THE EFFECT OF PLANT WATER POTENTIAL ON NITROGEN FIXATION OF SOYBEANS A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by JOHN NENE-OSOM AZU In partial fulfilment of requirements for the degree of Master of Science June, 1975 John N-0 Azu, 1975 University of Ghana http://ugspace.ug.edu.gh ' C g , 1 6 S \ ^ - ) A z 3 """""I R o rvv^ Q201571 University of Ghana http://ugspace.ug.edu.gh ABSTRACT THE EFFECT OF PLANT WATER POTENTLAL ON NITROGEN FIXATION OF SOYBEANS John Nene-Osom Azu, M.Sc. Supervisor: University of Guelph, 1975 Professor J.W. Tanner Field experiments at two sites, Elora and Arkell,were conducted to investigate the effect of plant water potential on the nitrogen fixation of two soybean cultivars, Vansoy and Anoka. The treatments were Vansoy and Anoka both under irrigated and non-irrigated conditions. The acetylene reduction technique was employed to estimate nitrogen fixation of nodules and measurements of plant water potential were made using the pressure chamber technique. Nitrogen fixation and plant water potential measurements were made at various stages during the entire growing period of the soybeans, and the results showed that water stress which was related to low plant water potential caused a reduction in nodule numbers, fresh weight and dry weight. There was evidence that moderate moisture stress mainly affected the amount of nodule tissue formed and not nitrogen fixing efficiency whereas severe stress led to reduction in both nodule mass and efficiency of fixation. The total seasonal amount of nitrogen fixed by Vansoy and Anoka was reduced substantially by water stress while high plant water poten­ tial resulting from irrigation led to large amounts of nitrogen fixed. Regression analysis of nitrogen fixed with plant water potential indicated that reduction in plant water potential was accompanied by University of Ghana http://ugspace.ug.edu.gh reduction in the amount of nitrogen fixed, thus showing a relationship between plant water potential and nitrogen fixation. Irrigation substantially increased seed yields of Vansoy and Anoka at Arkell, but there was no significant increase in either variety at Elora. University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation to Dr. J.W. Tanner for his invaluable guidance and encouragement throughout the course of this study and preparation of this manuscript. Special grati­ tude is also extended to Dr. K.R. Stevenson, Dr. T.E. Bates and Dr. D.J. Hume for their sound advice and assistance; and Mr. P. Gostovic and Q. van de Vrie for their technical aid. I wish to thank the Canadian International Development Agency for their scholarship provided through the Ghana Project and the National Research Council for funding the research. Finally the author thanks the many loved ones both home and abroad whose moral support kept him on in completing this research. i University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS INTRODUCTION ............................................. 1 LITERATURE REVIEW ........................................ 3 MATERIALS AND METHODS ............... 10 RESULTS AND DISCUSSIONS . 16 SUMMARY AND CONCLUSIONS .............................. 28 LITERATURE CITED ......................................... 45 APPENDICES............................................... 49 Page ii University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 1. Table 2. Table 3. Table 4. Table 5. Page Rainfall data in cm from June to September, 1974 (Elora, Ontario) ............................ Water potentials of bagged leaves of Vansoy and Anoka for 3 dates at the two locations ........ Water potentials at three different dates of different grafting combinations of Vansoy and Anoka ...................................... 40 Effect of irrigation on mean nodule dry weight, nodule size, ug ^-fixed per dry weight.hr, and amount of N£ fixed per hectare, day ............. ^ Effect of irrigation on seed yield, total nitrogen fixed, period of fixation and plant population for Vansoy and Anoka at the two locations .......... 2^ 43 44 iii University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Regression analysis of plant water potential with plant age for the different treatments at Elora and Arkell ............................. 30 Page Age: N2[C2H2]-fixing activity profiles showing the effect of irrigation on daily N2 IC2 H2 ]fixation, (Kgs N2 fixed per ha.day) by Vansoy and Anoka at the two locations ............................ Age: N2 [C2 H2 ]-fixing activity profiles showing the effect of irrigation on nodule number per plant for Vansoy and Anoka at the two locations .......... Age: N2 [C2H2 ]-fixing activity profiles showing the effect of irrigation on nodule dry weight (g) per plant for Vansoy and Anoka at the two locations .. Age: N2 [C2 H2 ]-fixing activity profiles showing the effect of irrigation on nodule size of Vansoy and Anoka at the two locations .................... Age: N2 [C2H2 ]-fixing activity profiles showing the effect of irrigation on fixation efficiency, (mg N2 - fixed per g dw.hr) of Vansoy and Anoka at the two locations .................................... 35 Daily variation of N2 [C2 H2 ] fixation of irrigated and non irrigated treatments at the two locations.. 36 Total or seasonal nitrogen fixed, (Kgs. per ha.) by Vansoy and Anoka under irrigated and non-irrigated conditions ................................... 37 Regression analysis of Kg. ^-fixed per ha.day with plant water potential for Vansoy and Anoka at the two locations .......................... 38 Regression analysis of per cent soil moisture with plant age for the different treatments at Elora and Arkell ............... 39 iv University of Ghana http://ugspace.ug.edu.gh LIST OF APPENDICES Appendix 1. Analysis of variance for different parameters of nitrogen fixation ............................ 49 Appendix 2. Age: C2H2]-fixing activity profile showing the effect of irrigation on ug N2 fixed per nodule.hr for Vansoy and Anoka.......................... 50 Appendix 3. Age: N2 [C2 H2l-fixing activity profiles showing the effect of irrigation on ug N2 -fixed per plant.hr by Vansoy and Anoka at the two locations ....... 51 Appendix 4. Age: -fixing activity profiles showing the effect of irrigation on nodule fresh weight (g) per plant for Vansoy and Anoka at the two locations ... 52 Appendix 5. Age: N2 [C2 H2 l-fixing activity profiles showing the effect of irrigation on fixation efficiency (ug N2 fixed per g. nodule Fresh weight.hr) for Vansoy and Anoka at the two locations ................... 53 Appendix 6. Regression analysis of mg N2 fixed per g nodule fresh weight.hr with plant water potential for the two cultivars at the two locations ............ 54 Appendix 7. Regression analysis of mg N2 fixed per plant.hr for Vansoy and Anoka at the two locations ......... 55 Appendix 8. Analysis of variance for final seed yield of Vansoy and Anoka soybeans .......................... 56 Appendix 9. Analysis of variance for water potentials of bagged leaves of Vansoy and Anoka for 3 dates at the two locations ....... 57 Appendix 10. Analysis of variance for water potentials of different grafting combinations of Vansoy and Anoka at three different dates ............... 