MODULATION PROMISCUITY OF SOYBEAN GENOTYPES. A DISSERTATION SUBMITTED TO THE DEPARTMENT OF SOIL SCIENCE, ■ m-- FACULTY OF AGRICULTURE IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF PHILOSOPHY, SOIL SCIENCE. BY AARON ATTUA GYAU B.Sc. (Hons.); Postgraduate Dip. (Education) DEPARTMENT OF SOIL SCIENCE UNIVERSITY OF GHANA*LEGON MAY, 2001 University of Ghana http://ugspace.ug.edu.gh 6 p 3 6 5 7 0 7 University of Ghana http://ugspace.ug.edu.gh DECLRARATION „ I hereby declare that, except for reference to work o f other researchers which have been duly cited, this work is the result o f my own original research and that this thesis has neither in whole or in part been presented for another degree elsewhere. (STUDENT) 5ROF. S.K.A. DA^TSO (SUPERVISOR) University of Ghana http://ugspace.ug.edu.gh DEDICATION my father, Aaron Agyei Attua and my entire family. University of Ghana http://ugspace.ug.edu.gh ABSTRACT The objective o f the study was to obtain information on the existence o f Bradyrhizobium japonicum strains in Ghanaian soils, evaluate their effectiveness with the view to improving nodulation, nitrogen fixation and yield potential o f soybean. Eight soil series were screened for nodulation capabilities o f soybean using six cultivars (four promiscuous and two non-promiscuous). The soils were Adenta, Akuse, Anyinase and Bekwai; the rest were Hatso, Nyigbenya, Nzima and Toje. Four cultivars nodulated in four soils and one in five soils. There was no nodulation in Anyinase, Bekwai and Nzima soils. Bragg, a non-promiscuous genotype, nodulated considerably well contrary to documented reports in the literature that non-promiscuous American soybean genotypes do not normally nodulate in tropical soils. Most Probable Number (MPN) counts carried out established some relationship between nodulation and bradyrhizobia population in the soils used for the studies. Symbiotic effectiveness test carried out on 60 selected isolates from the screening experiment showed that 15% o f the isolates were highly effective, 65% ineffective and 20% moderately effective. Inoculation studies were carried out on three soybean cultivars namely Bragg (Non- Promiscuous American genotype), Bengbie (Promiscuous) and TGx (Promiscuous) using five isolates from the screening experiment and two imported isolates from Thailand in the Bekwai University of Ghana http://ugspace.ug.edu.gh soil. Generally inoculation led to improvement in shoot dry matter and total N, although the levels were different among the cultivars and isolates and thus showing that plant genotypes and bradyrhizobia strains significantly influenced inoculation response. Inoculation and N fertilization response carried out on four soybean cultivars, Bragg, Bengbie and TGx and Non-nodulating soybean genotype, in Adenta and Bekwai soils showed better nodulation and percent N-fixed in Adenta than Bekwai. This could be attributed to the higher bradyrhizobia count in Adenta than in Bekwai.Total N fixed was however higher in Bekwai than Adenta. This means that other factors inherent in Bekwai had enhanced plant growth and total N accumulation, and hence total nitrogen fixed. Bekwai soil had higher nitrogen, organic matter and phosphorus and was thus more capable o f providing nutrients and plant requirement for better plant growth than Adenta. The higher nodulation, percent and total N fixed at the 10 kg N /ha rate than at 100 kg N /ha application could be attributed to the depressing or inhibitory effects that inorganic nitrogen fertilizers have on nodulation and nitrogen fixation. v University of Ghana http://ugspace.ug.edu.gh ACKNOWLEGDEMENT How can I thank you God Almighty for bringing me thus far, for words would not be sufficient. You have been with me every moment o f this work and at last the battle has been won by your grace. My heartfelt thanks and profound gratitude go to Prof. S.K.A. Danso for the inspiration, guidance and direction he offered throughout the course. He also deserve commendation for accepting to supervise the work, the keen interest he showed in the work, and as the Director o f the Ecological Laboratory, he placed the facilities o f the laboratory at my disposal to ensure the success o f this work. Dr. Kumaga deserves to be mentioned for availing his offices to us and also offering bits and pieces o f useful advice The senior staff, junior staff and students o f the Soil Science Department played their part to ensure the success o f the work. Prof. Laryea who was our course adviser and later became the Head o f Department was eager that we completed the work in good time. He was always available to solve any administrative problem with dispatch. Prof. Yaw Ahenkora and Dr. G.N.N. Dowuona were always prompting and encouraging us. Dr. M.K. Abekoe was always on hand to share his immense experience with us. Prof. Ametekpor, and Dr. S.G.K. Adiku (who came back at a latter stage o f the work) also urged us on. University of Ghana http://ugspace.ug.edu.gh Messrs. M.S. Elegba, J.A. Natenor, M. Sarquah, B. Anipa, N. Agyekum and Ashia (Driver) rendered very useful services, which aided my work tremendously. Messrs O. Adusei, M. Aggrey, D. Nkansah A. Sowah, F.O. Ababio and Miss Henrietta Mbeah deserve to be mentioned. The two Ph.D. students at the time, J.O. Fening and W. Dogbe helped in the supervision o f our work and we thank them for that. University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS Page Declaration....................................................................................................ii Dedication.....................................................................................................iii Abstract.......................................................... iv Acknowledgement.......................................................................................vi Table o f contents.......................................................................................... viii List o f Tables................................................................................................xii List o f figures............................................................................................... xiii CHAPTER ONE 1.0 Introduction............................................................................................... 1 1.1 Background............................................................................................... 1 1.2 Objective.................................................................................................. 5 1.3 Hypotheses................................................................................................5 CHAPTER TWO 2.0 Literature Review ................................................................................. 7 2.1 Background...............................................................................................7 viii University of Ghana http://ugspace.ug.edu.gh 2.2 Biological nitrogen fixation....................................................................8 2.2.1 Brief classification o f rhizobia........................................................... 10 2.2.2 Cross inoculation groups..................................................................... 10 2.2.3 Promiscuity o f soybean genotypes................................................... 11 2.2.4 Inoculation............................................................................................. 13 2.2.5 Effectiveness o f rhizobia strains. ................................................ 17 2.2.6 Soil rhizobia population..................................................................... 19 2.27 Nodule formation and development................................................. 20 2.2.8 Environmental factors affecting biological nitrogen fixation ....22 2.3 Soybean-the host plant....................................................................... 26 2.4 Measurement o f fixed nitrogen.............................................................29 2.4.1 Nodule assessment...............................................................................30 2.4.2 Dry matter yield................................................................................... 30 2.4.3 Total nitrogen difference method......................................................31 2.4.4 Acetylene reduction assay................................................................. 32 2.4.5 I5N- Methodology...............................................................................34 CHAPTER THREE 3.0 MATERIALS AND METHODS........................................................ 38 3.1 Soil sampling........................................................................................... 38 3.2 Screening o f soybean for nodulating capabilities............................38 3.2.1.Planting material.................................................................................. 38 3.2.2.Pot experiment......................................................................................41 University of Ghana http://ugspace.ug.edu.gh 3.2.3 Isolation o f bradyrhizobia..................................................................41 3.2.4.Authentication o f bradyrhizobia Isolates........................................ 42 3.3 Cross inoculation Studies....................................................................... 42 3.4 Assessment o f the Effectiveness o f bradyrhizobia Isolates...........43 3.5 Inoculation o f soybean genotypes with bradyrhizobia isolates., .46 3.6 Soybean response to inoculation and nitrogen fertilizer application......................................................................................................... 47 3.7 15N-Analysis...............................................................................................49 3.8 Counting o f rhizobia................................................................................. 50 CHAPTER FOUR RESULTS......................................................................................................... 52 4.1 Nodulation potential o f six soybean cultivars in eight Ghanaian soils......................................................................52 4.2 Estimation o f population o f indigenous soybean bradyrhizobia.. .54 4.3 Selecting effective strains o f soybean bradyrhizobia for nitrogen fixation......................................................................... 54 4.4 Cross inoculation groupings o f some selected legumes............... 60 4.5 Response o f some selected soybean cultivars to inoculation.............61 4.6 Effect o f nitrogen fertilization on nodulation, nitrogen fixation and yield o f Bragg, Bengbie and TGx in Bekwai and Adenta so ils.. .67 x University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE 5.0 Discussion.......................................................................................... 