources, rates
and methods of application as subplots. Three replications were used.
Two control treatments were included in each replication (uninoculated and no P,
and inoculated no P). Two legume crops, fababean (cv. Ackerperle) and dry bean (cv. Ul
111) were seeded with a John Deere 71 Flexiplanter. Except for the uninoculated control,
seed was coated with peat cultures containing recommended strains of Rhizobium (Nitragin
Co., Milwaukee, Wl).
In addition to the variables measured during the 1980 field experiment, pods were
counted and dry weight evaluated for dry bean in the third harvest, and dry bean and
fababean for the fourth harvest.
All plots were irrigated to field capacity one day prior to the second, third and fourth
harvests to allow easy excavation of roots and nodules.
Meteorological observations are reported in Tables 105 and 106 (Appendix).
29
Table 4. Summary of Contrast Comparison Coefficients, 1980.
Treatments*+N -N
Contrasts I 2 3 4 5 6 7 8 9 I 2 3 4 5 6 7 8 9I C1 I I I I I I I I I - I - I - I - I - I - I - I - I - I2 C2 +8 - I - I - I - I - I - I - I - I +8 - I - I - I -1 - I - I - I - I3 C3 0 I I I I - I - I - I - I 0 I I I I - I - I - I - I4 C4 0 - I I - I I - I I - I I 0 - I +1 - I +1 - I +1 - I +15 C5 0 I I - I - I I I - I - I 0 I I - I - I I I - I - I6 C6 0 I I - I - I 0 0 0 0 0 I I - I -.1 0 0 0 07 C7 0 0 0 0 0 I I - I - I 0 0 0 0 0 I I - I - I8 C8 0 - I I - I I 0 0 0 0 0 - I I - I I 0 0 0 09 C9 0 0 0 0 0 - I I - I I 0 0 0 0 0 - I I - I I
10 C1XC2 +8 - I - I - I - I - I - I - I - I -8 I . I I I I I I I11 C1X C3 0 I I I I - I - I - I - I 0 - I - I - I - I I I I I12 C1XC4 0 - I I - I I - I I - I I 0 +1 - I +1 - I +1 - I +1 - I13 C1XC5 0 I I - I - I I I - I - I 0 - I - I I I - I - I I I14 C1X C6 0 I I - I - I 0 0 0 0 0 - I - I I I 0 0 0 015 C1X C7 0 0 0 0 0 I I - I - I 0 0 0 0 0 - I - I I I16 C1X C8 0 - I I - I I 0 0 0 0 0 I - I I - I 0 0 0 017 Cl X Cg 0 0 0 0 , 0 - I I - I I 0 0 0 0 0 I - I I - I
*Treatment number refer to those of Table 1.
The field was similar to the one used in 1980. Soil samples were collected prior to
planting as previously described and results of chemical analyses are reported in Table 5.
The soil was a fine-silty, mixed family of Typic Haploborolls (Koala, 1982). The experi
mental area had been fallowed the previous year.
Statistical Analysis
Two separate analysis of variances were calculated. The experiment was first analyzed
as a split plot design in a factorial arrangement omitting the two controls. This analysis
allowed the determination of the N and P main effects and interactions. A second analysis
was performed as a split plot in randomized complete block design with the two controls
included. The nitrogen treatments served as main plots and all 10 subplots as independent
30
Table 5. Summary of Soil Properties at Experimental Site, 1981.
higher root N concentrations than orthophosphoric acid at the third harvest (P < 0.05)
but was less at maturity (Tables 122 and 123, Appendix). Increasing P supply increased
root N concentrations in triple superphosphate (contrast C7 ) but had no effect with ortho
phosphoric acid. Root N concentrations were higher when P was banded than when broad
cast. Root N concentrations declined gradually from the second harvest to maturity sug-,
gesting translocation to the reproductive organs.
Grain Yield
There were significant phosphorus sources X nitrogen as well as method of P applica
tion X nitrogen interactions (Tables 13 and 14). Triple superphosphate, banded application
increased fababean grain yield relative to the control on plants supplied with 100 Kg ha-1
of inorganic N. Broadcast phosphorus at 27 Kg ha-1 resulted in an initial decrease in grain
yield followed by an increase to the control level. On plants reliant on symbiotically fixed
N, banded application significantly decreased fababean grain yield at the low phosphorus
level followed by an increase to the control level. Broadcast application in that case did not
affect fababean grain yield. Banded and broadcast decreased grain yield relative to the con
trol when phosphorus was applied as orthophosphoric acid on plants supplied with inor
ganic N. However, banded application resulted in greater grain yield reduction than
broadcast.
39
Table 13. Effects of Phosphorus and Nitrogen Application on Fababean Final Forage and Grain Yields, and Grain Percent Total Nitrogen at Bozeman, Montana, 1980.
Table 15. Analysis of Variance for Fababean Shoot Dry Weight at Bozeman, Montana, 1981.
Source of Variation dfWeeks from Emergence
4 7 10 13
Mean Squares
N v s n o N I 0.0282 76.16 1189 9188Inoculated vs not inoculated I 0.2139 73.21** 7.97 6514.7**No P (inoculated) vs P (factorial) I 0.0320 10.64 74.15 185.7
Factorial 14 ,
P sources I 0.0252 12.61 42.38 3652*P levels I 0.0052 68.64* 1.802 559.7
Methods of application I 0.1302 9.72 435.0 360.2All 2, 3 and 4 factors interaction 11 0.4906'** 124.26** 2000.0** 11484.2**Error 36 0.0843 9.879 124.9 696.2
* and * * denote significance at the 5 and 1% levels, respectively.
table 16. Analysis of Variance for Fababean Root Dry Weight at Bozeman, Montana,1981.
Weeks from Emergence
Source of Variation df 4 7 13
Mean Squares
N vs no N I .0350 0.3183 37.29Inoculated vs not inoculated I .1409** .1361 88.45**No P (inoculated) vs P (factorial) I .0103 .0076 4.71
Factorial 14P sources I .0239 .0463 19.13P levels I .0001 .1938 13.76
Methods of application I .0380 .1964 1.96All 2, 3 and 4 factors interaction 11 .2743** 1.9827** 67.88**Error 36 .0159 .1347 11.86
* and * * denote significance at the 5 and I % levels, respectively.
Nodule Number
Mean squares and mean values are reported in Table 17 and Table 126 (Appendix)
respectively. Application of 100 Kg ha”1 N only slightly reduced fababean nodule number
at all harvests except at the second sampling (7 weeks from emergence) where a significant
decrease was observed. Increasing phosphorus supply generally increased nodule number
but this was not statistically significant in 1981. Triple superphosphate produced higher
42
Table 17. Analysis of Variance for Fababean Nodule Number at Bozeman, Montana, 1981.
Source of Variation dfWeeks from Emergence
4 7 13
Mean SquaresN v s n o N I 132.0 22970* 5762inoculated vs not inoculated I 1610.55 19912.08** 169932**No P (inoculated) vs P (factorial) I 1220.29 155.52 74135**
Factorial 14P sources I 261.3 6604* 2080P levels I 481.3 3658 18720
Methods of application I 216.7 728 19040All 2, 3 and 4 factors interaction 11 8433.0** 21543** 124559**Error 36 663.6 1296 8137
* and * * denote significance at the 5 and 1% levels, respectively.
nodule numbers than monoammonium phosphate probably due to nitrogen in monoam
monium phosphate. Nodule number was higher in banded than broadcast application at
most harvests.
Nodule Dry Weight
Fababean nodule dry weight followed the same pattern as nodule number (Table 18;
Table 127, Appendix). Nitrogen addition decreased nodule dry weight except only at the
second harvest (P < 0.01). This differs greatly from dry bean results where significant
decreases were obtained at all harvests. Increasing phosphorus supply increased nodule
weight at maturity (P < 0.01).
Shoot and Root N Concentrations
Mean shoot N concentrations decreased gradually from 4.8% at the first sampling date
to 2.9% at maturity (Table 128, Appendix). Nitrogen application did not increase shoot N
concentrations except at the third harvest (Table 19). Root N concentrations also decreased
from the first sampling to maturity. Root N concentrations were higher on plants reliant
on symbiotically fixed N at periods of optimum noduIatioh and N2-fixation (C2H4), third
43
Table 18. Analysis of Variance for Fababean Nodule Dry Weight at Bozeman, Montana 1981.