58 Page University of Ghana http://ugspace.ug.edu.gh INTRODUCTION Nitrogen fixation by a legume requires specific Rhizobium and suitable environmental conditions for nodule formation and activity. The amount of nitrogen that is fixed is determined by the compatability between the plant and the bacteria and by the total environment of which climate and host nutrition are probably the most important (Bell et al. 1971). Many observations have been made on the effects of moisture stress on root nodules of various legume species. These studies have been done mainly on number, size and weight of nodules to evaluate the effect of stress on the amount of nodule tissue that is formed. In many instances, nitrogen fixing activity has been considered to vary directly with nodule weight and large nodules were considered effective. This may not necessarily be true since nodules vary greatly in size and number depending on the plant and the Rhizobia. Recently, the acetylene reduction technique has been used to estimate the nitrogen fixation by legumes under moisture stress. Most of this work, however, has involved the use of detached nodules and the employment of one-time assays in growth rooms or chambers. In some cases, plants have been grown under optimum conditions and then water stressed over a period of time for measurement of the reduction in nitrogen fixation due to the stress. Thus, although water stress has been shown to affect growth, comparatively few studies have been done to evaluate its effect on nitrogen fixation under stress conditions in the field, because measure­ ments of the effect of plant and soil water potential on the symbiotic - 1 - University of Ghana http://ugspace.ug.edu.gh nitrogen fixing systems in the field have been difficult to obtain. The aim of this study was to determine the extent to which continuing exposure to favourable and unfavourable moisture conditions affected the nitrogen fixation of two soybean cultivars growing under field conditions. This information may provide a basis for the evaluation of a) the effect of plant water potential on nitrogen fixation, b) the contribution to the nitrogen nutrition of the plants made by the symbiotic system under moisture stress, and c) the extent to which this contribution might be improved through irrigation during periods of stress in the field. - 2 - University of Ghana http://ugspace.ug.edu.gh LITERATURE REVIEW Soybean (Glycine max L. Merrill) nodules, as well as those of other legumes, result from a complex interaction between the root cells and compatible Rhizobium species. The interaction begins when Rhizobia penetrate the epidermal cells and terminate when the aged remnants of the nodules are sloughed from the roots. Environmental factors can affect the successful establishment of an effective symbiosis between Rhizobia and their hosts at any or all of the following three stages: they may affect the occurrence, growth and survival of the root nodule bacteria; modify nodule formation; and affect the functioning of the formed nodule (Vincent 1965). Examination of accounts dealing with the effects of environmental factors reveal how difficult it can be to distinguish between those secondary effects which influence nitrogen fixation through their more generalized influence on the plant itself, and those that operate directly on the symbiosis. However, from a practical point of view, any factor which stands in the way of the full realization of a plant's fixation potential will be equally serious as to how much nitrogen the nodules can fix. The effects of climate on nodulation, establishment of seedlings and subsequent yield of annual legume crops have been studied in many parts of the world. The results indicate that these effects are likely to be important in all places where the climate is characterized by more or less severe and seasonally well-defined drought conditions as well as in areas with short periods of moisture stress during the growing season. Because the amount of nitrogen fixed by the nodules is a function - 3 - University of Ghana http://ugspace.ug.edu.gh of the central tissue volume and the active life of the tissue (Chen and Thornton 1940), the factors that determine the longevity of this tissue are extremely important. Many noticeable internal and external changes are observed in nodules under water stress (Sprent 1971). Of the various environmental factors affecting nitrogen fixa­ tion, water supply has been studied very little, except where it involves excessive water supply and reduced aeration under waterlogged conditions (Schwinghamer et al. 1970, Engin and Sprent 1973). Considerable attention has been directed to the interactions occurring at the root hair surface where compatible strains of Rhizobia invade the root hair and produce an infection thread (Fahraeus and Ljunggren 1968, Roughley _et al. 1970). However, less is known of inter­ actions under excess moisture or drought conditions, and much less is known of interactions which occur in the subsequent stages of nodule development, particularly when the Rhizobia are released from the infec­ tion thread into the host cell and change from vegetative rods into nitrogen fixing bacteroids, and during subsequent nitrogen fixation (Sprent 1973, Pankhurs et al. 1972). Wilson (1931) reported that drought adversely affected kidney bean (Phaseolus vulgaris) root nodules in that a reduction of soil moisture from 20% to 12.5% led to the shedding of about 36% of the root nodules. Other reports have also shown beneficial effects of irrigation on nodule number and size. Masefield (1952) found that there was a steady fall in the nodulation of Phaseolus vulgaris during the summer and attributed this to the progressive drying of the soil during the season. He also reported that the consistently higher nodulation observed in Northern Nigeria than in most places in the south - 4 - University of Ghana http://ugspace.ug.edu.gh was due to the heavier soils in the North which were much wetter during the growing season. Soils with a higher water table were also observed to result in legumes having greater nodulation. Masefield (1961) studied the effect of irrigation on the nodulation of Vicia faba, Pisum sativum and Phaseolus vulgaris at Oxford. Irrigation generally increased the size as well as the number of nodules and he concluded that it would be worthwhile to examine the possibility that irrigation may in suitable circumstances confer more advantage on nodulated legumes than on other crops due to its effect in causing an improvement in nodulation. Results of McKee (1961) showed that nodulation of birdsfoot trefoil (Lotus corniculatus L.) as measured by nodule number and size, and the presence of functional nodules, tended to decline with increas­ ing periods of dessication, and nodulation may be retarded or inhibited in soils subjected to dessication or alternate periods of moisture and drought. However, according to Vincent (1965), the suitability of a root hair for invasion might be adversely affected before the shortage of water sufficiently depresses the number of bacteria, and the damage so caused might persist even after the cells have recovered. Pate et^ al. (1969) pointed out that transport of fixed nitrogen out of the nodule required continuous access by the nodules to an external source of water. They suggested that if water is in short supply, the products of fixation might accumulate in the nodule and thus inhibit further functioning of the nodule. Diatloff (1967) examined the field nodulation of cowpea (Vigna unguiculata Walp.) and four other legumes under fluctuating moisture - 5 - University of Ghana http://ugspace.ug.edu.gh conditions, and concluded that there was loss of nodules during excessively wet or dry conditions. He attributed the nodule loss with decreasing moisture partly to removal of nodules by the shrink­ ing clay. Doku (1970) presented evidence to show that with increasing availability of moisture, there was a concomitant increase in the nodulation of cowpeas. Storage of nodules in water or immersion in water during assay have been shown to markedly decrease nitrogen fixing activity (Diatloff 1967, Hardy et al. 1968, Schwinghamer et al. 1970). Sprent (1969) indicated that acetylene reduction by detached nodules was sensitive to water, both in excess and deficiency. She concluded that immersion in water inhibited acetylene reduction by reducing the oxygen supply to the nodules. The findings agreed with Bergersen's (1962) report that, at low pO^> root nodule fixation diminished due to limited ATP supply. According to Sprent (1971a), when the fresh weight of detached nodules of soybean was reduced