76 5.1 Introduction...........................................................................................76 5.2.Nodulation potential o f soybean in Ghanaian soils.....................76 5.3 Cross inoculation................................................................................. 79 5.4 Symbiotic effectiveness...................................................................... 79 5.5 Response o f soybean to inoculation..................................................80 5.6 Response o f soybean to inoculation and nitrogen fertility...........83 5.7 Summary................................................................................................. 85 5.8 Conclusion..............................................................................................87 University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES. Table Page 3.1 Classification o f soils studied........................................................................................................ 40 3.2 Physicochemical properties o f soils used for the inoculation studies....................................48 4.1 Population o f bradyrhizobia nodulating soybean........................................................................54 4.2 Effectiveness grouping o f 60 soybean isolates selected from the screening experiment..................................................................................................................55 4.3 Effectiveness grouping o f 60 soybean rhizobia isolates from some Ghanaian soils.......................................................................................................... 56 4.4 Cross inoculation grouping o f 60 soybean isolates with cowpea and groundnut..................60 4.5 Number and dry matter wt. o f nodules formed by Bragg, Bengbie and TGx inoculated with seven soybean bradyrhizobia isolates........................................................ 62 4.6 Inoculation effects o f seven soybean Bradyrhizobium isolates on three soybean cultivars, Bragg, Bengbie and TGx in Bekwai soil............................................. 68 4.7 Number and dry weight o f nodules formed by Bragg, Bengbie and TGx in Bekwai and Adenta soils fertilized with 10 and 100 kgN/ha.........................................70 4.8 Shoot dry matter yield o f Bragg, Bengbie and TGx in Bekwai and Adenta soils fertilized with 10 and 100 kg N /ha .............................................................................. 72 4.9 Percent nitrogen fixed by Bragg, Bengbie and TGx in Bekwai and Adenta soils fertilized with 10 and 100 kg N /ha...........................................................................................72 4.10 Amount o f fixed and total N accumulated by Bragg, Bengbie and TGx in Bekwai and Adenta soils fertilized with 10 and 100 kg N /ha...................................... 73 4.11 Total nitrogen accumulated by Bragg, Bengbie and TGx in Bekwai and Adenta soils fertilized with 10 and 100 kg N /ha...................................................................................... *............. 74 XM University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES F igure Page Fig 3.1 Map o f Ghana showing the ecological zones and sites where the soils were sampled..........................................................................................................39 Fig. 4.1 Graph showing nodulation o f soybean in eight Ghanaian soil series....................... 53 Fig. 4.2 Sample o f soybean plants used for effectiveness studies...........................................59 Fig. 4.3 Inoculation effect o f selected soybean isolates on soybean cultivars...................... 64 University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE 1.0 INTRODUCTION 1.1 Background Nitrogen is frequently the most limiting nutrient for high crop yields in the tropics, where food production depends mostly on the natural fertility o f the soil. Closely associated with this problem is the global shortage o f dietary protein, which is most acute in the tropics, and where the cost o f animal protein products is often beyond the purchasing power o f many people. Protein deficiency has thus been linked to the predominantly carbohydrate diet o f the population in the tropics. 1.1.1 Inorganic Fertilizer Traditionally, nitrogen fertilizers have been used to improve nitrogen fertility o f the soil. However, there are many factors militating against the use o f nitrogen fertilizers. These include, high fertilizer cost due mainly to the high cost o f production and high transportation charges. The high production cost o f fertilizers is due primarily to the enormous amount o f fossil energy (crude oil) required for their production, a situation which has worsened in recent times o f escalating crude oil prices. In addition, the fact that these fertilizers are not manufactured locally and have to be in most cases imported from far away Europe and North America further inflates the cost. This situation is not likely to change favourably in the foreseeable future. In fact according to Bumb (1994), price hike o f fertilizer in Ghana was 29000% compared to what pertained a decade ealier, a situation that caused the reduction o f fertilizer use on rice by more than 60%. Apart from the initial high cost o f fertilizers, local infrastructure is in such a poor University of Ghana http://ugspace.ug.edu.gh state that, purchase, transport and delivery are serious drawbacks to sustainable food crop production in the tropics (Ayanaba, 1977). There are equally serious problems associated with actual application o f fertilizer in the field. Nitrogen fertilizers when applied in large quantities have the tendency o f causing pollution and in some cases acidification o f soils (Tisdale and Nelson, 1975). Low pH is potentially detrimental to agricultural production because it adversely affects the growth o f plants and the survival o f beneficial microorganisms in the soil. Low pH also disrupts the balance o f some soil nutrients. Also there is the possibility o f nitrogen fertilizers leaching beyond the root zone or being washed away by erosion, thereby rendering them unavailable to plants (Bartholomew, 1977). Furthermore, fertilizer elements can end up in water bodies causing ecological problems to aquatic life and also to humans. For example, nitrates from fertilizer that find their way through leaching and erosion into water bodies, cause algal blooms, consequently leading to oxygen depletion upon the death and decomposition o f the algae in such water bodies (Alexander 1971). It is also known that the consumption o f plants w ith excessive uptake o f nitrate can cause a disease called methaemoglobinaemia (Alexander 1971.and Keeney 1982). Finally large amounts o f energy (crude oil) could be saved and put to other uses i f alternatives or supplements to fertilizers were to be used. 1.1.2 Biological Nitrogen Fixation The problems associated with inorganic fertilizers that have been mentioned above warrant the search for other alternatives to nitrogen fertilizers. This need has led to the intensification o f research into biological nitrogen fixing systems as alternatives or supplements to chemical 2 University of Ghana http://ugspace.ug.edu.gh fertilizers, as recommended by Date (1975). Not only is biologically fixed nitrogen (BNF) inexpensive compared with inorganic fertilizers; fixed nitrogen is also not plagued with many of the disadvantages such as pollution and health hazards that are associated with the non-judicious use o f inorganic fertilizers. The escape o f biologically fixed nitrogen into the environment in the form o f nitrates is very minimal. Reports from FAO (1978) estimate that globally, biological nitrogen fixation contributes about three times as much nitrogen as supplied by inorganic fertilizer. It has to be recalled that natural nitrogen fixation pre-dates the use o f artificially made nitrogen fertilizers and there is a lot o f prudence in reverting to this source o f nitrogen in this era o f environmental consciousness when there is a lot o f aversion for most things chemical. Biological nitrogen fixation therefore appears to be the most promising alternative or supplement to inorganic fertilizers (Hardarson et. al., 1987). However, the amount o f nitrogen fixed in legumes depends on factors such as effectiveness o f the symbiosis between the host and the (Brady)Rhizobium strain, the species, and even cultivar o f the legume, among other things. In order to realize the maximum benefits o f the symbiosis, there is the need to ensure that the appropriate Rhizobium capable o f nodulating the host plant and fixing nitrogen is present in the soil. 1.1.3 Soybean Soybean occupies a premier position among crops, being the most important source o f both protein concentrates and vegetable oil (de Haen, 1994). In a situation where plant sources contribute about 70% o f the world’s protein needs, or even more in developing countries (Rachie and Roberts, 1974), the importance o f soybean cannot be overemphasised, de Haen, (1994) predicted that about 62^’million people would become seriously undernourished by the 3 University of Ghana http://ugspace.ug.edu.gh end o f the twentieth century, most o f them in tropical countries and that the demand for affordable protein and energy-rich food in these countries was already high and continued to increase. Soybean demonstrates exceptional potential for minimizing protein deficiency in both human and animal nutrition in tropical developing countries (Rachie and Roberts, 1974). Moreover, soybean and cowpea are widely grown in the tropics and the amount o f nitrogen fixed by these important plants is very substantial (Alexander, 1977). Soybean is an important source o f food, feed and oil. It is said to be a whole meal, because it contains all the major food requirements in satisfactory combinations. It produces high quality oil (about 21%) a protein content o f about 40% and carbohydrate o f about 34% (Scott and Aldrich, 1983). Soya oil is highly digestible and contains no cholesterol while the protein is superior with essential amino acid distribution similar to milk. It could therefore serve as a good substitute for meat and fish. In addition soybean is the most cultivated crop legume in the world in terms o f total production and international trade (Source: Humanity Development Library, Legon.). Since soybean is capable o f symbiotic nitrogen fixation with Bradyrhizobium, an intelligent manipulation o f the soybean-Bradyrhizobium relationship will go a long way in alleviating the problem o f food shortage in the tropics and protein deficiency in the whole world. There are two main soybean genotypes, the American soybean genotype also known as the non- 4 University of Ghana http://ugspace.ug.edu.gh promiscuous genotype and the Asian genotype referred to as the promiscuous genotype (Pulver et. al, 1978; Roughley et. al., 1980). The higher yielding American genotype does not nodulate readily with the indigenous bradyrhizobia in tropical soils, and therefore there is frequently the need to inoculate it for adequate nodulation and nitrogen fixation (Nangju, 1980; Pulver et. al. 1982). Cultivars o f Asian origin have however been reported to nodulate freely in many tropical soils (Anon, 1982). The main focus o f this study is to obtain information on the existence o f soybean bradyrhizobia in Ghanaian soils, to evaluate their effectiveness and to improve upon the nodulation, nitrogen fixing and yield potential o f both promiscuous and non-promiscuous soybean genotypes. 