Weeks from EmergenceSource of Variation df 4 7 13
Mean SquaresN v s n o N I .0012 1.707** .9400Inoculated vs not inoculated I .0234** 0 .4523** 6.7980**No P (inoculated) vs P (factorial) I .0002 0.0260 2.4294**
Factorial 14P sources I .0102* .1938** .1519P levels I .0002 .0624 .3745
Methods of application I .0008 .0137 .0052All 2, 3 and 4 factors interaction 11 .0302** .1558* 3 .5076**Error 36 .0019 .0157 .2434
* and * * denote significance at the 5 and I % levels, respectively.
Table 19. Analysis of Variance for Fababean Shoot %N at Bozeman, Montana, 1981.
Weeks from EmergenceSource of Variation df 4 7 10 13
Mean SquaresN vs no N I .0505 3.337 0.4472* 0.0187Inoculated vs not inoculated I 0.9747* 0.4896 0.2059 0.2809*No P (inoculated) vs P (factorial) I 0.0451 0.2721 0.2049 0.0001
Factorial 14P sources I .0070 .4163 2.516** 0.0075P levels I .0616 .0809 0.0020 0.0021 ,
Methods of application I .0080 .3350 0.1355 0.0154All 2, 3 and 4 factors interaction 11 .8300** 1.8085** 0 .9470** 0 .4821**Error 36 .1973 0.3480 0.1598 0.0803
* and * * denote significance at the 5 and 1% levels, respectively.
and fourth harvests, but were.not statistically significant (Table 20). Increasing P supply
also increased root N concentrations (Table 129, Appendix).
Shoot and Root P Concentrations
Shoot and root phosphorus concentrations were higher in the early stage of plant
growth and gradually declined with age. Plants reliant on symbiotically fixed N had higher
shoot and root P concentrations than plants supplied with 100 Kg ha*1 N. In general, there
44
was no significant difference due to P sources, levels and methods of application for both
shoot and root P concentrations (Tables 21 and 22).
Table 20. Analysis of Variance for Fababean Root %N at Bozeman, Montana, 1981.
Source of Variation dfWeeks from Emergence
4 7 13
Mean SquaresN vs no N I .1717 3.499 .1325Inoculated vs not inoculated I 3 .0120** 2 .4843** .9042**No P (inoculated) vs P (factorial) I 0.0186 0.3330 .0495
Factorial 14P sources I 0.0120 0.0105 .0059P levels I 0.0024 0.9436* .0809
Methods of application I 0.1519 ' 0.5105 .0221All 2, 3 and 4 factors interaction 11 1.4002** 2 .0866** 1.2224**Error 36 .1792 .1804 .0937
* and * * denote significance at the 5 and 1% levels, respectively.
Table 21. Analysis of Variance for Fababean Shoot: %P at Bozeman, Montana, 1981.
Weeks from Emergence
Source of Variation df 4 7 10 13
Mean Squares
N vs no N I .0031 .0004 .0020 .0002Inoculated vs not inoculated I .0017 .0004 .0001 .87X/I0-5No P (inoculated) vs P (factorial) I .0013 .0015 .0006 .0005
Factorial 14P sources I .0007 .0001 .0012 .0019*P levels I .0014 .0006 .24X10-S .35X10-5
Methods of application I .0015 .438X10-5 .94X10"6 .0004All 2, 3 and 4 factors interaction 11 0 .0062** .0040** .0011** .0031**Error 36 .0013 .0006 .0003 .0004
* and * * denote significance at the 5 and 1% levels, respectively.
45
Table 22. Analysis of Variance for Fababean Root %P at Bozeman, Montana, 1981.
Source of Variation dfWeeks from Emergence
4 7 13
Mean SquaresN v s n o N I .0015 .0053 .0026Inoculated vs not inoculated I .0015 .41X 10“4 .0002No P (inoculated) vs P (factorial) I .0010 .0001 .64X10"6 .
Factorial 14P sources I .0006 .23X10 4 .0003P levels I .59X1 O'6 .37X10"4 .0001
Methods of application I .15X10-6 .16X10-6 .35X1 O'6All 2, 3 and 4 factors interaction 11 .0082** .0035** .0020**Error 36 .0005 .0008 .0002
* and * * denote significance at the 5 and 1% levels, respectively.
Discussion on Fababean 1980 and 1981 Field Experiments
There is indication from the 1980 data that the rates of P used were too small to pro
duce the expected results in a high P fixing soil. Band application of soluble P fertilizers
often results in more efficient use of the fertilizer by the crop being grown than is obtained
with broadcast application (Sleight et al., 1984). In theory, band application reduces soil
fertilizer contact, resulting in less "fixation" of the P by the soil than would occur with
broadcast application. This leaves more P chemically available to the crop (Tisdale and
Nelson, 1975). There was substantially greater grain yield reduction as a result of banding
orthophosphoric acid than broadcasting. It is suggested that banding orthophosphdric acid
in this soil concentrated root development in the regions where P was applied and the
limited root development around the bands caused the plants to be less resistant to periods
of moisture stress that occurred at the end of July and early August (TAble 106, Appen
dix). Yost et al. (1979) described a similar situation on a Brazilian oxisol of low available
P. The fact that root dry weight did respond to P supply and that shoot weight was lower
in banded than broadcast treatments supports the above assumption. Nodule number, dry
weight and nitrogenase activity were much higher in fababean than dry bean but were
46
generally not affected by P supply in 1980 (Tables 7, 8, and 9) and this is attributed to the
low levels of P used. In experiments (1982, 1983 and 1984) where P supply was high, the
above parameters increased with increasing P applications. It is also suggested that the high
grain yield on plots not receiving combined N resulted from the high rate of symbiotic N2-
fixation of the fababean plants that had outperformed those supplied with 100 Kg/ha N as
ammonium nitrate. This is supported by the high nitrogenase activity values (43 jumole
C2H4 plant-1 h r 1 ) in the -N treatments (Table 9) as well as the shoot N concentrations
(Table 11).
Results obtained in the 1981 field experiment supported those of 1980. In 1981,
however, orthophosphoric acid was replaced by monoammonium phosphate and this might
explain the low nodulation obtained with this treatment (containing inorganic N) relative
to triple superphosphate. Phosphorus levels were also raised to 60 Kg/ha and 120 Kg/ha for
the low and high applications, respectively. This explains in part the positive responses of
shoot weight, nodule number and mass to P additions relative to 1980.
Effects of Placement and Source of P Fertilizer on Nodulation, N2-Fixation
and Growth of Dry Bean, 1980
Shoot Dry Weight
Shoot dry weight mean and contrast comparison values are reported in Tables 132
and 133 (Appendix). Shoot weight was generally not increased by the nitrogen rate used.
In fact, plant growth seemed to be negatively affected by nitrogen and was significantly
lower at the second harvest (day 44).
Phosphorus application increased shoot weight slightly at all harvests but a significant
difference was reached only at the third harvest (contrast C2 , P < 0.01). There was no
significant interaction between P and N treatments.
47
Triple superphosphate was more effective than orthophosphoric acid (contrast C3 )
but was significantly higher only at maturity (P < 0.01).
Seed banded application resulted in significantly higher shoot weight than broadcast
at the third (P < 0.05) and fourth (P < 0.01) harvests.
Root Dry Weight
Contrast comparison and root dry weight mean values are given in Tables 134 and
135 (Appendix). Nitrogen application had no effect on root dry weight while effects due
to P sources and levels were only evident at maturity (contrasts C2, C3, C5, C6 and C7),
Triple superphosphate was more effective than orthophosphoric acid at the third
harvest (contrast C2, P < 0.05). Maximum root weight was obtained at P2 level (contrast
C5) with both sources of P (contrasts C6 and C7). The significant contrast C1 X C7 (P <
0.01) at maturity suggests a positive interaction between P1 and N when phosphorus is
applied as triple superphosphate.