below 80% of its maximum value by dessication, irreversible changes occurred. Acetylene and nitrogen reducing activities ceased, the respiratory rates became very low and there were gross structural changes in the nodules. However, reduction of the water content down to 80% of the maximum fresh weight led to a proportionate slowing down of acetylene reduction with a concomitant reduction in respiratory activity. Kuo et aT. (1971) reported a decrease in the ratio of nitrogen fixed to carbon absorbed, with increasing soil water suction at each of the soil temperatures used and they attributed this to the effect of plant water stress on translocation of carbohydrates to the root nodules. - 6 - University of Ghana http://ugspace.ug.edu.gh They however added that plant water stress possibly had a direct effect on the nitrogen fixation within the nodule as reported by Sprent (1971a), who suggested that the direct effect may be aggra­ vated by reduced supplies of photosynthate from wilted leaves. Johnson (1971), in a preliminary investigation, observed a linear decrease in nitrogen fixation with decrease in plant water potential. Furthermore, a decrease in nodule weight, leghemoglobin content and acetylene reduction occurred in unwatered plants compared to watered controls. The results suggested a pronounced effect of soil water status as well as plant water potential on soybean nitrogen fixation. There is evidence to show that severe dehydration produced irreversible changes accompanied by loss of acetylene reduction due to structural changes within the nodule. Sprent (1971b), investigating the role of nodule cortical cells of soybeans, observed that apart from vascular traces, the cells are mainly vacuolate with active cytoplasm and are connected with each other and to cells of the infected region by numerous plasmodesmata, with a network of air spaces running through­ out the nodule. Using manitol, Sprent (1971c) observed that osmotically applied stress affected the outer cells of the nodule more quickly and more severely than the inner cells, with the vacuolate cells being more susceptible to stress than non-vacuolate cells. Loss of about 30% of their fresh weight resulted in breakdown of the cytoplasm. She there­ fore concluded that the vacuolate cells of the cortex play an integral part in the nitrogen fixing activity of the whole nodule. This gives an indication that, apart from the general effects of water stress on the plant and thus its indirect effects on nitrogen fixation (Kuo _et al. 19 71), there is a direct effect of stress on the nodule tissues. - 7 - University of Ghana http://ugspace.ug.edu.gh Working on whole plants of Vicia faba and Glycine max, Sprent (1972) reported that water stress reduced nitrogen fixation of soybean plants grown in pots and, provided the nodules retained some activity, recovery on watering was normally complete within hours, suggesting a transient effect of the stress. A field trial with Vicia faba showed a high degree of correlation between soil water content and nitrogen fixation. Observations on slow natural drying over a 6-week period showed that there was a progressive reduction in activity and irrigation led to the restoration of activity. The results showed that maximum nitrogen fixation occurred at about field capacity, above which activity was reduced due to aeration problems. Soybean nodules however could survive for several days under water while still attached to the parent plant, resuming their full rate of activity when the water was drained off. Results presented by Engin and Sprent (1973) indicated that during a period of water stress, the initial response of nitrogen fixation was a direct effect on the nitrogen fixing tissues. The severity of the response varied with the degree and duration of stress and provided that the stress was not too severe, complete recovery could be achieved. They concluded that longer periods of stress resulted in depression of nodule growth, and the impairment of efficiency resulted partly from the direct effects of stress on the nitrogen fixing tissues, but it could be supplemented by reduced respiration which may be a major factor in lowering nitrogen fixing efficiency in nodules. Very few studies have been done on the nitrogen fixing activity of different soybean cultivars under water stress in the field, espec­ ially the employment of developmental studies of a legume under stress to correlate nodule structure and development, and nitrogen fixing activity. - 8 - University of Ghana http://ugspace.ug.edu.gh Host of the work done to date has been on detached nodules, since the quantitative characteristics of the process in the field have previously been determined only with difficulty (Hardy jrt _al. 1971). At present it has been established that moisture stress profoundly affects the rate of nitrogen fixation and hence the total amount of nitrogen that is fixed in the field throughout the growing season. Unfortunately, there have been no reports showing ageiN^- fixing activity profiles under water stress conditions. Data of this nature, showing physiological and structural effects, are needed since water stress in the field during the growing season is comnon- place while activity profiles have been established for plants under optimum moisture conditions. As has been emphasized by Hardy et al. (1971),a complete profile of nitrogen-fixing activity is essential for a valid evaluation of the effect of any factor on nitrogen fixation because of the variation in nitrogen fixation during the season. Infor­ mation of this nature would offer a more realistic approach towards the appraisal of nitrogen fixation under water stress in the field. - 9 - University of Ghana http://ugspace.ug.edu.gh MATERIALS AND METHODS Two soybean cultivars, Vansoy and Anoka'*' were grown during the summer of 1974 at two locations, the Elora Research Station and the Arkell Research Station, both near Guelph. The Elora site is on a London loam soil which is imperfectly drained, and the Arkell site is on a well-drained Burford loam over gravel. Both tests consisted of four treatments each, two cultivars, Vansoy and Anoka, and two moisture regimes, irrigation and no irrigation. The four treatments were alloted to plots in a split-plot design with four replications. Irrigated and non-irrigated constituted the main plots and cultivars the sub-plots. One replication at the Arkell site had to be discarded due to poor emergence, hence three replications were used. Soybeans were planted in 30 row plots on May 30 at the Elora site and June 3 at the Arkell site, all in 6 m rows with 35 cm spacing between rows. 2 A commercial Rhizobium japonicum inoculum and D-L plus, (Diazinon = 15%, Lindane = 25%, Captan = 15%), an insecticide-fungicide, at the rate of 2.8 g (active) per 1 kg of seed were applied to the seeds in the planters just before planting. Weeds were controlled chemically by incorporating 1.1 kg/ha (active) of tri-fluralin before planting and 0.55 kg/ha (active) of linuron pre-emergence at both locations, but in Arkell, a field bindweed (Convolvulus arvensis) infestation was con­ trolled by hoeing. Obtained from Dr. J.W. Lambert, University of Minnesota. Hansen Inoculator Co., Milwaukee, Wisconsin 53209. 