1.2 Objectives. ❖ To assess whether inoculation is necessary for nodulation, increased nitrogen fixation and yield o f promiscuous and non-promiscuous soybean genotypes. ❖ To isolate bradyrhizobia from soybean genotypes with the aim o f identifying effective and competitive strains for inoculation studies. ❖ To examine whether some bradyrhizobial isolates from the promiscuous type would nodulate and be effective on the non-promiscuous genotype. 1.3 Hypotheses ♦♦♦ That highly effective and competitive Bradyrhizobium japonicum strains occur in soils in 5 University of Ghana http://ugspace.ug.edu.gh Ghana. That some B. japonicum isolates obtained from promiscuous soybean genotypes are capable o f nodulating non-promiscuous soybean genotypes That B. japonicum inoculation on seed or into soil is necessary for higher nodulation, increased nitrogen fixation and enhanced yield o f promiscuous and non-promiscuous soybean genotypes. University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Background. Soybean has great potential in the tropics as a source o f oil and high protein food (Ranga Rao et. al., 1982), and has also been identified as an affordable source o f protein and oils especially for rural dwellers in many African countries (Abaidoo, 1997). In Ghana, soybean is the most important source o f plant protein and edible oil (Awuku et. al. 1991, Giller and Wilson, 1993) The protein content obtained from soybean is about twice that o f meat, cowpea or limabean and four times that o f cereals (Awuku et al. 1991). It is therefore not surprising that Africa used to import soybean to meet its protein needs in the past (Abaidoo, 1997). However, the imports had to be curtailed due mainly to balance o f payment problems (Abaidoo, 1997). The only option left was for the African countries to resort to the production o f soybean locally. In Ghana, currently the crop is intensively cultivated within and around the Tono Irrigated Project site. Nitrogen has long been recognised, as the key to soil fertility and major constraint to crop production (Nye and Greenland, 1960). Large amounts o f nitrogen are required for good soybean production (Cattelan and Hungria, 1994). But the element is a very limited nutrient in tropical and African soils, and in Ghana the deficiency is widespread (Nye and Greenland, 1960) to the extent such that, local production o f the legume would become sustainable only when soil nitrogen is supplemented with nitrogen from fertilizer or biological nitrogen fixation (BNF) (Cattelan and Hungria, 1994). 7 University of Ghana http://ugspace.ug.edu.gh Several factors make the addition o f fertilizers to tropical soil a poor management practice. The efficiency o f fertilizer utilization by soybean is usually less than 50% (Cattelan and Hungria, 1994). Moreover, nitrogen fertilizer is very expensive, which explains why the dependence of soybean only on this source o f N would lower the profit o f farmers, at times they may not make any profit at all (Cattelan and Hungria, 1994) especially in Ghana which is a non-fertilizer producing country. Ecological problems have also been reported to be associated with fertilizer use. From the foregoing it is evident that N fertilizer does not hold much promise for Africa and the most sustainable nitrogen source is biological nitrogen fixation. 2.2 Biological Nitrogen Fixation. Biological nitrogen fixation refers to the symbiotic and non-symbiotic fixation o f atmospheric nitrogen by microorganisms into forms that plants can utilize, a process which raises high hopes o f meeting the high nitrogen demand o f the world (Dakora, 1977). Symbiotic nitrogen fixation involves mutual association between bacteria o f the genus Rhizobium and legumes, which results in the fixation o f nitrogen in the root nodules (Frobisher et. al., 1974). Neither the legume nor the bacterium is capable o f fixing nitrogen by itself. The rhizobia, when growing in nodules o f legumes, convert nitrogen from the air into organic forms. Thus by the combined action o f plant cells and bacterial cells, gaseous nitrogen is assimilated into simple nitrogenous compounds, such as amino acids and polypeptides in plants, bacteria and the surrounding soil (Frobisher et. a l, 1974). In the absence o f rhizobia, and combined nitrogen in the soil, legumes die. However, if the right types o f rhizobia are present, legumes are capable o f thriving in nitrogen deficient soil, and in so doing, they enrich the soil as well (Frobisher et. al., 1974). This 8 University of Ghana http://ugspace.ug.edu.gh form o f nitrogen fixation forms the basis for the recognition o f the importance and application o f legumes in agriculture as far back as the Greek Roman era (Tisdale and Nelson, 1956). Rhizobia are unique among soil microorganisms in their ability to form nitrogen-fixing symbiosis with legumes. To enjoy the benefits o f the symbiotic association, the rhizobia must exhibit saprophytic competence and also be able to out-compete other rhizobia for infection sites on legume roots (Somasegaran and Hoben, 1985). Generally rhizobia are capable o f existence in the soil for a long time without contact with the host plant. However, the rhizosphere is a zone o f increased microbial growth and activity compared to bulk soil. The roots o f leguminous plants secrete soluble, organic nitrogenous compounds and simple soluble carbon compounds such as amino acids, malic acid, pentoses and phosphorus for use by microorganisms and other plants. The sloughing off o f bark and root coverings and death o f roots provide a rich source o f carbohydrates and their derivatives to support a luxuriant flora o f nitrogen fixers (Frobisher et. al., 1974). A well nodulated crop such as red clover may introduce as much as 120 kg o f fixed N /ha into the soil. This nitrogen was estimated to cost around $600 in 1973 in the form o f commercial fertilizer (Frobisher et. al., 1974). In fact it has been estimated that globally, BNF contributes between 139 and 170 million tons of nitrogen to plants each year (Burns and Hardy, 1975), a figure that is about three times the world annual nitrogen fertilizer production o f 49.6 million tons (FAO, 1978). Duong et. al. (1984) have reported that biological nitrogen fixation facilitates the cultivation o f soybeans on commercial scale with reduced nitrogen fertilizer inputs. 9 University of Ghana http://ugspace.ug.edu.gh 2.2.1 Brief Classification o f Rhizobium. The root nodule bacteria o f the family Rhizobiaceae are genetically diverse and physiologically heterogeneous group o f microorganisms that used to be classified together (as Rhizobium) by virtue o f their ability to nodulate groups o f plants o f family Leguminosae. In 1982, Jordan classified rhizobia into two genera and named them Rhizobium and Bradyrhizobium based on their growth rate. Rhizobium strains are considered fast growers and Bradyrhizobium slow growers. Since then further work by taxonomists have led to the discovery o f three new genera that have been added to the two existing ones. These are Azorhizobium (Dreyfus et. al., 1988), Sinorhizobium (de Lajudie et. al., 1994) and Mesorhizobium (Lindstrom et. al., 1995). Soybean rhizobia belong to the genus Bradyrhizobium and have been referred to as Bradyrhizobium japonicum (Jordan 1982). 2.2.2 Cross Inoculation Groups A cross inoculation group refers to a collection o f leguminous species that develop nodules when exposed to bacteria obtained from the nodules o f any member o f that particular plant group. Therefore, a single cross-inoculation group ideally includes all host species that are infected by an individual bacterial strain (Alexander, 1977). Out o f the over 20 cross inoculation groups identified, only seven have achieved prominence, and six have been sufficiently classified to the species status (Alexander, 1977). But the six species are not entirely distinct. 10 University of Ghana http://ugspace.ug.edu.gh For example, the soybean and cowpea bacteria groups, commonly considered to be separate, contain many similar bacterial strains, and organisms isolated from soybean nodules frequently infect cowpea and vice versa, thus suggesting that at least some cowpea rhizobia may be varieties o f the soybean rhizobia (Alexander, 1977). Bacterial strains that invade legumes outside their particular class and plants that they infect are examples o f a phenomenon referred to as symbiotic promiscuity. The validity o f the cross-inoculation group system has not gone unchallenged because it has been found that many legumes are nodulated by rhizobia o f other host-bacterial groups. The effect is that, the integrity o f the cross inoculation concept as a system for determining relatedness among rhizobia strains has become compromised (Wilson, 1944; Bromfield and Barron, 1990) and is now in general disrepute. Its continued usage is on the basis o f convenience and agronomic significance (Graham et al. 1991). Also it has some practical use for selecting rhizobial strains with potential for inoculant usage for particular legume crops (Mpepereki et. al., 1996). 2.2.3 Promiscuity of soybean genotypes. Certain cultivars o f Glycine max are nodulated by some strains o f Rhizobium spp. (Cowpea miscellany) as well as their normal micro-symbiont, Bradyhizobium japonicum (Leonard, 1923; Sears and Carroll, 1927; Van Rensburg et. al., 1976; Roughley et. al., 1980 ). Pulver, et. al. (1978) also reported that locally-adapted cultivars o f G. max in Nigeria, e.g. Malayan, nodulated promiscuously and fixed nitrogen with indigenous rhizobia whereas cultivars bred in U.S.A. e.g. 11 University of Ghana http://ugspace.ug.edu.gh Bossier were more specific, nodulated sporadically, fixed little nitrogen and responded significantly to inoculation with B. japonicum. Nangju (1980), Pulver et. al. (1982) and Ranga Rao et. <2/.