Results obtained with root dry weight in dry bean must be interpreted carefully since
approximately the same volume of soil was excavated each time. A treatment that might
result in a more extensive root development might still show a low root weight value, indi
cating that all of the roots were not recovered. This is mainly true with treatments result
ing in more fibrous root systems.
Nodule Number, Dry Weight and Nitrogenase Activity
Nodule number was significantly reduced (P < 0.01) by nitrogen application at all
stages of plant growth (Table 23; Tables 136 and 137, Appendix). These results agree with
those of Cackett (1965), Gallagher (1968) and Graham (1978) obtained with dry bean.
Triple superphosphate increased nodule number by 11 percent on treatments not receiving
nitrogen relative to the control (also not receiving nitrogen) and orthophosphoric acid
lowered it by 12 percent (contrast C3). However, nodule dry weight (Table 24; Tables 138
48
and 139, Appendix) and nitrogenase activity (C2 H4 ) (Table 25; Tables 33 and 34, Appen
dix) were increased more by orthophosphoric acid than triple superphosphate in the -N
treatments. These results suggest that triple superphosphate influences nodulation by
increasing nodule initiation but not nodule growth. The specific activity data support this
hypothesis since there was a 9 percent reduction caused by triple superphosphate as
compared to orthophosphoric acid (Table 26).
Table 23. Effect of Source of P on Dry Bean Nodule Number at Bozeman, Montana, 1980.
Table 26. Effect of Source of P on Dry Bean Specific Activity at Bozeman, Montana 1980.
Days from Planting
Control Ortho I "P-N +N -N +N -N +N
Mmole C2H2 plant-1 hr 1 per unit dry weight of nodule in g24 36.94 37.98 58.09 37.16 52.23 26.7744 10.43 26.76 16.19 29.94 15.31 20.6564 6.04 6.14 8.79 24.54 8.10 9.6585 2.17 4.25 3.56 30.99 3.17 4.81
Broadcast application increased nodule number more than banded application except
at the final harvest but nodule dry weight was increased more by banded than broadcast
(Table 27). This suggests an increased nodule initiation as a result of broadcast which is
not followed by nodule development. This is again supported by the nitrogenase activity
with a 11 percent increase and the specific activity values with a 6.6 percent increase of
banded over broadcast applications.
Table 27. Effect of Banded (S) and Broadcast (B) Applications on Dry Bean Nodule Number, Dry Weight, N2 -ase and Specific Activities at Bozeman, Montana, 1980.
Days from Nodule Number Nodule Weight N2 -ase Specific ActivityPlanting S B S B S B S B
Nitrogenase activity for dry bean as influenced by P levels, sources, method of appli
cation and N treatments are reported in Table 140 (Appendix) and analysis of variance in
Table 141 (Appendix). Nitrogenase activity was very sensitive to N application. Applica
tion of 100 Kg N/ha reduced it drastically by 74.8, 72, 82.3 and 74.6 percent at the first.
50
second, third and fourth harvest, respectively. Even in treatments not receiving nitrogen,
nitrogenase activity in dry bean was minimal at the first and second sampling dates,
increased slightly at the third harvest and declined sharply thereafter until maturity (Table
25).
Nitrogenase activity was highly correlated with nodule number and nodule weight at
all harvests except for nodule weight at the first harvest (Table 28). These high correla
tion values suggest that nodule number and weight can serve as a qualitative indicator of
Nz -fixation activity in experiments where nitrogenase activity is not available when using
effective strains. No reliable correlations, however, were found between nitrogenase
activity and N concentrations in either shoot or root and specific activity. This is due to
the fact that these variables are highly influenced by soil fertility level and nitrogen appli
cation rates. These results are consistent with those of fababean in 1980.
Table 28. Correlation Coefficients of Dry Bean Nitrogenase Activity with Nodule Number and Weight, Shoot and Root %N and Specific Activity at Bozeman, Montana, 1980.
* and * * denote significance at the 5 and 1% levels, respectively.
Root Dry Weight
Root dry weight mean squares are reported in Table 31 and mean values in Table 151
(Appendix). Nitrogen fertilization only affected on root weight at maturity. Monoammon
ium phosphate increased root weight more than triple superphosphate at all harvests, but
54
was only statistically significant at maturity. Similar results were obtained from banded
and broadcast applications.
Table 31. Analysis of Variance for Dry Bean Root Dry Weight at Bozeman, Montana, 1981.
Source of Variation dfWeeks from Emergence
4 7 13
Mean SquaresN v s n o N I .0608 .0621 2.321*Inoculated vs not inoculated I .0080 .0014 .0002No P vs P (factorial) I .0054 .0845* .0540
Factorial 14P sources I . .0027 .0481 2.2320*P levels I .0001 .0320 .4901
Methods of application I .0044 .0001 .2625All 2, 3 and 4 factors interaction 11 .0755** .1884** 3.3087**Error 36 .0105 .0123 .4889
* and * * denote significance at the 5 and 1% levels, respectively.
Nodule Number
The effects of nitrogen application on nodulation paralleled those of 1980 expert
ment. Nodule number was reduced at all stages of plant growth (Table 152, Appendix) but
was not statistically significant probably because of overall poor nodulation (Table 32).
The most reduction occurred at 7 weeks after emergence at which date the +N treatments
reduced nodule number by 45 percent. The results did not show any significant difference
as related to phosphorus sources, levels and methods of application.
Shoot Nitrogen Concentrations
Table 33 indicates that nitrogen application increased shoot nitrogen concentrations
at the first and second harvests (P < 0.05). Monoammonium phosphate resulted in higher
55
shoot nitrogen concentrations at the second harvest, but was similar to triple superphos
phate at other harvests. This probably indicates a transitory effect of the N added in mono-
ammonium phosphate. Increasing phosphorus supply did not affect nitrogen concentra
tions and no significant difference was noted between methods of P applications.
Table 32. Analysis of Variance for Dry Bean Nodule Number at Bozeman, Montana, 1981.
Weeks from EmergenceSource of Variation df 4 7 13
Mean Squares .N vs no N I 45.07 18230 1402Inoculated vs not inoculated I 2250.95 2106.8 3570.8No P vs P (factorial) I 2518.07 1419.8 1716.5
Factorial 14P sources I 172.5 204.2 5229* .P levels I 3088 526.7 713
Methods of application I 58.5 35.0 1645All 2, 3 and 4 factors interaction 11 11843** 9353.6** 20148**Error 36 982.0 867.3 1092
* and * * denote significance at the 5 and 1% levels, respectively.
Table 33. Analysis of Variance for Dry Bean Shoot %N at Bozeman, Montana, 1981.
Weeks from EmergenceSource of Variation df 4 7 10 13
Mean SquaresN v s n o N I 2.285* 8.4600* 1.032 .2996Inoculated vs not inoculated I .0288 .5834* .0675 .0055No P vs P (factorial) I .0656 .0224 .2500 .3910*
Factorial 14 - ■ ■P sources I .0140 .6816* .3350 .0469P levels I .0752 .5334* .0213 .0363
* and * * denote significance at the 5 and 1% levels, respectively.
Shoot and Root P Concentrations
Increasing N and P supply did not increase shoot phosphorus concentrations (Table
35; Table 155, Appendix). Additionally, there were also no significant responses due to
phosphorus sources and methods of application. However, a general decline in shoot
phosphorus concentrations was observed from the first harvest to maturity, similar to
shoot and root nitrogen concentrations.
Increasing P supply increased root P concentrations at the second and final harvests.
At all levels of phosphorus supply, phosphorus concentrations in roots were much greater
in the -N treatments than those in the +N treatments. No significant differences existed
relative to P sources and methods of application even though broadcast application resulted
in higher P concentrations at later stages than banded application (Table 36).
57
Table 35. Analysis of Variance for Dry Bean Shoot %P at Bozeman, Montana, 1981.