2 - 10 - University of Ghana http://ugspace.ug.edu.gh At Elora, approximately 2.54 cm water were applied by sprinkler irrigation to the appropriate treatments on July 4, 11, 18, 23, August 2, 9, 15, 17 and 22. Irrigation was applied twice a week at Arkell by means of an oscillating garden sprinkler. Two sets of samples per treatment, each consisting of four plants, were harvested twice a week and the following measurements made on each sample: a) leaf water potential b) estimates of nitrogen fixation rates c) number of nodules d) fresh and dry weight of nodules Plant water potential was measured with a pressure chamber designed after that of Waring and Cleary (1968). For measurement to be taken, the petiole of a top, well exposed leaf was cut off and placed in a plastic bag covered with aluminized mylar tape to minimize transpiration. The petiole was then inserted into the foam rubber compression gland in the lid of the chamber. The leaf was quickly removed from the bag and put into the chamber and the lid bolted on, exposing the petiole to the out- 2 side. The pressure reading in dynes per cm required to produce bubbling on the cut petiole surface was recorded and converted into bars, the units of water potential. Rates of nitrogen fixation were estimated by means of acety­ lene reduction technique (Hardy _et aJ.. 1968). Excised soybean roots were harvested between 10 a.m. and 12 noon for the Arkell site and 12 noon and 2 p.m. for the Elora site. Nodules were harvested at approxi­ mately the same time of day to avoid diurnal variation in acetylene reducing activity (Stewart et al., 1967, Hardy et al. 1968). - 11 - University of Ghana http://ugspace.ug.edu.gh At each harvesting date, four plants were collected and decapi­ tated immediately prior to assay. The nodulated roots were put into 1252 ml plastic bags and heat sealed after driving out the air. The bags were filled to 0.2 atmospheres with a mixture of 10% acetylene and 90% Argon:oxygen:carbon dioxide (80:30:0.03) at a flow rate of 1800 ml/min for Argon and oxygen and 200 ml/min for acetylene using a gas proportionator. The puncture made by the proportionator needle was sealed with masking tape and checked for leaks. After 30 minutes incuba­ tion period, a 10 ml sample of gas from each bag was transferred into 10 ml evacuated blood sample tubes with a syringe and these samples were used for gas chromatographic analysis of their ethylene concentrations. Samples for standard curves were prepared in a similar fashion using various levels of pure ethylene, however the plastic bags contained only sand and no nodulated roots. A Varian Aerograph series 1520 gas chroma­ tograph with 100-120 mesh, Poropak N columns and equipped with dual flame ionization detectors was used. The 250 ul gas samples were analyzed in duplicate during each run. Ethylene peak heights obtained from the gas chromatographic analysis of field samples were compared with the standard curves of peak heights versus ethylene content of injected samples which were obtained each time a run was made. These values were converted into amounts of nitrogen fixed, ^ [ 0 2^], using a ratio of three moles of ethylene produced to one mole of ^ fixed (Johnson and Hume 1973). The amounts of Nj fixed during the growing season was calculated as: t mmoles C„H -*■ C.H./plant day x 28 mg N fixed = E — — -------------- z conversion factor of 3 - 12 - University of Ghana http://ugspace.ug.edu.gh where t is age in days and 28 is the molecular weight of N2* A daily variation of C2H2-reducing activity was measured by sampling two sets of four nodulated plants every three hours from 6 a.m. and 6 p.m. during three days in mid-August; August 16 for Elora and August 15 and 19 for Arkell. The results obtained were used to convert the amount of nitrogen fixed during one hour between 10 a.m. and 12 noon for Arkell and 12 noon and 2 p.m. for Elora into the amount of nitrogen fixed per day. Since night time fixation rates were not obtained in this work, adjustment for rates during the night was based on the data of Johnson (1971), where the average amount of nitrogen fixed from 6 p.m. to 6 a.m. was shown to be 83% of the daytime amount. It has been noted by Gibson (1969) that the rate of nitrogen fixation during the normal dark period could be as high as that during periods of illumination if a temperature of 30°C was maintained. Data presented by Hardy e_t al. (1968) also showed that although there is a close relation­ ship between light and nitrogen fixing activity, plants maintained on a 16 hour light and 8 hour dark cycle did not show a marked diurnal varia­ tion. Therefore the possibility exists that the correction of values of nitrogen fixed during the night based on Johnson's work may represent an underestimation. Semiweekly activities were integrated for the complete growth cycle to obtain a profile of N2[C2H2] fixation throughout the growing season. The nitrogen fixation rates were finally converted to g N2[C2H2] fixed per hectare.day based on populations of 81,000 to 123,000 plants/ha at Arkell and Elora (Table 3). These plant populations did not differ significantly between the treatments at either location. - 13 - University of Ghana http://ugspace.ug.edu.gh At each sampling date, the bags containing the nodulated roots were put into an ice box until root volumes were determined by water displacement. These volumes were subtracted from the volumes of the bags to obtain the volume of gas mixtures in each bag. The nodules were picked, counted and weighed. They were then dried at 80°C oven tempera­ ture to constant weight. Soil samples were also collected from root depths where the nodules were mostly concentrated at every sampling date for per cent moisture determinations. On October 3, three rows of each plot 3 meters long were hand harvested after making a stand count and threshed for seed yield determination. The yields were converted to 14% moisture. There was a frost on September 24 before Anoka could mature. On August 19th, 20th, 26th, 27th, 28th and September 3rd the lowest leaves on three plants per plot were bagged _in situ with plastic bags covered with aluminized mylar tape. On the subsequent days the leaf water potentials of bagged leaves were measured using a pressure bomb. The water potentials of the bagged leaves provide an index of the resistance to water movement through the root and stalk to the point of insertion of the leaf (Dube 1972) . The results obtained on leaf water potentials at the two locations and the bagging experiment gave an indication that Anoka was consistently at higher leaf water potential than Vansoy under droughty conditions. This led to an investigation in which grafting of Vansoy tops on Anoka roots, and vice versa were made. The different combina­ tions were Anoka, Vansoy, Anoka/Vansoy, Vansoy/Vansoy, Anoka/Anoka, and Vansoy/Anoka. - 14 - University of Ghana http://ugspace.ug.edu.gh The grafting was done on one week old seedlings planted on the 12th September in a growth room in pots filled with a sandy loam soil. On December 13 watering was ceased and the soil allowed to dry out. Six leaf water potential measurements were made on each treatment for three consecutive days, December 16, 17 and 18. - 15 - University of Ghana http://ugspace.ug.edu.gh RESULTS AND DISCUSSION The months of July and August of 1974 were particularly dry as shown by the distribution and amounts of rainfall at Elora (Table 1). In July, August and September 29, 21 and 80% of the normal rainfall was recorded. These months represent the major part of the growing season, and only 43% of the normal 23.5 cm of rainfall was recorded. Thus in the absence of irrigation, there was substantial water deficiency in the field throughout most of the growing season. On September 24, there was a killing frost with a grass minimum temperature of 15°C at Elora which terminated soybean growth. In general plant water potential values recorded under non­ irrigated conditions at Arkell throughout the season, (Fig. 1) were lower than those recorded for the same treatments at Elora, indicating the pronounced drought conditions at Arkell due to the sandy soils. The difference in water potential between irrigated and non irrigated treat­ ments was also greater at Arkell than at Elora. A unique relationship between the amount of nitrogen fixed and leaf water potential could not be obtained because of other confounding effects such as plant age. That is, the rate of nitrogen fixation was a function of the stage of growth of the plant and thus was not uniquely related to leaf water potential. However comparison of Figs. 1 and 2 shows that