(1982) established that, although soybean has great potential in the tropics as a source o f oil and high protein food, adequate nodulation and nitrogen fixation o f high yielding cultivars bred in North America required inoculation with the appropriate Bradyrhizobium japonicum when grown in soils in which soybeans had not been previously cultivated. The indication is, that the American soybean genotypes have specific bradyrhizobia species requirement and the compatible populations are seldom available in soils where the crop has not been previously grown, a demonstration o f the classical cross-inoculation concept, where soybean would not nodulate with cowpea rhizobia population. By contrast, those resulting from breeding programmes possessing the promiscuity genotype, nodulate freely in many tropical soils (Anon, 1982). The American soybean types are therefore referred to as non-promiscuous genotypes. The Asian soybean genotypes on the other hand are able to nodulate promiscuously with native rhizobia and have therefore been referred to as promiscuous soybean types. The specific relationship that exists between the legume and the rhizobia should be carefully considered as one o f the key factors affecting the amount o f nitrogen fixed. While soybeans for example require B. japonicum for effective nodulation and nitrogen fixation (Caldwell and Vest, 1968), others such as cowpea nodulate effectively with a range o f indigenous bradyrhizobia populations (Sellschop, 1962) referred to as Bradyrhizobium species. 12 University of Ghana http://ugspace.ug.edu.gh 2.2.4 Inoculation For optimum nitrogen fixation to be achieved in legumes, especially in soils where such legumes have not existed or been cultivated traditionally, there is the need to inoculate with specific effective strains o f rhizobia. Soybean is exotic to Africa (Hardley and Hymowitz, 1973) and generally considered to be a new crop in most tropical countries (Cattelan and Hungria, 1994), therefore their B. japonicum is either not present in the soil or they occur in very low populations (Cattelan and Hungria, 1994; Anon, 1975). It has been observed that where soybean has not been previously grown, there is generally a response to inoculation with B. japonicum especially with non-promiscuous cultivars (Cattelan and Hungria, 1994). Even where nodulated legumes are grown, there is a school o f thought that there is the need to inoculate subsequent crops as an insurance against inoculation failure. This is done on account o f the fact that populations o f rhizobia decline rapidly in soils in the absence o f the host plant, particularly in highly acidic soils. With low population, the expectation is that inoculation with efficient strains would increase crop yield. B. japonicum inoculation is therefore a necessary prerequisite for adequate nodulation and nitrogen fixation o f soybeans in Africa. But inoculation in Africa has not been without problems. In fact in the past, inoculation in Africa was generally considered not feasible since many countries were not sufficiently equipped to cope with the demands associated with the handling and usage o f inoculum in the tropics (Ayanaba, 1977). 13 University of Ghana http://ugspace.ug.edu.gh Some o f the factors that militated against inoculation were: • Lack o f laboratory facilities for production, maintenance and distribution o f inoculants. • The absence o f skilled personnel to man the inoculum production laboratories. • Low survival rate o f imported rhizobia strains in adverse tropical climate. • Poor transportation and storage facilities (Ayanaba, 1977). Over the years there have been some improvements in the requisite factors and conditions for inoculant production (Abaidoo, 1997), to the extent that some African countries such as Zambia and Uganda have successfully developed inoculum facilities which have promoted inoculum use in soybean production (Anon, 1993; Anon, 1996). Nevertheless, the fact still remains that, the level o f inoculation in Africa is rather very low and unsatisfactory. A condition which might be partly due to lack o f education and demonstration o f the importance o f inoculation to legume crop productivity. It is also worthy o f mention that, the factors alluded to earlier as having contributed to inoculation failure in Africa are, by and large, much prevalent in many parts o f Africa. Even though Rhizobiologists may view the inability o f Africa to adopt inoculation as a means o f increasing legume crop productivity as a disappointing development, most farmers and agronomists prefer conditions and circumstances that make inoculation unnecessary and irrelevant. Perhaps, while the solution to the inoculation problems in Africa was being sought, albeit quite slowly, the breeding o f soybean genotypes that would be susceptible to the nodulation with indigenous bradyrhizobia population already present in African soils was resorted to as an alternative means o f improving soybean production (Abaidoo, 1997). This 14 University of Ghana http://ugspace.ug.edu.gh approach is predicated on reports that some cowpea-miscellany rhizobia were capable o f nodulating American soybean genotypes (Leonard, 1923; Sears and Caroll 1927). Furthermore, it had been observed that local soybean varieties in Nigeria and Tanzania (Pulver el. al. 1985) and Thailand (Na Lampong, 1976) nodulated freely with bradyrhizobia in the soils and did not respond to B. japonicum inoculation. This was in contrast with the non-promiscuous American soybean cultivars which nodulated sporadically, fixed little nitrogen and responded significantly to B. japonicum inoculation (Pulver et. al., 1985). Pulver et. al. (1982) also reported that the indigenous bradyrhizobia that nodulated the local and American soybean cultivars in Nigeria were Bradyrhizobium spp. Furthermore, they attributed the insignificant response to inoculation exhibited by local soybean cultivars to their incompatibility with B. japonicum strains and the presence o f the more compatible indigenous bradyrhizobia in Nigerian soils. Kueneman et. al. (1984) also observed that the marginal response o f adapted soybean cultivars to Bradyrhizobium japonicum inoculation was indicative o f the fact that Bradyrhizobium spp were capable o f maintaining high soybean yields without inoculation or fertiliser nitrogen. Motivated by these findings, Researchers at the International Institute o f Tropical Agriculture (IITA) in Nigeria adopted, as guiding principle, the capability o f soybean to nodulate effectively with indigenous Bradyrhizobium spp but not with B. japonicum (Kueneman et. al., 1984). A working principle which operates on the following basic assumptions: • That the improved soybean genotypes designated as Tropical Glycine cross (TGx), nodulate freely and effectively with Bradyrhizobium spp. populations. • That the Bradyrhizobium spp. nodulating TGx exist abundantly in all African soils, therefore, soybean yields could not be limited by BNF without inoculation and inorganic 15 University of Ghana http://ugspace.ug.edu.gh fertilizer N. From the above principles and assumptions, it is not surprising that soybean breeding programmes in Africa for over two decades tended to rely on effectiveness o f indigenous Bradyrhizobium spp. to supply N to meet soybean requirements (Abaidoo, 1997). Later research findings have however shown that, nitrogen fixation by indigenous bradyrhizobia could not meet the N demands o f all soybean genotypes in all locations o f cultivation. There were instances where Pal and Norman (1987) measured yield increases from fertilizer application in Northern Nigeria contrary to claims by Pulver et. al. (1985) that TGx soybean cultivars did not respond to N application; Pal and Norman (1987) therefore recommended the application o f inorganic N fertilizer in split-applications at several growth and developmental stages o f soybean to obtain maximum yields. Furthermore, they observed that the ability o f TGx soybean cultivars to maintain moderate yields without fertilizer application or inoculation depended on the type o f cultivar as well as the location o f cultivation; an observation which has been confirmed by Okereke and Eaglesham (1992) and Abaidoo (1997). According to Abaidoo, field trials o f soybean conducted in Nigeria (unpublished data) as well as observation from soybean fields in N igeria and Ghana indicated that TGx soybean genotypes failed to nodulate adequately in some farmers fields though they had been previously cropped with cowpea, groundnut, bambara groundnut and pigeon pea. The fact that these tropical legumes had been previously cultivated on those fields may suggest the presence of adequate Bradyrhizobia spp. in the soil to infect the soybean genotypes that were planted. One can therefore surmise that, high soybean productivity can be achieved by establishing the 16 University of Ghana http://ugspace.ug.edu.gh presence o f indigenous Bradyrhizobium spp. and/or B.japonicum populations in all locations of interest in Africa and ascertaining, their compatibility and effectiveness on various soybean genotypes. Where these native rhizobia species prove efficient in optimum nitrogen fixation, there may not be the need for inoculation. On the other hand, where they are found not to be efficient, there may be the need to introduce (through inoculation) more efficient and compatible strains selected either from a few local or imported bradyrhizobia. This idea is based on reports that, in situations where native rhizobia are often more competitive, yet less efficient nitrogen fixers than introduced strains (Johnson et. al., 1964; Bergerson et. al., 1971; Van Schreven, 1971) the native rhizobia tend to colonize most o f the available sites for nodule formation. Also they form nodules that fail to sufficiently satisfy the nitrogen needs o f the host because such symbiosis is ineffective in terms o f nitrogen fixation. In other cases however, the introduced strains fail to obtain response due to the presence o f high population o f efficient indigenous strains present in the soil (Sellschop, 1962; Ayanaba and Nangju, 1973). For inoculation to yield its desirable objective in soils with large but ineffective rhizobia, there is the need for the introduced inoculum to be more competitive (Obaton, 1975). Looking at the other scenario where efficient native strains already exist, inoculation cannot offer any special advantage. 