Weeks from EmergenceSource of Variation df 4 7 10 13
■ Mean SquaresN v s n o N I .0017 .0026 .0021 .0074*inoculated vs not inoculated I .0058* .16X10-4 .68X10-= .0001No P vs P (factorial) I .0061* .62X10-= .0004 .0005
Factorial 14P sources I .21X10-4 .79X10"S .0020 .0005P levels I .0004 .0003 .0002 .0003
Shoot N Concentrations. There was no consistent trend on shoot N concentrations as
a function of P application (Tables 79 and 80). Shoot N concentrations were generally
higher on plants reliant on symbiotic N than those receiving fertilizer N. This is consistent
with the results obtained in 1982 and supports the assumption that plants dependent on
symbiotic N can accumulate more N than those receiving 75 Kg/ha (1983) but not up to
200 Kg/ha (1982) of fertilizer N.
Grain Yield. Table 81 indicates that P supply nonsignificantly increased seed yield.
Plants reliant on symbiotically fixed N had higher seed yield than those supplied with 75
Kg/ha of combined N. These results contrast with those obtained in Canada by Sosulski
and Buchan (1978) and are consistent with the 1982 experiment. Both years results
support the hypothesis that N2 fixation on nonfertilized plants was able to supply more N
than plants fertilized with 75 Kg/ha of combined N or supplied N as effectively as those
receiving 200 Kg/ha of combined N resulting in higher or equal seed yield respectively.
These results also demonstrate the importance of seed inoculation.
Fteld Experiment 1984
Shoot and Root Dry Weight. Mean squares reported in Table 82 indicate that there------------------------ ------------------
were no significant effects of P and N on green pea shoot dry weight. The mean values,
however (Table 83), showed that shoot weight was increased with increasing P levels and
that N supplied plants had lower shoot dry weight than those reliant on symbiotic N. The
trend of P and N effects on shoot weight is consistent with the 1982 and 1983 experiments
even if statistically significant effects were not observed.
Root dry weight was not affected by either P or N application except for N at the
first harvest (Tables 84 and 85). Similar trends were observed when nodule dry weight was
added to the root dry weight (Tables 86 and 87).
1 0 7
T a b le 7 9 . A n a ly s is o f V a r ia n c e a n d O rth o g o n a l P o ly n o m ia ls fo r G re e n Pea S h o o t % N ,a t B o ze m a n , M o n ta n a , 1 9 8 3 .
Weeks from Emergenceof Variation df 5 7 9
Blocks 3 .1214Mean Squares
.1187 .0743P-Ievels 3 .1927** .0654 .1893
Linear I .0228 .0361 .0578Quadratic I .0977 .1600* .2627Cubic I .4575** .0001 .2475
N-Ievels 3 .1510* .0388 .2831Linear I .0428 .0805 .5040Quadratic I .4084** .0008 .0977Cubic I .0038 .0551 .2475
NXP 9 .0499 .0322 .0672Error 45 .0373 .0364 .1501
*P < 0.05; * * P < 0.01.
Table 80. Mean Values of Green Pea Shoot %N Averaged over N and Averaged over I Levels, Respectively, at Bozeman, Montana, 1983.
Weeks from Emergence
T reatments 5 7 9
% NP-Ievels
Po 4.14 3.90 2.40P, 4.35 3.78 2.19P2 4.11 3.76 ,2.38
Table 81. Mean Values of Green Pea Seed Yield Averaged over N and Averaged over P Levels, Respectively, Kg/ha at Bozeman, Montana, 1983.
P-Ievels Yield (Kg/ha) N-Ievels Yield (Kg/ha)
Po 2057 N0 2203P1 2196 Ni 2103P2 2128 N2 2011P3 2117 N3 2180
LSD .05 N.S. LSD .05 N.S.
Nodule Dry Weight. Phosphorus application increased nodule dry weight and was
highly significant at the final harvest (Tables 88 and 89). Increasing N application signifi
cantly decreased nodule dry weight at all harvests. The effects of P and N on nodule dry
weight are consistent with those of 1983. Significant ,NXP interactions on nodule dry
weight were recorded at the second and final harvests (Table 88).
Shoot N Concentrations. Phosphorus and N had no significant effect on shoot N
concentrations except at the first harvest in which plants reliant on symbiotic N had higher
(P < 0.01) shoot N content than those receiving 75 Kg/ha of fertilizer N (Tables 90 and
91). This observation confirm the results of 1982 and 1983 that the pea plants reliant on
symbiotic N can accumulate N as effectively as fertilized plants.
Assessment of the Role of P in Green Pea Nodulation and N2 Fixation
Results based on the 1982, 1983 and 1984 experiments showed:
First, that the NxP interaction on green pea shoot weight was usually positive (Tables
67, 71 and 82). This suggests that P might not be directly involved in N2 fixation. These
results contrast with those obtained with faba bean where no significant interactions were
observed.
Second, that increasing P supply increased, nonsignificantly, shoot N concentrations
in 1982 but was not consistent in 1983 and 1984.
1 0 9
T a b le 8 2 . A n a ly s is o f V a r ia n c e and O rth o g o n a l P o ly n o m ia ls fo r G re e n Pea S h o o t D ryW e ig h t a t B o ze m a n , M o n ta n a , 19 8 4 .
W eeks fro m E m erg en ce
of Variation df 4 6 8
Mean SquaresBlocks 3 0.3825 13.80 95.08P-Ievels 3 0.5927 18.11 74.31
Linear I 0.7050 20.08 35.45Quadratic I 0.9702 28.46 185.00Cubic I 0.1029 5.77 2.54
N-Ievels 3 2.6950 22.76 9.83Linear I 0.4636 0.90 3.12Quadratic I 0.2209 6.50 0.32Cubic I 7 .399** 60.88* 26.05
T a b le 8 4 . A n a ly s is o f V a r ia n c e an d O rth o g o n a l P o ly n o m ia ls fo r G re e n Pea R o o t D ryW e ig h t a t B o zem a n , M o n ta n a , 1 9 8 4 .
Source Weeks from Emergenceof Variation df 4 6 8
Blocks 3 0.0935Mean Squares
0.2200 0.1873 'P-Ievels 3 0.0225 0.0722 0.0269
Linear I 0.0137 0.1877* 0.0208Quadratic I 0.0405 0.0129 0.0410Cubic I 0.0134 0.0158 0.0189
N-Ievels 3 0 .0958** 0.0476 0.0697Linear I 0 .2096** 0.0314 0.0016Quadratic I 0.0207 0.1097 0.1980Cubic I 0.0570 0.0016 0.0095
T a b le 8 6 . A n a ly s is o f V a r ia n c e a n d O rth o g o n a l P o ly n o m ia ls fo r G re e n Pea N o d u le + R o o tD ry W e ig h t a t B o z e m a n , M o n ta n a , 1 9 8 4 .
Source Weeks from Emergenceof Variation df 4 6 8
Blocks 3 0.0732Mean Squares
0.1963 0.1828P-Ievels 3 0.0174 0.0510 0.0494
Linear I 0.0042 0.1121 0.0146Quadratic I 0.0420 0.0366 0.1122Cubic I 0.0060 0.0043 0.0215
N-Ievels 3 0.0514 0.0331 0.0582Linear I 0.0891 0.0006 0.0285Quadratic I 0.0650 0.0490 0.1425Cubic I 0.0000 0.0500 0.0035
T a b le 8 8 . A n a ly s is o f V a r ia n c e an d O rth o g o n a l P o ly n o m ia ls fo r G re en Pea N o d u le D ryW e ig h t a t B o ze m a n , M o n ta n a , 1 9 8 4 .
g o u rc e ________________ W eeks fro m E m erg en ce
of Variation df 4 6 8
Mean SquaresBlocks 3 0.0019 0.0027 0.0014P-Ievels 3 0.0013 0.0067 0.0069**
Linear I 0.0025 0.0113* 0.0006Quadratic I 0.0000 0.0056 0.0200**Cubic I 0.0363** 0.0031 0.0003
N-Ievels 3 0.0296** 0.0393** 0.0154**Linear I 0 .0257** 0.0410** 0.0179**Quadratic I 0 .0103** 0.0121* 0.0044Cubic I 0.0528** 0.0650** 0.0240**
t a b le 9 0 . A n a ly s is o f V a r ia n c e an d O rth o g o n a l P o ly n o m ia ls fo r G re en Pea S h o o t N itro g e nC o n c e n tra tio n s a t B o ze m a n , M o n ta n a , 1 9 8 4 .