a relationship clearly exists between water potential and N fixation on a day to day basis throughout the season. This relationship however, could not be established statistically since there were only two moisture levels used. - 16 - University of Ghana http://ugspace.ug.edu.gh It is evident that plants under irrigated treatments were at high leaf water potentials and high fixation rates while those under non­ irrigated treatments had low leaf water potentials and low nitrogen fixation rates (Figs. 1 and 2). The effect of irrigation on the number of nodules per plant is shown in Fig. 3. There was a progressive increase in nodule numbers due to irrigation. At Elora there were 42 and 38% reductions in mean nodule number per plant of Vansoy and Anoka, respectively (Table 2) under stress compared to the irrigated treatments. At Arkell, there was 32% reduction in nodule number per plant for Anoka but nodules on Vansoy were not significantly reduced. It has been shown by McKee (1961) that nodulation tended to decline with increasing periods of dessication and may be retarded or inhibited on soils subjected to alternate periods of moisture and drought. There was a practical effect of irrigation on the nodulation of soybean as has been reported by Fred _et al. (1932) and Diatloff (1967). Results of moisture level effect on nodule dry weight and size (mg fresh weight/nodule) are shown in Fig. 4 and 5 respectively. At both locations there was a steady increase with time in nodule dry weights due to irrigation where the water potential of plants was high. The rate of increase was, however, smaller for the moisture stressed treatments and there were no differences between the two cultivars under either high or low water potentials. The reduction in mean nodule dry weight due to stress (Table 2) was 56 and 45% for Vansoy and Anoka respectively at Elora and 48 and 50% for Vansoy and Anoka at Arkell. - 17 - University of Ghana http://ugspace.ug.edu.gh There was no difference in mean nodule size between the treat­ ments at Arkell (Table 2). At Elora, however, irrigation increased the mean nodule size of Vansoy and caused a decrease in Anoka. Irrigation generally increased the number and weight of nodules, as reported by Masefield (1961) and Johnson (1971) . The changes in efficiency of fixation of the two cultivars due to moisture levels are shown in Fig. 6. The efficiency of fixation measured as ug N^-fixed per g nodule dry weight.hr was not significantly affected by the different treatments at Elora. There was however a cultivar x moisture level interaction at Arkell (App. 1), where the water stress level was higher. The mean fixation efficiency of each cultivar was significantly lower under the non-irrigated conditions (Table 2). There were 55 and 31% reductions in efficiency under water stress for Vansoy and Anoka respectively. There is an indication that, apart from the reduction in the amount of nodule tissue (Fig. 4), there was a reduction in efficiency due to high water stress conditions at Arkell (Fig. 6). Johnson (1971) observed that fixation efficiency of stressed plants showed no reduction compared to control until after 15 days of drying and this may explain why there was no such reduction at Elora where the stress level was not as high as it was at Arkell. This suggests that efficiency was depressed significantly under severe dessication of the plants or nodules. Engin and Sprent (1973) also reported similar reduction in fixation efficiency and they suggested that this resulted partly from the direct effect of stress on the existing nitrogen fixing tissue and may be supplemented by effects on the distribution of respiratory products within the nodules. It appears - 18 - University of Ghana http://ugspace.ug.edu.gh that with longer stress periods, a larger proportion of the existing nitrogen fixing tissue is damaged beyond complete recovery and severity of this response varies with the degree and duration of the stress. Sprent (1971) observed that severe water stress led to a breakdown of the cytoplasm of vacuolate cells of the nodule cortex and concluded that they played an integral role in nitrogen fixation of the nodule. Other workers have suggested that the reduction in fixation efficiency may be due to the effect of plant water stress on the translocation of sugars to the root nodules (Kuo and Boersma 1971, Lawrie and Wheeler, 1973). Pate et al. (1969) also suggested that under water stress condi­ tions the products of fixation may accumulate in the nodule and this may inhibit further functioning of the nodule. Bergersen (1962) and Sprent (1969, 1971a) have shown that res­ piration is frequently a primary limiting factor in nitrogen fixation and in their recent work, Sprent (1972) and Engin and Sprent (1973) suggested that the depression in respiration due to moisture stress affected energy yielding processes and could be a means by which nitrogen fixation was inhibited. The results of this present work suggest that the depression of fixation efficiency due to water stress is dependent on the degree and duration of stress, and under moderate moisture stress, significant depression does not occur. There was a gradual reduction in fixation efficiency from nodule initiation to the end of the season and with nodule mass increases for all treatments at both locations. Similar observations have been made by Gibson (1967), Bond (1936), Stewart (1962), Johnson (1971) and Minchin and Pate (1973). There are indications from all these reports that as nodule mass increased with plant age, the volume of degenerated - 19 - University of Ghana http://ugspace.ug.edu.gh tissue increased and this added to the non-fixing cortex. Therefore although there was an increase in the relative nodule volume it led to a decrease in fixation efficiency. Thus the gradual loss of activity with time was due to ageing and build-up of senescent bacteroid tissue. However, the accentuated reduction in non-irrigated treatments at Arkell suggests that dessication was a major effect since the irrigated plants maintained higher efficiencies. Data on activity per nodule.hr (App. 2) and activity per plant.hr (App. 3) also show that nodules of plants under irrigated conditions were supporting nitrogen fixation at higher rates than those under stress. Fig. 7 shows the results of daily response of N^Cc^H^] fixed for the different treatments from data for one day August 15 and 16 for Arkell and Elora respectively at the stage in the season when fixation was at a maximum. These results were used to convert the amount of nitrogen fixed during sampling time into amount of nitrogen fixed per day. These values were then used for adjustment of fixation from 6 a.m. to 6 p.m. to account for diurnal variation. The procedure used to adjust for night time fixation has been described in the materials and methods. Activities expressed as g.N^-fixed per ha.hr did not change appreciably during the day at Elora, with the exception of Anoka irrigated and Vansoy non-irrigated which decreased from morning to afternoon. At Arkell however, activities for Vansoy and Anoka irrigated followed the pattern of increase in light intensity and thus availability of photosynthate through the day. This indicates a correlation between light intensity and nitrogen fixation as observed by Johnson (1971). - 20 - University of Ghana http://ugspace.ug.edu.gh Vansoy and Anoka non-irrigated were fixing nitrogen at very low rates and did not change to any extent during the day. Hardy et_ al. (1968) observed that excess moisture and/or low light intensity eliminated the normal diurnal variation in fixation. The results for Elora suggest that since water was applied to the irri­ gation treatments the day before measurements were made, there is a possibility that this eliminated the normal daily variation since excess moisture causes a reduction in nitrogen fixation (Schwinghamer et al. 1970). Results of the non-irrigated