2.2.5 Effectiveness of Rhizobia Strains. Rhizobia have generally been categorized into three groups according to their ability to fix nitrogen. These are, effective, moderately effective and ineffective. Far back in 1888, Hellriegel achieved fame by producing conclusive evidence that the apparent nitrogen fixation in leguminous plants occurred only when the plant was furnished with 17 University of Ghana http://ugspace.ug.edu.gh root nodules. Further research over the years have revealed that the ability o f rhizobia to induce and form nodules on their compatible legume host is not a sufficient condition for nitrogen fixation to proceed, though it is a necessary precondition (Giller and Wilson, 1993). Singleton and Stockinger (1982) asserted that the strains o f Rhizobium present in the soil might range from highly efficient symbionts (effective strains) to those that are capable o f nodule formation but unable to reduce atmospheric nitrogen (ineffective strains). Effectiveness according to them followed a normal, distribution pattern. From the forgoing, one can safely deduce that, while some rhizobia fix nitrogen in large quantities, others fix partially and yet others may live in nodules as non-fixing parasitic forms. Evidently, there is the need therefore to do a thorough screening o f rhizobia in the soil for effectiveness before embarking on any measure o f inoculation process. Symbiotic effectiveness of indigenous rhizobia population is therefore an important parameter for the selection o f strains for inoculant production. Also, it is a primary factor for the determination o f incidence and magnitude o f legume response to inoculation (Singleton and Travers, 1986,Thies et. al., 1991). To conclude, one may re-echo the conviction held by Bromfield and Ayanaba (1980) as well as Danso (1988), that, the presence in the soil o f the appropriate rhizobial strains that are highly effective is a prerequisite for nitrogen fixation in legumes and that where these are either absent or ineffective, rhizobial inoculation is necessary to ensure nitrogen fixation. There is the prevalence o f ineffective indigenous strains in the average soils against which inoculant strains have to compete for nodulation (Owiredu, 1980). Many researchers associated some legume failures with the large population o f ineffective native rhizobia present in soils (Leonard, 1930; Johnson et. al., 1964; Holland, 1970; Labandera and Vincent, 1975). In soils 18 University of Ghana http://ugspace.ug.edu.gh with large population o f inefficient native strains, the strong competitive ability o f the native strains accounts for the lower rate o f nodules formed by the introduced strains, often accounting for between only 0 and 17% o f all the nodules formed (Johnson et. al., 1964 and Ham el. al., 1971). 2.2.6 Soil Rhizobia Population Nodulation and nitrogen fixation occur in legumes when the appropriate rhizobia arepresent in the soil and in adequate numbers. Also, population density o f indigenous rhizobia contributes immensely towards competition for nodule occupancy and response to inoculation. Information on rhizobia numbers would go a long way in helping rhizobiologists to asses the need or otherwise for inoculation among other things. However, data on the population sizes o f cowpea and soybean rhizobia in tropical soils arelacking (Munlogoy and Ayanaba, 1986). The limited available information suggests a population range o f 103 to 104 rhizobia per gram soil for native food legumes (Zengbe, 1980; Munlongoy and Ayanaba, 1986; Danso and Owiredu, 1988). According to Danso (1992), by most standards, these numbers should be adequate for nodulation. Estimates o f bacteria numbers vary according to the means o f determination (Alexander 1977). Enumeration o f rhizobia is normally done by the most probable number (MPN) method (Alexander, 1965) using plastic pouches (Weaver and Frederick, 1972). Other methods include the immunofluorescence technique (Schmidt et. al., 1968). The most probable number infection test is based on the assumption that organisms are randomly distributed and that the presence o f one Rhizobium cell is capable o f inducing nodulation on an appropriate host (Woomer et. al., 1988). 19 University of Ghana http://ugspace.ug.edu.gh 2.2.7 Nodule Formation and Development. Rhizobium infects legume roots leading to the development o f nodules within which nitrogen fixation takes place. Nodule formation is a multistep process with four main stages: (1) Pre-infection (2) Infection and nodule organogenesis (3) Nodule functioning and maintenance (Vincent, 1980), and (4) Nodule senescence. Pre-infection starts when rhizobia are attracted by chemotaxis to the organic compounds (flavonoids) excreted by root hairs (Turgeon and Bauer, 1985; Nap and Bisseling, 1990; Gerahty et. al., 1992). The flavonoids are host specific and stimulate the multiplication o f rhizobia besides acting as chemo-attractants and cause the rhizobia to become attached to the root hairs. Each flavonoid switches on nod genes in the bacterial cell, which causes it to synthesize Nod factors. Root hair deformation or curling occurs (Bauer, 1981) under the influence o f Nod factors (Hirsch, 1992). Dazzo et. al. (1984) suggested that the adhesion o f the bacterial symbiont to the root surface is a critical step prior to the successful infection phase o f the nodule development process. Following the attachment o f the rhizobia to the root hair, invagination o f the root hair takes place and the bacteria penetrate the root hair epidermis and enter the plant. Penetration is an active process during which the bacteria enter the plant through the tip o f the root hair (Bauer, 1981; Dazzo et. al., 1984) provided these bacteria are the suitable strains. Penetration o f root hair appears to be under control o f both Rhizobium and the plant (Bauer, 1981). Only a small and variable proportion o f the host root hairs become infected and about 60-99% o f the infections abort during the process (Dart, 1977). It has also been observed that only certain discreet portions o f the root are susceptible to infection (Bauer, 1981;). Mature 20 University of Ghana http://ugspace.ug.edu.gh roots are however generally found not to be susceptible to infection (Dart, 1977). The penetration o f the root hair is followed by the formation o f a hypha-like infection thread in which the rhizobia multiply enormously. The infection process causes cells in the root cortex to divide to form the nodule primodium. The infection threads enter the primodial cells and bacteria are released into the plant cell cytoplasm. Each bacterium becomes a bacteroid, and undergoes fission. The presence o f the bacteria stimulates multiplication o f plant cells around that area, with the resultant formation o f a nodule tissue. The rhizobia within the nodule receive their nutrient supply from the plant. During senescence, the supply o f carbohydrate to the nodules is reduced. Nitrogenase activity and leghaemoglobin content decline and the nodule degenerates. There are two main types o f nodules; determinate and indeterminate. Generally, temperate legumes form indeterminate nodules while tropical legumes form determinate ones (Vance, 1983). Determinate nodules have a fixed life span as against the indeterminate ones, which grow for an infinite period o f time. The type o f nodule formed depends on the plant and not the Rhizobium strain (Dart, 1977). There are different shapes and sizes o f nodules, determined by soil conditions and Rhizobium-plant variety interaction (Lynch and Wood, 1989). The size may vary from 1mm. to over 1cm. The shapes are global, cylindrical, peanut, elongate and lobed, flattened or collaroid. Lynch and Wood (1989) have attributed the failure o f bacteroids to persist in nodules as a major cause o f nodule ineffectiveness. Generally, ineffective nodules have much shorter life span and o f smaller size than those which are effective, but they are in greater number than the effective ones. The leghaemoglobin content o f the effective nodules far exceed the ineffective ones and this is reflected in the interior colour o f the effective being more pink while the ineffective range 21 University of Ghana http://ugspace.ug.edu.gh from le ss p in k to g reen in co lo u r (L ynch and W ood , 1989). 2.2.8 Environmental Factors Affecting Biological Nitrogen Fixation. Factors affecting nitrogen fixation have been extensively studied. The outcome o f the studies generally shows that environmental factors affect the legume and Rhizobium individually or both components o f the association as a whole. They may affect plant growth, the symbiotic association directly (Sprent et. al., 1988) or they may affect the biochemistry o f fixation indirectly. Environmental factors may be biological, chemical and physical. For purposes o f this discussion they would be considered under biotic and abiotic factors. Abiotic factors include moisture, aeration, nutrients, soil temperature, pH and salinity among others. Biotic factors result from competition and antagonism from other organisms living with the microsymbiont in the soil. Considering the plant and the rhizobia, since water is a major component o f protoplasm, adequate supply must be available for their vegetative development. But where moisture becomes excessive, microbial proliferation as well as root development are suppressed because the oversupply limits gaseous exchange and available oxygen supply (Alexander, 1977). Soil moisture may affect biological nitrogen fixation indirectly through plant growth, and directly through infection (Sprent, 1979) and nodule characteristics. Sinclair et. al. ( 1987 ) have shown that nitrogen fixation rates are more sensitive to moisture than any other plant physiological process ( Alexander, 1977). Lack o f oxygen to the host legume roots in waterlogged 22 University of Ghana http://ugspace.ug.edu.gh environment may also result in reduced acetylene reductase activity and nodulation (Witty et. al., 1986). The detrimental effects o f desiccation and high temperature on cowpea and soybean Rhizobium survival in soils are well documented (Boonkerd and Weaver, 1982; Hartel and Alexander, 1984; Munlongoy et. al., 1981). On the other hand, there is the evidence that Rhizobium has the ability to survive in desiccated soils (Van Schreven, 1970; Foulds, 1971; Danso, 1977). It is believed that the bacteria were able to achieve this due to their dependence on hygroscopic water surrounding the soil particle (Giltner and Langworthy, 1916). Soil temperature affects the survival o f Rhizobium and Bradyrhizobium in soil, with lower temperatures being more favourable than