g o u rc e ______________ W eeks fro m E m erg e n c e
of Variation df 4 6 8
Blocks 3 .0177Mean Squares
.1014 .0181P-Ievels 3 .0156 .0135 .0131
Linear I .0263 .0015 .0090Quadratic I .0189 .0352 .0189Cubic I .0015 .0038 .0113
N-Ievels 3 .1131** .0102 .0672Linear I .0008 .0300 .0070Quadratic I .0564 .0002 .0189Cubic I .2820** .0003 .1758
at all harvests (Table 92). Shoot" weight was increased by more than 81, 47, 53 and 100
percent by the highest P rate used (210 Kg/ha) relative to the control at the first, second,
third and fourth harvests, respectively (Table 171, Appendix). Nitrogen application also
increased shoot weight during the entire growing season (P < 0.01). It was noted that at
low P level, N application did not increase shoot dry weight. At high P level, however,
shoot weight of plants supplied with fertilizer N was higher than nonfertiIized ones. This
interaction effect was significant at maturity (P < 0.01). These results are consistent with
the dry bean 1980 and 1981 experiments but differ from those of fababean and green
pea. In fababean, plants reliant on symbiotic N had higher shoot dry weight than those
supplied with 200 Kg/ha of fertilizer N after nodulation and N2 fixation were effective 10
weeks from emergence. Green pea plants were also shown to be N self-sufficient in soils
of low to medium soil fertility. The dry bean shoot weight results confirm earlier reports
(Cackett, 1965; Gallagher, 1968; Robinson et al., 1974) that dry bean N2 fixation is very
ineffective in meeting the plants needs.
1 1 5
T a b le 9 2 . A n a ly s is o f V a r ia n c e fo r D ry Bean S h o o t D ry W e ig h t as A ffe c te d b y P S u p p lyan d M o d e o f N N u tr i t io n a t B o ze m a n , M o n ta n a , 1 9 8 2 .
Sourceof Variation df
______________Weeks from Emergence_________ ■__4 8 10 12
average yield of 3067 Kg/ha. These results are similar to those of 1980. High P level
increased grain yield of plants in the -N regime but not those in the +N regime. At low P
level, however, grain yield was higher in the +N regime. This is illustrated by the highly
significant NXP interaction (P < 0.01). Total rainfall of 1982 growing season was similar
to 1980 and both years are considered dry. It is then concluded that under dryland farm
ing conditions of limited soil water availability, early maturing bean varieties and/or timing
of planting that will allow the plants to escape the period of water stress prevailing at the
end of July and early August (at Bozeman) might be as important as soil fertility consider
ations. This hypothesis is supported by the negative correlation (existing between grain
yield and shoot dry weight. Nitrogen fertilized plants had highejf shoot dry weight but
lower grain yield. The high grain yields obtained with dry bean in these experiments show
that this crop has a great potential as a rotation crop and may compete successfully with
wheat in dryland agriculture.
1 1 8
Field Experiment 1983
Shoot and Root Dry Weights. Table 96 indicates that P application had a significant
effect on dry bean shoot dry weight only toward maturity (9 and 11 weeks from emer
gence). The effect of P at these sampling dates was linear as shown in Table 96 and by the
response surface in Figure 17 for the 9 weeks from emergence sampling. Nitrogen addi-
. tions, however, did not have any influence on shoot weight, presumably due to moisture
limitations.
Root dry weight increased with increasing P level and was significant at the third and
fourth harvests (Tables 98 and 99). ■
Nodule Number. Low levels of P (60 and 120 Kg/ha) increased, nonsignificantly,
nodule number relative to the control at the first (Fig. 18) and second (Fig. 19) harvests
(Tables 100 and 101). At the highest rate of P used (180 Kg/ha), however, nodule number
was decreased. This is illustrated in the response surfaces of Figures 18 and 19. At the third
and fourth harvests, nodule number was lower on treatments receiving P fertilizer except
for the application of 60 KgAia of P at the third harvest. This differs from earlier results
obtained at all sampling dates and the decrease was highly significant except at maturity.
Nodule number was reduced by 67, 73, 53 and 54 percent at each successive harvest on
plants supplied with fertilizer N. The above relationships are expressed in the following
equations:
At the first harvest,
5. Y = 21.00 + 0.96 P - 4.61 N ** R2 = .17
At the second harvest,
6. Y = 42.17 0.97 P - 8.33 N ** R2 = .19
At the third harvest,
7. Y = 39.05 - 0.73 P - 6.28 N ** R2 = .18
1 1 9
T a b le 9 6 . A n a ly s is o f V a r ia n c e a n d O rth o g o n a l P o ly n o m ia ls f o r D r y Bean S h o o t D ry' W e ig h t a t B o ze m a n , M o n ta n a , 19,83.
Source Weeks from Emergenceof Variation df 5 7 9 11
Blocks 3 17.39 74.89Mean Squares
52.07 120.2P-Ievels 3 9.172 38.38 420 .3** 2088*
Linear I 24.28* 20.62 1122** 5201**Quadratic I 2.194 52.83 138.5 702Cubic I 1.041 41.70 .85 360
N-Ievels 3 3.385 8.52 142.7 189.9Linear I 6.053 12.10 3.71 16.91Quadratic I .180 13.11 423.0* 308.5Cubic I 3.923 .35 1.47 244.3
Figure 17. Shoot weight (g/2 plants) of 9 week-old dry bean plants in response to P and N applications at Bozeman, Montana, 1983.
121
T a b le 9 8 . A n a ly s is o f V a r ia n c e a n d O rth o g o n a l P o ly n o m ia ls fo r D ry B ean R o o t D ryW e ig h t a t B o ze m a n , M o n ta n a , 1 9 8 3 .
Source Weeks from Emergenceof Variation df 5 7 9 11
Figure 18. Nodule number per 2 plants of 5 week-old dry bean plants in response to P and N applications at Bozeman, Montana, 1983.
F ig u re 1 9 . N u m b e r o f n o d u les p er 2 p la n ts o f 5 w e e k -o ld d ry bean p la n ts in response to Pa n d N a p p lic a tio n s a t B o ze m a n , M o n ta n a , 1 9 8 3 .
1 2 3
T a b le TOO. A n a ly s is o f V a r ia n c e a n d O rth o g o n a l P o ly n o m ia ls f o r D r y Bean N o d u le N u m b er a t B o ze m a n , M o n ta n a , 1 9 8 3 .
Source ________________Weeks from Emergenceof Variation df 5 7 9 11
The relatively poor nodulation in dry bean accounted for much of the variability observed
in 1983. The fact that nodule number decreased at the highest P rates and with plant age
was problably related to moisture limitations. Shoot dry weight was higher with high P
rates. However, unlike in fababean and green pea crops where this resulted in higher
nodule number, in dry bean the increased shoot weight likely caused more soil water deple
tion around the root zones of high P treatments and this negatively affected nodulation. It
is well documented that water stress is a major factor affecting nodulation and N2 fixation,
particularly in dry bean (Day et al., 1980; Sprent, 1972; Sprent and Bradford, 1977). At
the end of the growing season, soil water was probably depleted in all treatments (Table
106, Appendix) and as a result, plants receiving P were more affected. This would explain
the negative slopes found with P in the above regression equations. The lower correlation
values also suggest that factors other than P and N might be controlling dry bean nodula
tion in this experiment. Nodules were in general small and were not removed for dry
weight evaluation.
Shoot N Concentrations. Shoot N concentrations were not affected by increasing P
supply but were significantly increased by N application at all harvests (Tables 102 and
103). These results are in contrast with those obtained with fababean and green pea in
which shoot N concentrations were increased by P but not by N fertilizer. In fababean and
green pea, nodulation and thus N2 fixation was high and accounted for the high N con
tent in plants reliant on symbiotic N. Therefore, there was no difference between the two
1 2 5
T a b le 1 0 2 . A n a ly s is o f V a r ia n c e an d O rth o g o n a l P o ly n o m ia ls f o r D r y Bean S h o o t % N a tB o ze m a n , M o n ta n a , 1 9 8 3 .