treatments at both locations also indicate that under moisture stress where normal photosynthesis is hampered, fixation activity may represent utilization of stored photo- synthate and thus elimination of the photosynthesis-dependent variation often observed under optimum conditions. Fig. 2 shows the average amount of nitrogen fixed per day over the season for the different treatments. These were obtained from conversion of daily nitrogen fixation rates to kg. C^ H^ ] fixed/ha. day based on plant populations (Table 3) of the different treatments. There was a progressive increase in overall daily amounts of nitrogen fixed per hectare.day for all treatments until late pod filling stage, which was followed by a decrease as the plants aged. Greater amounts of nitrogen were fixed daily by irrigated treatments at both locations. At Elora the reductions in mean daily amounts of nitrogen fixed (Table 2) due to moisture stress were 64 and 56% in Vansoy and Anoka respectively compared to the irrigated treatments. At Arkell water stress significantly reduced mean daily fixation by 73 and 79% in Vansoy and Anoka respectively. The period of fixation (Table 3) was different for the two locations and ranged from 64 to 82 - 21 - University of Ghana http://ugspace.ug.edu.gh days at Arkell and 54 to 64 days at Elora. Thus, although treatments at Arkell maintained low fixation rates, they continued through a longer period of time. Cumulative or total seasonal nitrogen fixed for the different treatments (Fig. 8 and Table 3) were obtained by summing the daily nitrogen fixation rates of each treatment. The results for Elora (Table 2) indicate that water stress resulted in 56 and 55% reductions in the total amounts of nitrogen fixed over the season for Vansoy and Anoka respectively. The reduction was more severe at Arkell due to the dry sandy soils and there was 74 and 76% reduction in seasonal fixation for Vansoy and Anoka. Thus, high plant water potential due to irrigation increased the total nitrogen fixed, and the extent of reduction due to water stress was dependent on the severity of the stress. Comparison of seasonal fixation for the two locations (Fig. 8 and Table 3) shows that total fixation of irrigated treatments at Arkell were 22 and 33% higher for Vansoy and Anoka than the corresponding treatments in Elora, although the daily nitrogen fixation (Fig. 2) was higher at Elora. This could be attributed to the prolonged period of fixation at Arkell (Table 3). The amounts of nitrogen fixed (Fig. 2) show that under long periods of drought interrupted by rainy days in the field, non-irrigated plants maintained a low but sustained nitrogen fixation activity. This may be due to a depression of nodule initiation and growth and/or the fact that the nodules, after prolonged stress were unable to recover significantly to the level of non-stressed ones depending on the severity and duration of the stress (Engin and Sprent 1973). - 22 - University of Ghana http://ugspace.ug.edu.gh There was no significant difference in fixation efficiency between the treatments at Elora and so reduction in total fixed under water stress did not result from depression of efficiency (Fig. 6). The data on nodule dry weights (Fig. A) however suggest that the differ­ ences in amounts of ^ ^ 2 !! ] fixed between irrigated and non-irrigated treatments at Elora could be largely explained by the differences in nodule dry weights and thus the amount of nitrogen fixing tissue since there was a significant reduction in nodule weight and no reduction in efficiency. Therefore under low plant water potential at Elora,initial bacteroid tissue development appeared to be more sensitive to moisture stress than nitrogen fixing efficiency. The data for Arkell suggest that under severe stress both reduction in nodule dry weight and impairment of efficiency contributed to the reduction of total N2^-C2H2^ fixe*^j University of Ghana http://ugspace.ug.edu.gh FIX ED / ha ( ac cu m ul at ed ov er se as on E I o r a A r k e 11 Days after planting Days after planting University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Fig. 9 Regression analysis of Kg. N^-fixed per ha.day with plant water potential for Vansoy and Anoka at the two locations. Elora Vansoy Y = 2.464458345 + (0.275845085X) + (-0.029796739X2) Anoka Y = 1.22833872 + (0.210174418X) + (-0.02118273162X2) Arkell .4399* .3077** Vansoy Anoka Y = -1.961090078 + (0.9548441289X) + (-0.744509X2) + (0.00158396X3) .4099* Y = -1.047208802 + (0.6152462239X) + (-0.522005X2) + (0.00188576X3) .2008* University of Ghana http://ugspace.ug.edu.gh FI X ED /h a. d ay B A R S £ A rke ll B A R S University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Fig. 10 Regression analysis of per cent soil moisture with plant age for the different treatments at Elora and Arkell. Elora Vansoy irr. Anoka irr. Vansoy no irr. Anoka no irr. Y Y Regression Equation Non Sig. Non Sig. -29.88262873 + (2.464474658X) + (-0.040905366X2) + (-0.000208506X3) -27.14044205 + (2.305780108X) + (-0.0038455512X2) + (0.0001972104X3) .32** .29** u> VO Arkell Vansoy irr. Anoka irr. Vansoy no irr. Anoka no irr. Non Sig. Y = -60.30341750 + (4.116630586X) + (-0.06733657614X2) + (0.00034388911X3) .26** Y = 16.17266943 + (-0.07854166923X) .11* Y = -93.6628542 + (5.790183939X) + (-0.09966342259X2) + (0.0005424285X3) .33** University of Ghana http://ugspace.ug.edu.gh 20 16 12 8 4 0 20 16 12 8 4 0 I o r a Vansoy irr. no irr. Anoka irr. no irr. 30 r k e I I 40 50 60 70 80 Days after planting 90 100 30 40 50 60 70 80 Days after planting 90 100 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh - 40 - Table 1. Rainfall data in cm from June to September, 1974 (Elora, Ontario) Date June July August September 1 0 0 0.36 0.83 2 0 Trace 0.13 0 3 0 Trace 0.10 0 4 0 0.43 0 0 5 0 0 0 0 6 0.31 . 0 0 0 7 0 0 0 0 8 0.1 0 0 0 9 0 0 0.05 0 10 Trace 0 0 0 11 0.25 0 Trace 0.66 12 0.025 0 0.1 0.05 13 0 0 0 0 14 0.58 0.13 0 0 15 1.65 0 0 0 16 0 0 0.05 0 17 0.25 0.48 0 0.94 18 2.21 0 0 0 19 0.71 0 0 0.25 20 0.25 0 0 0 21 0.025 0 0 0 22 0 0 0 0 23 0 0 0.03 0 24 0 0 0 0.23 25 0.10 0.53 0 0.46 26 0.23 0 0.36 0 27 0 0 0 0.53 28 2.62 0.03 0 1.63 29 0.61 0.64 0 0.05 30 0 0.08 0.05 0.28 31 Trace 0 0.48 Trace Total 9.93 2.31 1.70 2.54 Normal 7.62 7.87 8.05 7.62 Source: Agrometeorology Section, Department of Land Resource Science, University of Guelph. University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Table 2. Effect of Irrigation on mean nodule dry weight, nodule size, ug ^-fixed per dry weight.hr, and amount of N2 fixed per hectare.day. Nodule dry weight g/plant Nodule size gram f.w/ nodule Nodule number per plant ug N2 fixed per gram dry weight Kg No fixed per hectare, day Elora Vansoy irr. * 0.27 a 17.5 b 43 a 572.5 a 0.75 a Anoka irr. 0.29 a 18.3 b 48 a 522.3 a 0.48 a Vansoy no irr. 0.12 b 13.5 c 25 b 585.8 a 0.27 b Anoka no irr. 0.16 b 21.8 a 29 b 464.8 a 0.21 b Arkell Vansoy irr. 0.21 ab 22.3 a 30 ab 875.3 a 0.67 a Anoka irr. 0.24 a 23.2 a 38 a 669.0 a 0.63 a Vansoy no irr. 0.11 c 18.4 a 20 b 392.0 b 0.18 b Anoka no irr. 0.12 c 15.8 a 26 b 458.9 b 0.13 b Treatment means followed by the same letter do not differ significantly according to Duncan's Multiple Range Test at the 5% level. University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Table 3. Effect of irrigation on seed yield, total nitrogen fixed, period of fixation and plant population for Vansoy and Anoka at the two locations. Seed yield (kg/ha) Seasonal N2 ~fixed (kg/ha) Period of fixation (days) Plant population/ha Elora Vansoy irr. ft 2240 a 48.1 a 54 123212 a Anoka irr. 1654 b 30.3 b 61 96143 a Vansoy no irr. 2205 a 21.0 be 56 120412 a Anoka no irr. 1929 b 13.7 c 65 85875 a Arkell Vansoy irr. 1016 a 61.9 a 73 120723 a Anoka irr. 561 b 44.5 ab 64 115812 a Vansoy no irr. 270 c 15.8 be 76 80897 a Anoka no irr. 385 be 10.5 c 82 84631 a Treatment means followed by the same letter do not differ significantly according to Duncan's Multiple Range Test at the 5% level. University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh - 43 - Table 4. Water potentials* of bagged leaves of Vansoy and Anoka for 3 dates at the two locations. Date Elora Aug. 28 Aug. 29 Sept. 4 (bars) Vansoy irr. -4.7 b -5.8 a -4.8 a Anoka irr. -3.9 b -5.2 a -4.5 b Vansoy no irr. -8.0 b -11.4 b -8.0 c Anoka no irr. -5.2 a -7.2 c -6.1 ^ Arkell Aug. 20 Aug. 21 Aug. 27 Vansoy irr. -6.4 a -7.4 a -4.9 a Anoka irr. -4.6 a -6.2 a -3.7 ab Vansoy no irr. -18.3 b -18.1 b -9.4 b Anoka no irr. -14.9 c -16.7 b -6.2 c means of three measurements figures in the same column followed by the same letters do not differ significantly according to Duncan's Multiple Range Test at the 5% level. University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh - 44 - Table 5. Water potentials at three different dates of different grafting combinations of Vansoy and Anoka. Dec. 16 Dec. 17 Dec. 18 (bars) Anoka Vansoy ** -10.4 a -1 1 . 