high temperatures (Bowen and Kennedy, 1959; Danso and Alexander, 1974; Danso, 1977). High soil temperatures may also affect survival of inoculated Rhizobium in tropical soils (Bowen and Kennedy, 1959). Hungria and Franco (1993) observed that nodules formed by effective strains at high temperatures (35 and 38°C) were ineffective. Hardarson et. al. (1989) and Wadisirisuk et. al. (1989) reported that deep placement o f nodules enhanced nitrogen fixation in soybean. Piha and Munns (1987) also suggested that deeper-placed nodules might be more active in nitrogen fixation when top soil temperatures are high because the sub-soil temperatures are lower than the top-soil temperatures. Strains o f rhizobia differ in their ability to withstand high temperatures (Bowen and Kennedy, 1959; Marshall, 1956). The differences in tolerance may be due to adaptation o f rhizobia to hot environment (Tuzimura et. al., 1963), age and number o f cells, (Fred et. al., 1932) and type o f soil (Marshall and Roberts, 1963). The seasonal fluctuations o f rhizobial population in tropical soils is the result o f high soil temperatures, large variations o f soil moisture and low level o f 23 University of Ghana http://ugspace.ug.edu.gh organic matter (Obaton, 1975). Soil acidity affects plant growth as well as bacterial occurrence and the survival o f Rhizobium. Highly acidic or alkaline conditions tend to inhibit many common bacteria as the optimum for most species is near neutral pH. Nevertheless, there are reported cases o f soils o f pH 3 containing bacteria (Alexander, 1977). Low pH is common in many tropical soils and tends to severely limit the survival o f introduced rhizobia and legume nodulation (Danso 1977; Danso and Alexander, 1974; Vincent and Waters, 1954). Acid and aluminium stresses are essentially bacteriostatic (Munns and Keyser, 1981). Aluminium toxicity is o f importance to growth and survival o f cowpea rhizobia (Hartel and Alexander, 1983). In 1984, F.A.O. reported that acidity, calcium, aluminium and manganese concentrations interact and affect both bacterial growth, root-hair infection and plant growth. Salinity has been known to cause permanent water stresses due to high osmotic pressure and making some nutrients such as phosphorus, molybdenum, iron, boron, manganese and zinc unavailable to both legume plant and the rhizobia. The availability o f nutrients is essential for growth and multiplication o f rhizobia (Fred et. al., 1932). Nevertheless, lack o f nutrients does not cause the rapid death o f rhizobia in the soil (Chen and Alexander, 1971; Danso and Alexander, 1974) High levels o f available soil nitrogen have been shown to sufficiently supply the nitrogen need o f nitrogen fixing plants and as a result inhibit biological nitrogen fixation. 24 University of Ghana http://ugspace.ug.edu.gh There have been many studies o f the effects o f combined nitrogen on the physiology o f the Rhizobium-legame symbiosis. One study by Anderson (1956) gave the indication that biologically fixed nitrogen and combined soil nitrogen seem to produce the same response in nitrogen fixing plants and the interaction between them is negative. It has also been established that large amounts o f applied nitrogen reduce root-hair infection (Munns, 1968; Dazzo and Brill, 1978), nodule number (Dart and Mercer, 1965), nodule mass (Summerfield et. al., 1977), the nitrogen fixing activity o f nodulated roots (Gibson, 1974), and the total amount o f nitrogen fixed, (Alios and Bartholomew, 1959). The degree o f inhibition however varies with the form o f nitrogen compound (Dart and Wilson, 1970), the cultivar (Gibson, 1974), the species (Alios and Bartholomew, 1959), strain o f Rhizobium (Pate and Dart 1961) and nutritional conditions (Pankhurst, 1981). At low levels, the effects o f applied nitrogen on symbiosis may be stimulative (Gibson and Nutman, 1960; Gibson, 1974). Phosphorus deficiency is acute in soils o f very low or very high pH (Sanyal et. al., 1990). Both the host legume and Rhizobium require phosphorus for the normal establishment and functioning o f the nitrogen fixing symbiosis (Robson et. al., 1981). Many biological factors affect the rhizobial population in the soil. Some microorganisms may antagonise Rhizobium by producing antibacterial metabolites. Others such as protozoa and bacteriophages act as predators and parasites respectively on rhizobia (Danso et. al., 1975; Barnet, 1980; Roughley, 1985); while others may act as competitors. Root-nodule bacteria are known to persist better in sterile than non-sterile soil (Van Schreven, 1970; Danso and 25 University of Ghana http://ugspace.ug.edu.gh Alexander, 1974). One can therefore deduce that biological agents are implicated in the decline o f rhizobia numbers in the soil. Microorganisms add many compounds to their environment, some o f which are produced to ward o ff potential competitors, parasites and predators. For instance Holland and Parker (1966) obtained a toxic water extract which inhibited Rhizobium trifolii. There are however reports o f stimulation o f some rhizobia by some soil microbes (Dixon, 1966). Some isolates o f bacteria and actinomycetes were found to stimulate strains o f Rhizobium trifolii (Hattugh and Luow, 1966). Among Rhizobium strains, it is reported that mixed inocula produced higher yields o f shoot dry weight and total nitrogen in red clover than single strain inoculation (Hofer, 1945), showing some synergism among the strains. 2.3 Soybean-The Host Plant. Soybean (Glycine max. L. Merrill) is a member o f the Leguminosae family and o f the order Papilonaceae. It is considered to have its origin in Northeastern China, although the genus has two major centres. One in Eastern Africa, the second in Australasia region with a secondary centre in China. Since its domestication around 11th century BC in China soybean has been a staple food in eastern Asia (Hymowitz, 1970). It remained confined to Asia until the beginning o f the 20th century when U.S.A. developed it into a major commercial crop. According to Anon (1982), all over the world soybean is the most important source o f commercial oil and grain legume crop. Soya flour is being incorporated into weaning foods (Annan, 1998) and in Ghana this constitutes one o f the major uses o f soybean. Soya flour can also be used to improve the protein content o f bread and gari (a staple food in most parts o f West Africa and Ghana). Soybean can be processed into avariety o f edible products such as soya milk, soyabean streak, soya sauce soya 26 University of Ghana http://ugspace.ug.edu.gh flour and soybean yoghurt. There are reports that milk is being processed from soybeans in Ghana (Annan, 1998). Soybean is the most cultivated crop in terms o f total production and international trade. In the United States o f America it is the second most valuable crop, surpassed only by maize. The world’s production has more than doubled over the last 20 years or so with global production in the latter part o f the 1980s exceeding 100 million tons annually (Source: Humanity Development Library, Legon.), The wet subtropics provide the best climate for the cultivation o f soybean with annual temperatures o f around 25°C and optimal rainfall o f between 500 and 750 mm. The plant is extremely photoperiodic , most varieties flower with day-light less than 14 hours a day. Reduced growth and yield become prevalent when daylight is less than 12 hours. Soybean is mainly cultivated for its seeds, commercially grown for human consumption, livestock food and the extraction o f oil. The fruit is normally a short hairy pod, which it varies from 2 to 10cm in length and 2 to 4cm in width according to variety. Being an annual its quick growing habit and easy cultivation lend itself to subsistence farming, a farming system very much predominant in West Africa and the tropics as a whole (Source: Humanity Development Library, Legon.). It has been mentioned earlier that large amounts o f nitrogen are required for good soybean production. For a yield o f 3000 kg/ha, 231 kg o f N is required (Borkert and Sfredo, 1994). Soybean can use nitrogen released by mineralization, residual soil nitrogen, fertilizer N or 27 University of Ghana http://ugspace.ug.edu.gh atmospheric nitrogen, which is converted to usable form in root nodules through symbiotic relationship between B. japonicum and soybean (Borkert and Sfredo, 1994). Like all legumes, the value o f soybean lies in its ability to grow under a wide range o f environment and may do well on poor soils without the need for supplemental nitrogen. The reason being that, though the soil is the primary source o f N for many crops, soybean obtains 65-86% o f its needs through the symbiotic process (Borkert and Sfredo, 1994). Therefore in most areas where soybean is now grown, production would be impractical without efficient symbiotic nitrogen fixation (Borkert and Sfredo, 1994). But soybean does not nodulate satisfactorily in West Africa, perhaps due to the fact that it was recently introduced into the Sub-region. (Anon 1975; Cuttelan and Hungria 1987; Ayanaba, 1977). However there are reports o f inoculation response by selected rhizobial strains (Bromfield and Ayanaba 1980; Owiredu and Danso, 1988; Annan 1998). Dennis (1975) reported o f inoculation response in three Ghanaian soils and predicted soybean to become a promising legume in terms o f nitrogen fixation. Work done by Owiredu and Danso (1988), also in Ghana, showed no nodulation in uninoculated Jupiter soybean (American genotype). However, few nodules were formed on Williams, also an American genotype, grown in the same soil. This seems to suggest that there are possible differences in the nodulating capacities o f non-promiscuous soybean genotypes to nodulate with native Bradyrhizobium in tropical soils (Kueneman et. al., 1984). According to Danso (1977), Danso and Alexander (1974) and Vincent and Waters (1954), low pH (a prevalent phenomenon in tropical soils) severely limits the survival o f introduced rhizobia and legume nodulation. Other reports by Haque et. al. (1980) in Sierra Leone, on the other hand, attributed poor nodulation in soybean to high soil temperature 28 University of Ghana http://ugspace.ug.edu.gh and moisture stress. High acidity is common in many tropical soils and can be a stress to soybean. The specific causes o f poor plant growth on acid soils may vary with soil pH, clay mineral type and amount, organic matter content and kind, level o f salts and particularly, with plant species or genotype (Foy et. al., 1978). Tropical soils should undoubtedly be limed in order to produce high yields. For example, Owiredu and Danso (1988) observed nodulation increase o f 13 times when lime was pelleted on the inoculated seeds compared with direct-seed inoculation only treatment. 