Source ________________Weeks from Emergenceof Variation df 5 7 9 11
ammonia can pick up an H+ on the acidic side of the bacteroid membrane, carry it across
as a neutral complex, then release it to OH" on the other side, destroying both the proton
gradient and the membrane potential. The dissipation of the proton gradient would pre
vent or reduce the formation of ATP necessary to reduce the nitrogenase enzyme. Also the
destruction of the membrane potential brings about the suppression of low potential
electrons that might be necessary to reduce ferreddxin or flayodoxin. One or both of these
mechanisms would give a satisfactory explanation for the inhibitory effects of NH4"1" in
legume infection, root hair curlings, nodule initiation and development and nitrogenase
synthesis. An advantage of the proposed theory is that it involves the high energetics of
N2-fixation that no other theory has implicated up to now.
Many of the facts reported in the literature on the effects of NH4+ on N2-fixation
provide circumstantial evidence for the above theory. Harper and Gibson (1984) found
that higher external NO3-IeveIs were more inhibitory to nodule appearance even though
NO3- uptake rates were similar in various soybean X rhizobium strain combinations; they
concluded that the external concentration of NO3- rather than the rate of NO3- uptake
appeared to have a major effect on the initial stages of nodulation. Maintaining the con
centration of NO3- in the solution following appearance of nodules greatly retarded or
prevented the development of nitrogenase activity. Urea also represses nitrogenase syn
thesis when provided as an N source. However, repression by nitrate or urea has been
shown to involve their conversion to NH4+ since mutants of A. vinelandii and K. pneu
moniae which lack nitrate reductase activity escape repression by nitrate but remain sus
ceptible to repression by nitrite or NH4+ (Kennedy and Postgate, 1977).
Drozd et al. (1972) also reported that the concentration of NH4"*" necessary to
repress nitrogenase synthesis depends on the bacteroid population density. Consequently,
at high cell densities, a higher concentration of repressor is required. If NH4* is considered
133
as an uncoupler (or ion ionophore), its action does not involve the destruction of the nitro-
genase enzyme and would explain observations made by Houward (1980) that the bacter-
oids of pea nodule, in which acetylene reduction has been severely inhibited by combined
nitrogen, retain their nitrogenase capacity and show the same nitrogenase activity as do
bacteroids from untreated nodules, provided they are supplied with exogenous ATP and
reductant. Results based on split root and placement experiments also support the above
model. Virtamen et al. (1955), for example, found that nodulation was inhibited on the
root system exposed to combined nitrogen, but was unaffected on the part growing in
nitrogen free media. Raggio et al. (1965) reported that nitrate in contact with the nodulat-
ing area of excised roots inhibited nodulation, while nitrate fed into the root did not.
However, it has been shown that under certain conditions, some amino acids can
repress nitrogenase synthesis as effectively as NH4*. L asparte and L glutamipe plus L-
aspartate both at I mg/ml completely repressed nitrogenase synthesis (Shanmugam and
Morandi, 1976). Since many amino acids act as uncouplefs of phosphorylation, the above
observations would still be consistent with the proposed theory. However, it would not
explain the fact that repression by these amino acids is still observed in a number of
mutant strains of K. pneumoniae which escape NH4+ repression. These derepressed strains
do not assimilate added NH4+ to any significant extent. Revertants which regain Asm+
phenotype are susceptible once more to NH4+ repression (Shanmugam and Morandi, 1976).
These observations suggest that more than one mechanism might be involved in NH4+
inhibition of nodule initiation, development and function in legumes. The fact that fertil
izer N had very different effects on nodulation and N2-fixation by the three different
species of host plants suggests that there are indeed more than one mechanism for the
inhibition.
134
CHAPTER 5
SUMMARY AND CONCLUSIONS
In 1980 and 1981, nodule number, dry weight and nitrogenase activity (1980) were
much higher in fababean than in dry bean but were generally not affected by P supply due
to the low levels of P used. Nodulation and nitrogenase activity were very sensitive to N
application in dry bean and nitrogenase activity was reduced by 75, 72, 82 and 75 percent
at the first, second, third and fourth harvests respectively. In fababean, however, percent
reductions were 47, 60, 62 and 51.
Excellent positive linear correlations between acetylene reduction rates and nodule
number and mass were found with both fababean and dry bean plants and can serve as a
qualitative indicator of N2 -fixation activity in experiments where nitrogenase activity is
not available.
In the fababean plant, the results showed that noduIation and N2 Tixation reached a
maximum at pod filling and remained constant until pod filling was complete and then
showed a decline (Table 9). This high rate of symbiotic N2-fixation resulted in high grain
yield on plots not receiving combined N that had outperformed those supplied with 100
Kg ha-1 N as ammonium nitrate. In dry bean, however, maximum nodulation and N2-
fixation reached a maximum during pod set and declined rapidly during the final weeks of
growth (Table 15). Therefore, N2Tixation (C2H4 ) was very limited and plants not receiv
ing nitrogen are considered nitrogen deficient. However, plots not receiving combined N
resulted in significantly higher grain yields than those receiving 100 Kg ha-1 . It was sug
gested that N fertilizer prolongs vegetative growth, thereby inducing water stress. These
results reflect the main difference between dry bean and fababean and suggest that one
135
possible improvement on dry bean N2 -fixation will be to select for plants having a higher
and longer plateau of nitrogenase activity.
In fababean, broadcasting P gave greater grain yield than banding when orthophos-
phoric acid was the P source. Banding orthophosphoric acid concentrated root develop
ment in regions where P was applied and the limited root development around the bands
caused the plants to be less resistant to periods of moisture stress that occurred at the end
of July and early August (at Bozeman). When P was applied as triple superphosphate,
banded application was more effective.
In dry bean, grain yield was increased when orthophosphoric acid was applied in band
relative to broadcast and the converse was true for triple superphosphate.
Seed ,inoculation is necessary in Montana soils for effective nodulation and N2 -fixa
tion in dry bean and fababean. However, under dryland conditions, nodulation and N2-
fixation may not limit dry bean grain yield. Factors such as water stress caused by the
vigorous growth of well nodulated plants may be the limiting factor for grain yield.
In 1982, fababean plants reliant on symbiotically fixed N were as efficient as plants
receiving 200 Kg/ha of fertilizer N in increasing shoot weight and shoot N concentrations.
Phosphorus supply increased shoot weight but did not significantly affect the relative dis
tribution of dry matter between the above and the underground portion of the plant. Root
weight was significantly increased by P supply but not by mode of N nutrition. Active root
nodules utilize significant quantities of photosynthate for nodule growth and N2-fixation
and this reduces considerably root extension and growth. Nodule number and dry weight
were significantly reduced by combined N at all harvests.
In 1983 and 1984, shpot and root weights were increased by P application. Nodule
number and nodule dry weight also increased with increasing P supply. However, these
increases did not precede increases in shoot weight. The positive effects of P on shoot
1 3 6
weight and Modulation did not translate into a significant higher grain yield. Usually there
were no NXP interactions for the measured variables.
Green pea shoot and root dry weights were increased with increasing P supply in 1982.
Nitrogen fertilization increased shoot weight by 35, 26, 15 and 18 percent at the four har
vests and indicates that the ability of green pea plants reliant on N2-fixation to compen
sate for fertilizer N is lower than fababean plants. Nodule number and dry weight were
increased with increasing P supply but paralleled effects on shoot dry weight. Nodule num
ber and dry weight were significantly reduced by fertilizer N, suggesting that unlike in faba
bean, NO3--N is highly inhibitory to green pea Modulation and N2-fixation. However, grain
yield obtained by inoculation in dryland agriculture was equal to that obtained from N
fertilization. Results obtained in 1983 and 1984 were similar to those of 1982.
Phosphorus and N increased significantly dry bean shoot and root dry weights in
1982. Few nodules developed on plants reliant on symbiotic N and almost none in treat
ments receiving 200 Kg ha-1 N of inorganic N. Therefore, P supply did not have any effect
on nodule number and dry weight. In contrast to fababean and green pea, N concentra
tions in plants supplied with fertilizer N were higher than those reliant on symbiotic N.
The poor Modulation in 1982 as compared to 1980 and 1981 experiments illustrates that
great variability existed between growing seasons as well as a poor ability of dry bean
plants to meet their genetic yield potential in the field without the addition of fertilizer N.