0 a -11.7 a Vansoy Vansoy -1 0 . 6 a -9.6 b -10.4 b Anoka -8 . 2 b -7.9 c -8 . 6 c Anoka Anoka -8 . 1 b -8.3 d -9.0 c Vansoy -10.4 a -10.3 e -10.9 b Vansoy Anoka -7.9 b -7.7 c -8.4 c * means of six measurements figures in the same column followed by the same letters do not differ significantly according to Duncan's Multiple Range Test at the 5% level University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh - 45 - LITERATURE CITED Bell, F. and P.S. Nutman. 1971. Experiments on nitrogen fixation by nodulated legumes in Biological Nitrogen Fixation in Natural and Agricultural Habitats (Ed. T.A. Lie and E.G. Mulder). Plant and Soil, Special Volume, pp. 231-264. Bergersen, F.J. 1962. The effects of partial pressure of oxygen upon respiration and nitrogen fixation of soybean root nodules. J. Gen. Microbiol. 29: 113. Bond, G. 1936. Quantitative observations on the fixation and transfer of nitrogen in the soya bean, with special ref­ erence to the mechanism of transfer of fixed nitrogen from Bacillus to host. -Ann. Bot. 50: 559-587. Chen, H.K. and H.G. Thorton. 1940. The structure of "ineffective nodules" and its influence on nitrogen fixation. Proc. Roy. Soc. (Lond.) B 129: 208-229. Diatloff, A. 1967. Effect of soil moisture fluctuation on legume nodulation and nitrogen fixation in a black earth soil. Qd. J. Agric. Anim. Sci. 24: 315-321. Doku, E.V. 1970. Effect of day-length and water on nodulation of cowpea (Vigna unguiculata (L.) Walp.) in Ghana. Exptl. Agric. 6 : 13-18. Dube, P.A. 1972. Studies of plant water relationships of different com lines. Ph.D. Thesis, University of Guelph. Engin, M. and J.I. Sprent. 1973. Effects of water stress on growth and nitrogen fixing activity of Trifolium repens. New Phytol. 72: 117. Fahraeus, G. and H. Ljunggren. 1968. Pre-infection phase of the legume symbiosis in The Ecology of Soil Bacteria, An Inter­ national Symposium, University Press, Liverpool, pp. 396-421. Fred, E.B., I.L. Baldwin and E. McCoy. 1932. Root nodule bacteria of leguminous plants. Monograph, University of Wisconsin Studies in Science 5: 194-195. Gibson, A.H. 1967. Physical environment and symbiotic nitrogen fixation. V. Effect of time of exposure to unfavourable root temperatures. Aust. J. Biol. Sci. 20: 1105-1117. Gibson, A.H. 1969. Physical environment and symbiotic nitrogen fixation. VII. Effect of fluctuating root temperature on nitrogen fixation. Aust. J. Biol. Sci. 22: 839-846. Hardy, R.W.F., R.D. Holsten, E.K. Jackson and R.C. Bums. 1968. The C2H2 -C2 H4 assay for N2 fixation: laboratory and field evaluation. Plant Physiol. 43: 1185. University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh - 46 - Hardy, R.W.F., R.C. Burns, R.R. Hebert, R.D. Holsten and E.K. Jack­ son. 1971. Biological nitrogen fixation: A key to world protein in Biological Nitrogen Fixation in Natural Agricul­ tural Habitats (Ed. T.A. Lie and E.G. Mulder). Plant and Soil, Special Volume, pp. 561-590. Hsiao, T.C. 1973. Plant responses to water stress. Ann. Rev. Plant Physiol. 24: 519-570. Johnson, H.S. 1971. Effects of nitrogen sources, organic matter and water stress on soybean nitrogen fixation and yield. M.Sc. Thesis, University of Guelph. Johnson, H.S. and D.J. Hume. 1973. Comparisons of nitrogen fixation estimates in soybeans by nodule weight, leghaemoglobin content and acetylene reduction. Can. J. Microbiol. 19: 1165-1168. Lambert, J.W. 1971. Registration of Anoka Soybeans. Crop Science 11: 135. Kuo, T. and L. Boersma. 1971. Soil water suction and root temperature effects on nitrogen fixation in soybeans. Agron. J. 63: 901-904. Lawrie, A.C. and C.T. Wheeler. 1973. The supply of photosynthetic assimilates to nodules of Pisum sativum L. in relation to the fixation of nitrogen. New Phytol. 72: 1341-1348. Masefield, G.B. 1952. The nodulation of annual legumes in England and Nigeria: Preliminary observations. Emp. J. of Exp. Agric. 20: 79. Masefield, G.B. 1961. The effect of irrigation on nodulation of some leguminous crops. Emp. J. Exp. Agric. 29: 51-59. McKee, G.W. 1961. Some effects of liming, fertilization and soil moisture on seedling growth and nodulation in birdsfoot trefoil. Agron. J. 53: 237-240. Minchin, F.R. and J.S. Pate. 1973. The carbon balance of a legume and the functional economy of the root nodules. J. Exp. Bot. 24: 259-279. Pankhurs, C.E., E.A. Schwinghamer and F.J. Bergersen. 1972. The structure and acetylene-reducing activity of root nodules formed by a Riboflavin-requiring mutant of Rhizobium trifolii. J. Gen. Micro. 70: 161-177. Pate, J.S. 1958. Nodulation studies in legumes. II. The influence of various environmental factors on symbiotic expression in the vetch (Vicia sativa L.) and other legumes. Aust. J. Biol. Sci. 11: 496. University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh - 47 - Pate, J.S., B.E.S. Gunning and L.G. Briasty. 1969. Ultrastructure and functioning of the transport system of the leguminous root nodule. Planta (Berlin) 85: 11-34. Roughley, R.J., P.J. Dast and P.S. Nutman. 1970. The infection of Trifolium subterraneum root hair by Rhizobium trifolii. J. Exp. Bot. 21: 186-194. Rogers, H.T. 1971. Soybean production - recent research findings: Site selection and importance of water. Bulletin Agric. Exp. Station, Auburn University 413: 11-16. Roponen, I.E. and A.I. Virtanen. 1968. The effect of the prevention of flowering and on the vegetative growth of inoculated pea plants. Physiol. PI. 21: 655. Schwinghamer, E.A., H.J. Evans and H.D. Dawson. 1970. Evaluation of effectiveness in mutant strains of Rhizobium by acetylene reduction relative to other criteria of N2 fixation. Plant and Soil 33: 192-212. Sprent, J.I. 1969. Prolonged reduction of acetylene by detached soybean nodules. Planta (Berlin) 8 8 : 372-375. Sprent, J.I. 1971a. The effect of water stress on nitrogen-fixing nodules. I. Effects on the physiology of detached soybean nodules. New Phytol. 70: 7-17. Sprent, J.I. 1971b. The effect of water stress on nitrogen-fixing nodules. II. Effects on the fine structure of detached soybean nodules. New Phytol. 71: 443-450. Sprent, J.I. 1971c. The effects of water stress on nitrogen-fixing nodules. III. Effects of osmotically applied stress. New Phytol. 71: 451-460. Sprent, J.I. 1972. The effects of water stress on nitrogen-fixing nodules. IV. Effects on whole plants of Vicia faba and Glycine max. New Phytol. 71: 603. Sprent, J.I. 1973. Growth and nitrogen fixation in Lapinus arboreus as affected by shading and water supply. New Phytol. 72: 1005. Stewart, W.D.P. 1962. A quantitative study of fixation and transfer of nitrogen in Alnus. J. Exp. Bot. 13: 250-256. Stewart, W.D.P., G.P. Fitzgerald and R.H. Burris. 1967. In situ studies on N2 fixation using the acetylene reduction technique. Proc. Natl. Acad. Sci. U.S. 58: 2071-2078. Waring, R.H. and B.D. Cleary. 1968. Plant moisture stress: Evalua­ tion by pressure bomb. Science 155: 1248-1254. University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh - 48 - Wilson, J.K. 1931. The shedding of nodules by beans. J. Amer. Soc. Agron. 