2.4 Measurement of fixed nitrogen Soon after the discovery o f biological nitrogen fixation, several attempts were made to measure the amounts o f nitrogen fixed by plants. Out o f these attempts emerged several methods for the estimation o f fixed nitrogen. The relevance o f these measurements is to gather information on gains and losses o f nitrogen in agricultural soils since scarcity and excessive amount o f nitrogen are o f great interest to agriculture and the environment. Scarcity o f nitrogen significantly affects crop yield because it is a major nutrient required by plants. On the other hand excess nitrogen in the soil leads to undesirable environmental consequences (Danso, 1995). In addition the measurement o f biological nitrogen fixation is an important prerequisite in determining how environmental factors could be manipulated to optimize nitrogen fixation. Several methods have been devised for the measurement o f biological nitrogen fixation but it has to be mentioned that some o f these methods are qualitative and hence o f little use for 29 University of Ghana http://ugspace.ug.edu.gh quantitative purposes (Danso, 1985). Several publications have discussed various methods for measuring BNF (Chalk, 1985; Danso, 1985; Danso, 1988; Danso e t al., 1993; Denison et. al., 1983; Kumarasinghe et. al., 1992; Shearer and Kohl, 1986 and Streeter, 1979). For purposes of this review, a brief account o f some o f these methodologies would be discussed. 2.4.1 Nodule Assessment. Nitrogen fixation in legumes takes place exclusively within nodules. Therefore the quantity and mass o f nodules have been relied upon as indirect evidence o f nitrogen fixation and to some extent the magnitude o f nitrogen fixed (Westermann and Kolar, 1978; Weber, 1966). Nodule assessment is a quick and inexpensive method that does not require any highly skilled labour. Initial screening programmes involving several Rhizobium strains and legume varieties usually employ this quick method. However, it is not a valid technique because it is now a well-known fact that not all the nodules formed on a variety or species o f legumes fix similar amounts o f nitrogen. Some nodules are effective; others are moderately effective and the rest ineffective. Therefore the method cannot estimate the exact amount o f nitrogen fixed. 2.4.2 Dry matter yield (DMY) This is also another indirect way o f estimating nitrogen fixed. Crop yield increases are due to the provision o f nitrogen through fertilizers in nitrogen deficient soils (Rennie et. al., 1982; Deibert et. al., 1979; Weber, 1966). In nitrogen free medium containing all other nutrients, dry matter yield increases are attributable to nitrogen fixed and this is the principle on which the method is based. Some studies have reported o f reliable estimates o f nitrogen fixation by the DMY method (Haydock et. al., 1980). The method is also useful in the initial screening o f large 30 University of Ghana http://ugspace.ug.edu.gh numbers o f plants for nitrogen fixation. It’s usefulness however is diminished in soils with high nitrogen, where plant yields may already be near their peak (Fried, 1978). In addition, significant yield responses are sometimes not attained in the presence or absence o f nitrogen fixation because other limiting factors besides nitrogen do not permit the nitrogen fixed to be translated into increased yields. The method is not sensitivity enough for the detection o f small differences in nitrogen fixation (Hardarson et. al. 1984). Another limitation is, that the method cannot be used on different varieties or species, as potential yields could genetically be different even under identical conditions. 2.4.3 Total Nitrogen difference (TND) method. This is one o f the oldest and simplest methods and has provided many valuable estimates o f nitrogen fixation, upon which several management practices have been based (Danso, 1995). It measures BNF as the difference between the total nitrogen contents o f plants that fix nitrogen and those that do not derive nitrogen from fixation. Ndfa = Total N (fixer) - Total N (non fixer) Where Ndfa is nitrogen derived from fixation. The method is based on the assumption that, both the nitrogen fixing and non-fixing plants absorb equal amounts o f soil nitrogen (Rennie and Rennie, 1983). This assumption may not hold in all situations since different plants may rarely have similar root morphological and physiological properties as well as absorbing their nitrogen from similar depths (Danso et. al., 31 University of Ghana http://ugspace.ug.edu.gh 1986). This is a major limitation o f the method (Danso, 1985). On the other hand several reports have demonstrated high correlation between TND estimates and those made by other BNF determination methods which are more sophisticated and expensive, including the most widely accepted 15N labelling technique (Legg and Sloger, 1975; Broadbent et. al., 1982). Studies by Patterson and LaRue (1983) and Rennie (1984) demonstrated that the TND method gives reliable estimates o f nitrogen fixed in plants grown in soils in which initial nitrogen content is low because in such situations BNF is high compared with soil nitrogen uptake. 2.4.4 Acetylene Reduction Assay (ARA) Technique This is also an indirect method for estimating biological nitrogen fixation. The main rationale underlying the procedure is that nitrogenase, the enzyme in procaryotic organisms involved in nitrogen fixation also converts acetylene to ethylene (Dilworth, 1966). Today, the ARA technique, as has been modified for current usage, is based on procedures outlined by Hardy and other researchers (1968). The method involves incubating the sample to be analysed in gas-tight chamber containing 0.03-0.1% (v/v%) acetylene for a few minutes to several hours. A t the end of the incubation period, a gas sample from the incubation vessel is injected into a gas chromatograph fitted with a Porapak N or P column and assayed for ethylene production (Hardy et. al., 1973). The amount o f ethylene produced could be used as a measure o f nitrogenase or nitrogen fixing activity. On the other hand, the quantity o f ethylene formed can be converted into total amount o f nitrogen fixed by multiplying ethylene produced by a conversion factor o f 3 (Hardy et. a l , 32 University of Ghana http://ugspace.ug.edu.gh 1973). The rationale is, that stoichiometrically, three pairs o f electrons are used up during the conversion o f N2 to NH4, compared to a single pair o f electrons used in the conversion o f acetylene to ethylene, i.e., N2+ 8+ +12ATP + 6e—> 2NH4+ + 12 ADP + 12Pi (1) C2H2 + 2H+ + XATP + 2 e ^ C2H4 + ADP + Xpi (2) The following are the advantages o f the ARA technique: ❖ The process is facilitated in view o f the fact that no end products other than ethylene are produced. ❖ The ethylene produced is stable and storable, so it is possible to analyse the gas samples later. ❖ It is a highly sensitive and a less costly method. The problems associated with ARA have been reviewed by many authors (Danso, 1985; Denison et.al., 1983; M inchin et. al., 1983; Witty and Minchin, 1988), some o f which have been listed below: ❖ The technique involves short term assays whiles the BNF that it measures proceeds over long duration in crops. Therefore ARA measurements have to be extrapolated to cover periods over which no measurements were made. ❖ ARA measurements are normally not done on whole plants growing in the field. In the preparation o f the plant samples, disturbances suffered by the nitrogen fixing system induce increased resistance to the flow o f oxygen into nodules which adversely affects the rate o f 33 University of Ghana http://ugspace.ug.edu.gh acetylene conversion into ethylene (Minchin et. al., 1986; Witty and Minchin, 1988). ❖ The samples used for ARA assays consist o f uprooted plants and mostly some o f the active nodules are lost in the uprooting process. This affects the amount o f ethylene produced since it depends on the proportion o f active nodules remaining on the plant after uprooting. As a result o f these shortcomings many reviewers and editors often reject papers that base their interpretations on ARA measurements (Vessey, 1994). 2.4.5 15N-MethodoIogy The 15N methods have been classified into three main forms. These are: (1) The !5N labelled gas method, (2) The 15N isotope dilution method and (3) The A value method (Danso, 1995). The underlying principle behind all these methods is, that the nitrogen fixing plants are grown in soils or atmosphere containing l5N /14N ratio which is different from i5N /I4N ratio (of 0.3663%) in the atmosphere. During fixation the incorporation o f nitrogen from the atmosphere results in a different 15N /i4N in plant tissues from that o f the soil or any other substrate on which the plant grows. The 15N isotope dilution method has two approaches (1) The 15N natural abundance method where the inherent I5N /I4N ratios in some soils is higher than in the atmosphere (Amarger et. a l , 1979). Various nitrogen turnover processes such as denitrification, which has preference for 14N over I5N, bring about the higher 15N /14N ratio. The second approach is where I5N-enriched inorganic or organic nitrogen is deliberately 34 University of Ghana http://ugspace.ug.edu.gh added to soil to artificially widen the differences between the 15N /14N ratio o f soil nitrogen and that o f the atmosphere (Fried and Middelboe, 1977). In both cases the l5N /I4N ratio o f the plant tissue is lowered during the assimilation o f unlabelled nitrogen (Danso et. al., 1993) by the plant. However, in the case o f the natural abundance the 15N enrichment is rather very low, and calls for the use o f highly sophisticated mass spectrometers well outside the reach o f many laboratories in developing countries. The more the amount o f nitrogen fixed the greater the dilution o f 15N /14N. The 15N-enriched fertilizer approach requires less sophisticated analytical capability; also, it is subject to fewer errors than the natural l5N abundance approach (Ledgard et. al., 1985). Therefore it is the most widely used in nitrogen fixation studies. There are some practical problems and in some cases improper usage that have been identified with the use o f the 15N techniques. These have been revealed in the course o f research on the increasing reliance on the technique. Questions being asked in the midst o f all these criticisms have led to a closer examination o f the methodology with the view to rectifying some o f the anomalies o f the techniques (Chalk, 1985). In some cases