Also the selection of early maturing bean varieties and/or timing of planting that will allow
the plants to escape the period of water'stress prevailing at the end of July and early
August (at Bozeman) might be as important as soil fertility considerations. The high grain
yields obtained with dry bean suggest that this crop represents a good alternative to cereal
production in Montana dryland agriculture. The results of 1983 and 1984 confirmed those
of 1982.
137
A set of criteria were used to evaluate the involvement of P in nodulation and N2 -
fixation and the results suggested that P supply increased nodulation and N2-fixation in
fababean, dry bean and green pea by stimulating the host plant growth rather than by
affecting nodule initiation and function.
A hypothesis is proposed to explain the inhibitory effects of ammonia on nodulation
and nitrogenase activity. In the proposed model, ammonia acts as an uncoupler or ion
ionophore and dissipates the electrochemical proton gradient created by the bacteroid
respiratory chain. The dissipation of the proton gradient would prevent or reduce the for
mation of ATP necessary to reduce the nitrogenase enzyme. More importantly, the des
truction of the membrane potential suppresses the low potential electrons that might be
necessary to reduce ferredoxin or flavodoxin. These mechanisms would give a satisfactory
explanation for the inhibitory effects of NH4*" in legume infection, root hair curlings,
nodule initiation and development and nitrogenase synthesis. The fact that fertilizer N
had very different effects on nodulation and N2 -fixation by the three different species of
host plants suggests that there may be more than one mechanism for the inhibition. The
effects of NH4* on nodulation and N2 -fixation on the three species of host plants can be
summarized as follows:
Species NH4+ Effects on Nodulation and N2-Fixation
dry bean severe
green pea moderate
fab a b e an n o n e to s lig h t
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139
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149
APPENDIX
Table 105. Average Monthly Temperatures Recorded at Experimental Site.
Average Maximum__________ _______ Average Minimum_________ _________ Average DailyMonth 1980 1981 1982 1983 1984 1980 1981 1982 1983 1984 1980 1981 1982 1983 1984
T a b le 1 0 7 . C a lib ra t io n E q u a tio n s R e la tin g T o ta l P e rc e n t N D e te rm in e d b y th e N itro g e nA u to a n a ly z e r a n d th e In fra -R e d A n a ly z e r T e s t V a lu e s fo r F ab a b e a n , G reen Peaand Dry Bean.
T a b le 1 0 8 . E ffe c ts o f P a n d N A p p lic a t io n s on F ab a b ea n S h o o t D ry W e ig h t, g p e r P la n t, asa F u n c tio n o f T im e a t B o ze m a n , M o n ta n a , 1 9 8 0 .
C1 X C2 .1145 .0144 .1508 183.2000*C1 X C3 .0006 .5184 .1122 23.6700C1 X C4 .0342 .0001 3.9010 22.2300C1 X C5 .0006 .0225 .3660 .4692C1 X C6 .0113 .0512 .3960 6.6980Ci X Cf .0200 .0002 .0512 2.6220C1 X Cg .0145 .6962 1.3610 32.8000C1 X C9 .0200 .6728 2.6450 .8845IP < 0.05; * * P < 0.01.'Contrast designations refer to those of Table 3 and are the same for all tables.
1 5 4
T a b le 1 1 0 . E ffe c ts o f P an d N A p p lic a tio n s o n F ab a b ea n R o o t D ry W e ig h t, g P la n t- 1 , as aF u n c tio n o f T im e a t B o ze m a n , M o n ta n a , 1 9 8 0 .
C l X C 2 .0000 .2147 22.5800** 33.1400C1 X C3 .0012 .0361 4.6440 40.0700C1 X C4 .0002 .4624 .0000 .3600C1 X C5 .0156 1.0610* .1190 .0009C1 X C6 .0181 .0800 .1985 13.6200C1 X C2 .0018 1.3780** .8712 13.3100C1 X Cg .0025 .0512 .1985 3.9760C1 X C9 .0008 .5408 .2048 8.0800
* P < 0 .0 5 ; * * P < 0 .0 1 .
1 5 5
T a b le 1 1 2 . E ffe c ts o f P a n d N A p p lic a t io n s on F ab ab ean N o d u le N u m b e r as a F u n c tio n o fT im e a t B o z e m a n , M o n ta n a , 1 9 8 0 .
C1 X C2 301.9000 1411.0 1240.0 3965.0C1 X C3 , 66.0200 817.9 1853.0 1109.0C1 X C4 1.2660 1.2100 107.1 392.0C1 X C6 28.89 28.09 2814.0 585.6C1 X C6 75.0300 16.25 567.8 5.12C1 X C7 1.1250 12.00 2621.0 1022.0C1 X C8 47.5300 . 75.64 1.804 677.1Cl X Cg ' 72.000 51.01 176.7 3.92
* P < 0 .0 5 ; * * P < 0 .0 1 .
1 5 6
T a b le 1 1 4 . E ffe c ts o f P a n d N A p p lic a t io n s on F ab ab ean N o d u le D ry W e ig h t as a F u n c tio no f T im e a t B o ze m a n , M o n ta n a , 1 9 8 0 .
Table 115. Contrast Comparisons for Fababean Nodule Dry Weight as a Function of Time at Bozeman, Montana, 1980.
Contrasts
C1
C2
C3
C4
C5
C6
C7
CsC9
C1 X C2
C1 X C3
C1 X C4
C1 X Cs C1 X C6
C1 X C7
C1 X Cg C1 X C9
Days from Planting
24 days 44 days 64 days 35 days
Mean Squares
.0218* 2.1750** 25.95** 36.68**
.2162X10"2 .07487* .5590 .6978
.1082X10"2 .1473** .0454 .0818
.1179X1 O' 1 * * .2484** 2.0240** 2.0340**
.2304X10"4 .03199 .0731 . .0515 .
.8192X10"4 .04749 .1814 .0772
.5120X10"5 .001225 .0019 .0019
.9267X10"2* * .2569** 1.5540** 1.9011*
.3346X10"2 * .03917 .5861* .4069
.6367X10"3 .03922 .2291 .1695
.9797X1 O' 3 .1051** .0019 .09178
.1296X10"4 .02216 .2211 .1422
.7396X10-4 .06227* .0101 .0219
.5056X10"3 .08586* .09228 .0267
.1066X10"3 .0036 .0211 .1388
.1843X10"3 .05139 .2688 .1998X10"5
.3485X10"3 .00262 .02149 .2828
* P < 0 .0 5 ; * * P < 0 .0 1 .
1 5 7
T a b le 1 1 6 . E ffe c ts o f P a n d N A p p lic a tio n s o n N itro g e n as e A c t iv ity ( C2 H 4 ) o f F ab ab eanas a F u n c tio n o f T im e a t B o ze m a n , M o n ta n a , 1 9 8 0 .
Days from Planting24 days 44 days 64 days 85 days
Treatm ents -N +N -N +N -N +N -N +NAtmole C2H4 p la n t" 1 h r -1
Ci X C2 10.4900* 33.3700 19.9500 .1320Cl X C3 .4160 41.7300 .0064 .0196Cl X C4 3.1860 .6889 5.1530 .0225Ci X C5 .0020 3.5720 43.6900* .0324Cl X C6 .3612 3.2000 .8978 .0181Ci X c? .2888 19.9100 70.5700** .1512Ci X C3 .0545 14.1000 .8712 .0545Cl X Cg 7.6050* 24.2900 17.1700 .0005
* P < 0 .0 5 ; * * P < 0 .0 1 .
1 5 8
T a b le 1 1 8 . E ffe c ts o f P a n d N A p p lic a t io n s on S p e c ific N itro g e n as e A c t iv i ty fo r F ab ab eanas a F u n c tio n o f T im e a t B o zem a n , M o n ta n a , 1 9 8 0 .
Days from Planting24 days 44 days 64 days 85 days
Treatm ents -N +N -N +N -N +N -N +NAimole C2H4 p lant 1 h r " 1 per g nodule d ry w eight
Cl X C2 269.20 1787.00 23.20 145.80Ci X C3 . 91.98 857.30 190.70 56.02Cl X C4 10340.00 977.20 1386.00 2329.00*Cl X C5 1837.00 845.60 25.30 1683.00Cl X C6 771.10 482.70 67,75 136.80Ci X C7 7811.00 366.90 235.40 2146.00Ci X Cg 15.74 119.00 128.30 324.11Cl X Cg 19560.00* 1109.00 1708.00* 2524.00*
* P < 0.05; * * P < 0.01.