23: 670-674. Vincent, J.M. 1965. Environmental factors in the fixation of nitrogen by the legume. In Soil Nitrogen Ed. W.V. Bartho­ lomew and F.C. Clark. Amer. Soc. Agron., Madison, Wisconsin, pp. 384-435. University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Appendix 1. Analysis of variance for different parameters of nitrogen fixation. Mean squares Source df Mean nodule dry weight Mean nodule size Mean nodule number ug N2 fixed per dry wt. Kg N2 fixed per ha. day Elora Reps 3 0.0004 2.4582 3.8208 8672.72 0.0071 Irrigation (I) 1 0.0731* 0.3025 1292.4025* 1958.10 ** 0.5576 Error (a) 3 0.0127 6.6784 41.3042 35206.9 0.0063 Cultivars (C) 1 0.0046 ** 82.810 81.9025 29326.60 0 .1 1 1 1 * C x i 1 0.0004 ** 57.0025 0.2025 5005.61 0.0487 Error (b) 6 0.000883 1.7821 18.3558 20631.12 0.0130 Arkell Reps 2 0.0004 23.211 62.054 20458.07 0.0615 Irrigation (I) 1 0.0341* 95.147 ** 338.248 360772.57 0.7174* Error (a) 2 0.0006 11.390 2.435 51997.67 0.0238 Cultivars (C) 1 0.0015 2.297 163.76* 14561.03 0.0058 C x I 1 0.0007 9.135 3.4240 55906.32* 0.00003 Error (b) 4 0.0016 10.790 20.697 7118.9 0.0235 ■c-VD University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Appendix 2. Age: ^[^I^j-fixlng activity profile showing the effect of irrigation on ug fixed per nodule.hr for Vansoy and Anoka. Elora Vansoy irr. Anoka irr. Vansoy no irr. Anoka no irr. Regression Equation R Y = 30.4416961670 + (-1.60167217X) + (0.027277626IK2) + (-0.000142435048X3) .3863** Y = 14.3616790771 + (-0.736386538X) + (0.0127645619X2) + (-0.0000677456992X3) .2850** Y = 9.1061096191 + (-0.489575803X) + (0.00923783705X2) + (-0.0000522168411X3) .2413* Y = -5.74072265625 + (0.226364374X) + (-0.00164620532X ) .02349 ** Arkell Vansoy irr. Anoka irr. Vansoy no irr. Anoka no irr. Y = -15.7073669434 + (0.538332462X) + (-0.00351343141X ) Y = -13.1220493317 + (0.456715941X) + (-0.00296930526X2) Y = -2.79789448 + (0.130891681X) + (-0.000941722887X2) Y = -0.155953502655 + (0.0861692429X) + (-0.00063875434X2) .2980** .2502** .1515** .1511** University of Ghana http://ugspace.ug.edu.gh 43 2 1 0 5 4 3 2 1 o r a Days after planting ke I I Days a fte r p lan ting University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Appendix 3. Age: ixing activity profiles showing the effect of irrigation on ug N^-fixed per plant.hr by Vansoy and Anoka at the two locations. Elora Vansoy irr. Anoka irr. Vansoy no irr. Anoka no irr. Regression Equation R Y = 1838.51074219 + -99.5081940X + (1.70037270X2) + (-0.00885314122X3) .4598** Y = 1382.76464844 + (-73.3696594X) + (1.23889256X2) + (-0.00638875738X3) .3763** Y = 306.775878906 + (-18.2508698X) + (0.344090641X2) + (-0.0019083065X3) .3512* Y = -246.600585938 + (8.70087051X) + (-0.0607023090X2) .2322** Arke11 Vansoy irr. Anoka irr. Vansoy no irr. Anoka no irr. Y = -601.45751953 + (19.715305X) + (-0.125148594X ) Y = -766.416015625 + (24.9071198X) + (-156767130X2) Y = -90.25772174 + (3.48179245X) + (-0.02378099X2) Y = -85.4902191162 + (3.17231274X) + (-0.020269412X2) .3420** .2939** .1709** .1077** University of Ghana http://ugspace.ug.edu.gh FIX ED /p la nt .h r D ays a fter planting A rkell University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Appendix 4. Age: ^[^t^j-fixing activity profiles showing the effect of irrigation on nodule fresh weight (g) per plant for Vansoy and Anoka at the two locations. Elora Vansoy irr. Y = Anoka irr. Y = Vansoy no irr. Y = Anoka no irr. Y = Regression Equation R -0.259750366211 + (-0.00355275651X) .8046* -0.14295959472 + 0.00408695638X .8533** -5.54009914398 + 0.130586326X .6252** -4.642592198 + (0.2160761669X) + (-0.003143527264X2) + (0.000016036X3) .7647** Arkell Vansoy irr. Anoka irr. Vansoy no irr. Anoka no irr. Y = 29.6501464844 + (-1.59395599X) + 0.0256945118X2+ (-0.0001076387X3) .7938* Y = 70.015411377 + (-3.73911953X) 4- (0.0609764345X2) + (-0.00028271391X3) .8612** Y = -4.69505786896 + (0.123773277X) .5103** Y = -5.57343482971+ (0.143117309X) .6724** University of Ghana http://ugspace.ug.edu.gh N O D UL E FR ES H W EI G H T, g/ pl an t Days after planting A r k e I I Days a fter planting University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Appendix 5. Age: ixing activity profiles showing the effect of irrigation on fixation efficiency (ug Fixed per g. nodule Fresh weight.hr) for Vansoy and Anoka at the two locations. Elora Regression Equation Vansoy irr. Y = 254.773956299 + (-1.73316002X) Anoka irr. Y = 326.062744141 + (-3.02380848X) Vansoy no irr. Y = 458.754394531 + (-4.40615082X) Anoka no irr. Y = 308.709228516 + (-2.89061451X) Arkell Vansoy irr. Y = 316.11425781 + (-2.33445835X) Anoka irr. Y = 325.568115234 + (-2.65125847X) Vansoy no irr. Y = 396.14233984 + (-3.97680092X) Anoka no irr. Y = 660.290283203 + (-12.4964046X) + (0.0605155937X3) .0841** .2401** .2098** .3333** .1333** .2264** .3599** .4445* University of Ghana http://ugspace.ug.edu.gh FI XE D /g no du le fre sh w ei gh t* hr E I o r a Days after planting A r k e 11 Days after p lanting University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Appendix 6 . Regression analysis of mg N2 fixed per g nodule fresh weight.hr with plant water potential for the two cultivars at the two locations. Elora Vansoy Anoka Arkell Vansoy Anoka Regression Equation Y = 429.6495946 + (-96.1433246X) + (15.987601X2) + (-0.749122X3) Non Sig. Y = -256.1085125 + (123.8900558X) + (-9.640262282X2) + (0.20557981X3) Y = -43.04547758 + (33.2793996X) + (-1.3733883X2) .2116* .4363* .3029** University of Ghana http://ugspace.ug.edu.gh FI XE D /g no du le fre sh w t. hr 0 -2 -4 -6 -8 -10 -12 -14 B A R S B A R S University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Appendix 7. Regression analysis of mg ^ fixed per plant.hr for Vansoy and Anoka at the two locations. Elora Regression Equation Vansoy Y = 338.39526 + (37.3158405X) + (-3.98921025X2) Anoka Y = 241.6444 + (32.6465909X) + (-3.4487266X2) Arkell Vansoy Anoka Y = 3567049924 + (170.5966306X) + (-13.37502108X2) + (0.286506483X3) Y = -265.4806249 + (150.868066X) + (-12.47441034X2) + (0.277918487X3) .3976* .2180* .4753** .4051** University of Ghana http://ugspace.ug.edu.gh E lo r a .5 r .4 .3 .2 .1 Z -0 J 0 - 2 - 4 -6 -8 -10 -12 -14 a- B A R S O A r k e 11 B A R S Vansoy Anoka University of Ghana http://ugspace.ug.edu.gh 1University of Ghana http://ugspace.ug.edu.gh - 56 - Appendix 8. Analysis of variance for final seed yield of Vansoy and Anoka soybeans. Source df Mean Squares F .05 Cal. Elora Reps 3 17.31 0.51 Irrigation (I) •> 1 13.00 0.38 Error (a) 3 33.90 Cultivars (C) 1 168.13 39.10 C x i 1 21.78 5.07 Error (b) 6 4.30 Arkell Reps 49.05 & * Irrigation 19.65 34.47 Error (a) 0.57 & & 5% -k Cultivars 144.50 30.55 C x i 55.24* 11.68** Error (b) 4.73 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh - 57 - Appendix 9. Analysis of variance for water potentials of bagged leaves of Vansoy and Anoka for 3 dates at the two locations. Source df Elora Reps 2 Irrigation (I) 1 Error (a) 2 Cultivars (C) 1 C X I 1 Error (b) 4 Arkell Reps 2 Irrigation (I) 1 Error (a) 2 Cultivars (C) 1 C X I 1 Error (b) 4 Mean squares Aug.28 Aug.29 Sept.4 5.93 2.14 0.57 15.87 43.32 17.04 1.27 0 . 0 1 2 . 2 1 9.72 17.28 3.74 3.2 9.72 1.84 0.63 0.26 0.013i Aug.20 Aug.21 Aug.27 20.57 4.81 0.063 369.63 337.08 48.00 19.88 21.16 0.13 20.28 5.07 8.67 1.63 0.003 0.85 1.69 2.34 0.417 F.05% Calc. Aug.28 Aug.29 Sept.4 A.7 210.0 0.256 12.A9 4332.0 7.71 15.A2 66.46 274.07 5.0 37.38 136.37 Aug.20 Aug.21 Aug.27 1.04 0.227 0.485 18.59 15.92 369.23 12.02 2.17 20.82 0.96 0.0013 2.05 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh - 58 - Appendix 10. Analysis of variance for water potentials of different grafting combinations of Vansoy and Anoka at three different dates. Source df Mean squares F.0.5% Calc. Dec. 16 17 18 Dec. 16 17 18 Reps 5 0.696 1.24 1.29 2.37 2.27 3.01 Treatments 5 10.48 10.95 11.33 35.58** 20.01** 26.25** Error 25 0.294 0.547 0.428 Total 35 University of Ghana http://ugspace.ug.edu.gh