1 5 9
T a b le 1 2 0 . E ffe c ts o f P a n d N A p p lic a t io n s o n F ab a b ea n S h o o t T o ta l P e rc e n t N itro g e n asa F u n c t io n o f T im e a t B o zem a n , M o n ta n a , 1 9 8 0 .
C 1 X Cg .0034 .0349 .0617 .1835C1 X C2 .0132 .0441 3.7060** .0156C1 X C4 .0056 .0144 .2652 .0030C1 X C5 .1482 .0081 1.8630** .0020C1 X C6 .2664 .0313 2.0000* * .2592C1 X C7 .0008 .0925 .2664 .1984C1 X Cg .0061 .0925 .0722 .5832Cj X Cg .0338 .2244 .2113 .4704
* P < 0 .0 5 ; * * P < 0 .0 1 .
1 6 0
T a b le 1 2 2 . E ffe c ts o f P a n d N A p p lic a t io n s on F ab a b ea n R o o t T o ta l P e rc en t N itro g e n as aF u n c t io n o f T im e a t B o ze m a n , M o n ta n a , 1 9 8 0 .
C l X C 2 .2304* .0032 .0049 .6058**C i X C 3 .0025 .0004 .1444 .0006C i X C 4 .0784 .0676 .0100 .0056C l X C 5 .0841 .0576 .2601* .1806C i X C 6 .0481 .0221 .0000 .2113*C i X C 7 .0365 .0365 .5304** .0200C i X Cg .1404 .1200 .0000 .0061C l X Cg .0005 .0005 .0221 .0338
?P < 0 .0 5 ; * * P < 0 .0 1 .
161
T a b le 1 2 4 . E ffe c ts o f P R ates , Sources a n d A p p lic a t io n M e th o d s a n d N o n F ab ab eanS h o o t W e ig h t as a F u n c tio n o f T im e a t B o z e m a n , M o n ta n a , 1 9 8 1 .
T a b le 1 2 6 . E ffe c ts o f P R ates , S ources an d A p p lic a t io n M e th o d s a n d N o n F ab ab eanN o d u le N u m b e r as a F u n c tio n o f T im e a t B o ze m a n , M o n ta n a , 1 9 8 1 .
60 MP S .1133 .1733 .5200 .1867 .8533 .8100120 MP S .0700 .0833 .6100 .2000 1.743 1.257
60 TP S .1333 .1267 .5767 .1900 1.077 1.257120 TP S .1500 .1600 .6900 .2933 1.063 .760060 MP B .1267 .0967 .5033 .1700 1.223 1.123
120 MP B .1067 .0900 .4467 .2567 1.113 1.16360 TP B .1167 .1067 .6533 .3133 1.450 1.237
120 TP B .1267 .1733 .8433 .3500 1.257 1.087
CV (%) = LSD (.05)
38.30.0726
31.30.2076
41.10.818
1 6 3
T a b le 1 2 8 . E ffe c ts o f P R ates , S ources a n d A p p lic a t io n M e th o d s a n d N o n F a b a b e a n S h o o tT o ta l P e rc en t N itro g e n as a F u n c tio n o f T im e a t B o ze m a n , M o n ta n a , 1 9 8 1 .
Table 129. Effects of P Rates, Sources and Application Methods and N on Fababean Root Total Percent Nitrogen as a Function of Time at Bozeman, Montana, 1981.
T a b le 1 3 0 . E ffe c ts o f P R ates , S ources an d A p p lic a t io n M e th o d s and N o n F ab ab ean S h o o tP% as a F u n c tio n o f T im e a t B o ze m a n , M o n ta n a , 1 9 8 1 .
T a b le 1 3 2 . E ffe c ts o f P a n d N A p p lic a tio n s on D ry Bean S h o o t D ry W e ig h t, g p er p la n t, asa F u n c tio n o f T im e a t B o ze m a n , M o n ta n a , 1 9 8 0 .
Cl X Cg .0007 .0608 .0374 .0201Ci x C3 .0000 .0729 .0009 .2162Cl X C4 .0064 .1764 .2209 .1482Cl X Cs .0000 .3364 .0784 .5550Cl X C6 .0008 .2381 .0313 .0061Cl X C7 .0008 .1105 .0481 1.280**Cl X C8 .0032 .3961 .0481 .1201Cl X Cg .0032 .0013 .1985 .7938*
* P < 0 .0 5 ; * * P < 0 .0 1 .
1 6 7
T a b le 1 3 6 . E ffe c ts o f P a n d N A p p lic a t io n s on D ry Bean N o d u le N u m b e r as a F u n c tio n o fT im e a t B o ze m a n , M o n ta n a , 1 9 8 0 .
C1 X C2 8.27 19.51 13.44 28.00C1 X C3 28.89 2.25 2.25 3.52C1 X C4 13.14 1.56 4.00 1.89C 1 X Cs 123.80** 90.25* 52.56 .77C1 x C6 24.50 13.78 24.50 5.28C1 X C7 116.30** 94.52* 28.12 1.13C1 X C3 24.50 26.28 .50 7.03Cj X C9 101.5* 47.53 12.50 21.12
* P < 0 .0 5 ; * * P < 0 .0 1 .
1 6 8
T a b le 1 3 8 . E ffe c ts o f P a n d N A p p lic a t io n s on D ry Bean N o d u le D ry W e ig h t as a F u n c tio no f T im e a t B o ze m a n , M o n ta n a , 1 9 8 0 .
C1 X C2 .5929X104 .5040X10-3 .1406X10-2 .4242X10’-2C1 X C3 .1024X10-4 .6996X10-3 .12 7 2X 1 0 1 .8911X10-2C1 X C4 .5625X10-4 .3142X10-2 .4331X 10 '1 .9018X10-'C1 X C5 .9610X IO-5 .1034X10-2 .3819x10-2 .6641X10-'C1 X C6 .1428X10-3 .5534X10-2 .4431X 10"1 .1651C1 X C7 .5724X10-4 .8364X10-3 .1516X10-' .1752X10-2C1 X C8 .9245X10-3 .2333X10-3 .2499X10-2 .2035X10-2Cl X Cg .5724X10-4 .4095x10-2 .5969X10"' .2207
* P < 0 .0 5 ; * * P < 0 .0 1 .
1 6 9
T a b le 1 4 0 . E ffe c ts o f P a n d N A p p lic a tio n s on N itro g e n a s e A c t iv ity ( C 2 H 4 ) o f D ry Beanas a F u n c tio n o f T im e a t B o ze m a n , M o n ta n a , 1 9 8 0 .
C1 X C2 .1849 .3383 .2533 .1320C1 X C3 .0064 .0702 .1156 .0196C1 X C4 .0784 .0462 .0625 .0225C1 X Cs .0064 .1806 .1521 .0324C1 X C6 .0013 .0013 .6612 .0181C1 X C7 .0061 . .4050 .0685 .1512C i X Cg .6844** .1200 .0841 .0545C i X C 9 .1861 .4232 .0041 .0005
* P < 0 .0 5 ; * * P < 0 .0 1 .
170
Table 142. Effects of P and N Applications on Specific Nitrogenase Activity for Dry Beanas a Function of Time at Bozeman, Montana, 1980.
C l X C 2 .0387 .7000** .0067 .8680*C l X C 3 .0784 .0484 .1980* .3306C l X C4 .0529 .0036 .2550* 1.2660**C l X C 5 .0841 .4624* .2256* 1.1130**C l X C 6 .1860 .2112 .0200 1.0800**C i X C 7 .0005 .2521 .6613** .2048C i X C 8 .0613 .1200 .0722 .5512Cj X C 9 .0061 .0685 .1984* .7200*
* P < 0.05; * * P < 0.01.
172
Table 146. Effects of P and N Applications on Dry Bean Root Total Percent Nitrogen as a Function of Time at Bozeman, Montana, 1980.
Days from Planting24 days 44 days 64 days 85 days !