however, the limitations seem to have arisen from general lack o f understanding o f some o f the basic principles o f the methods (Vose and Victoria, 1986). The accuracy o f the BNF measurements using the isotope dilution approach depends on how the 15N /I4N ratio assessed by the reference plant reflects that o f the soil- derived N. This is the greatest source o f error, especially for many studies where prior selection o f suitable reference plant was not done or where the criteria for selection were not followed satisfactorily (Fried et. al., 1983). In certain cases even prior selection may not work because the reference plant does 35 University of Ghana http://ugspace.ug.edu.gh not perform satisfactorily under all environments (Chaiwanakupt et. al., 1991; and Danso, 1991). There are situations where Danso (unpublished data) observed that seeds supposed to be non-nodulating isolines, upon inspection, nodulated profusely in some soils. Therefore the selection o f an appropriate reference crop for each nitrogen fixing crop is o f great importance (Fried et. al., 1983; Wagner and Zapata, 1982) A suitable reference plant must fulfil the following conditions: ❖ It should not fix N under the conditions o f the study otherwise the nitrogen fixation estimate made shall be underestimated by the extent to which the reference crop fixed nitrogen. ❖ Both reference and nitrogen fixing plants should take up N from a similar zone even though they do not necessarily have to absorb the same quantity o f soil N. ❖ Both reference and N fixing plants should have similar physiological growth patterns and both should be harvested at the same time. ❖ The tolerance levels o f both reference and nitrogen fixing plants to crucial environmental stresses should be similar to ensure that conditions affecting active uptake o f nitrogen of both plants would almost be equal. Typical examples o f some reference plants that have been used to assess soil 15N /14N ratios have been listed below: ■ A non-nodulating legume isoline (Ruschel et. al., 1982) ■ An uninoculated legume (Fried et. al., 1983). This is valid only in soils lacking effective Rhizobium strains for the legume in question. 36 University of Ghana http://ugspace.ug.edu.gh ■ Legume inoculated with ineffective Rhizobium strains for the legumes in question. * A non-legume, non-fixing plant e.g. cereal (Wagner and Zapata, 1982; Fried and Broeshart, 1975). The advantages derived from the I5N methodology are that: Currently the 15N soil enrichment technique is the most reliable for measuring N fixed in the field (Duhoux and Dommergues, 1985; Duque et. al., 1985; Hardarson et. al., 1984 , Legg and Sloger, 1975; Rennie, 1982; Ruschel et. al., 1979, 1982; West and Wedin, 1985). The method is very useful because, at a single harvest, it can measure the integrated amounts o f nitrogen assimilated by both green-house and field-grown plants as well as measuring the nitrogen contributed from soil or fertilizer sources (Danso, 1985; Fried et. al., 1983; Vose and Victoria, 1986), thus making it possible to manipulate plants or nitrogen fixing systems for maximum nitrogen fixation. The disadvantages o f the method include the relatively high cost o f 15N fertilizers and equipment. There is also the need for highly skilled technicians to do the l5N analysis. Recently the cost o f 15N fertilizers has gone down dramatically, and also, relatively inexpensive equipment such as micromass and emission spectrometer have been developed, solely for15N analysis. 37 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE 3.0 MATERIALS AND METHODS. 3.1 Soil Sampling. Soil samples collected from three ecological zones o f Ghana were used to assess nodulation capabilities o f soybean. The local names o f these soils were, Adenta, Akuse, Anyinase and Bekwai soil series. The rest were Hatso, Nyibenya, Nzima and Toje series (Table 3.1). Adenta, Hatso, Nyibenya and Toje soils were collected from the University o f Ghana Farms at Legon (Coastal Savanna zone, Fig 3.1). Bekwai and Nzima series were obtained from the University o f Ghana Research Station at Okumaning near Kade in the Eastern region Semi-Deciduous Forest Zone, Fig 3.1). Akuse series was collected from the Agricultural Research Station o f the University o f Ghana at Kpong, also in the Eastern Region (Coastal Savanna zone, Fig 3.1), while Anyinase series was collected from Anyinase near Axim in the Western Region (Rainforest Zone, Fig 3.1). All the soils were sampled from a depth o f 0-15 cm from uncultivated soil. Each soil was air- dried and stones, roots and other plant parts contained in the soil removed. They were then pulverised using pestle and mortar, and sieved through a 2mm-mesh sieve. 3.2 Screening of soybean for nodulation capabilities. 3.2.1 Planting Material. Six soybean cultivars were screened for their nodulation potential in eight Ghanaian soils. They were made up o f four promiscuous and two non-promiscuous genotypes. 38 University of Ghana http://ugspace.ug.edu.gh C Semi-deciduous Forest Zone D Forest-Savanna Transitional Zone E Guinea Savanna Zone Eg. 3.1 Map o f Ghana showing the ecological zones and sites where the soils were sampled. 3*T University of Ghana http://ugspace.ug.edu.gh Table 3.1 Classification of soils Studied. SOIL SERIES CLASSIFICATION LOCAL USD A Adenta Savanna Ochrosol Typic Paleustalf Nyigbenya Savanna Ochrosol Lithic Rhodustalf Hatso Savanna Ochrosol NA Akuse Tropical Black Earths Typic Calciustert Anyinase Forest Oxisols Ultisols Toje Savanna Ochrosol Typic Rhodustalf Bekwai Red Forest Ochrosols Rhodic Paleudult Nzima Brown Forest Ochrosols Rhodic Paleudult Source: Extracted from Ph. D. & M.Phil. theses o f Fening and Dowuona respectively. 40 University of Ghana http://ugspace.ug.edu.gh Four o f the soybean genotypes were obtained from seed store o f the Crop Science Department o f the University o f Ghana and two o f the genotypes obtained from the Savanna Agricultural Research Institute (SARI) in Nyankpala, near Tamale in the Northern Region o f Ghana. 3.2.2 Pot Experiment. The six soybean cultivars were planted in the eight soils using plastic pots, 28cm high with top interior diameter o f 25cm and bottom interior diameter o f 15cm. Three holes, each o f diameter 0.5cm were perforated at the base o f the pots to allow for drainage o f excess water, but with the base covered with filter paper to prevent loss o f soil. Each pot contained 1kg o f soil. The seeds were surface-sterilised with 70% alcohol and rinsed with six washes o f sterile distilled water before planting them at four per pot. Three days after germination seedlings were thinned to two per pot. There were three replicates for each soil and for each cultivar, using the split-treatment design, with soils being the main treatment. The experiment was carried out in the greenhouse and watered twice a day with distilled water. Harvesting was done 5 weeks after planting and nodulation assessment carried out after washing the roots under a gentle stream o f water to free them o f all soil particles. The nodules on each plant were counted and recorded, and some removed and used for Rhizobium isolation. 3.2.3 Isolation of Bradyrhizobia Two nodules from each plant were taken from the screening experiment. They were surface sterilised by immersing in 70% alcohol in small beakers for 3 minutes and in 0.1% mercuric 41 University of Ghana http://ugspace.ug.edu.gh chloride also for 3 minutes, after which the nodules were rinsed with 6 washes o f sterile distilled water, as recommended by Somasegaran and Hoben (1985). The sterilised nodules were pulverized with a pair o f heat sterilised, blunt tip forceps in a large drop o f sterile distilled water in a petri dish. A loopful o f the nodule suspension was streaked on yeast mannitol agar and incubated at 28°C.Afiter restreaking colonies growing on YEM agar plates were transferred into eppindorf tubes containing agar, labelled, and stored in a refrigerator at 4°C for future work. 3.2.4 Authentication of Bradyrhizobia Isolates. This was done to ensure that the isolates were pure cultures, and were thus still capable o f forming nodules on soybean roots. Soybean seeds were surface sterilised and pregerminated on 1% (w/v) water agar and planted in growth pouches (Somasegaran and Hoben, 1985). Each sterlised 2-plants/unit growth pouch was inoculated with 1ml broth culture o f an isolate (Somasegaran and Hoben, 1985). Those that were not inoculated served as control. The pouches were arranged randomly on a wooden rack with the experimental set-up in the greenhouse and the plants supplied with 50ml N-free nutrient solution (Broughton and Dilworth, 1970). The plants were harvested 28 days after planting, and the roots examined for the presence or absence o f nodules. 3.3 Cross Inoculation Studies. Specificity and promiscuity in symbiosis were studied in cross inoculation experiments. The ability o f the soybean isolates to nodulate cowpea and groundnut varieties was examined. The 42 University of Ghana http://ugspace.ug.edu.gh seeds o f these legumes (cowpea and groundnut) were carefully selected by hand sorting. The aim was to obtain viable, undamaged, clean seeds o f reasonably uniform sizes and colour for planting so as to reduce, as much as possible, variability among the seeds. The seeds were surfaced sterilised by immersion in 70% alcohol for 3 minutes and rinsed with 6 washes o f distilled water. They were also immersed in in 0.1% mercuric chloride for 3 minutes and rinsed with 6 washes o f distilled water, then allowed to imbibe water by soaking in distilled water for 1 hour and then rinsed twice. Pregermination was done by transferring the seeds aseptically onto 1% (w/v) water agar in petri dishes and incubated at 28°C for 2 or 3 days, after which they were planted in sterilised growth pouches containing sterile N-free nutrient solution (Somasegaran and Hoben, 1985). Each growth pouch was inoculated with 1ml suspension o f YMA broth culture o f one o f the isolates, which had been grown in culture bottles on a shaker for 5 days. Uninoculated growth pouches were set up as control. The growth pouches were randomly arranged on wooden racks in a greenhouse. The plants were supplied with sterile N-free nutrient solution throughout the growth period. Harvesting was done after 5 weeks and the plants examined and scored for the presence or otherwise o f nodules. 3.4 Assessment o f the Effectiveness of Bradyrhizobia Isolates. This experiment was done using one cultivar o f soybean named Bengbie. The growth medium was sand obtained from the Densu riverbed. The sand was flooded with 2N hydrochloric acid solution and left to stand for 3 days in large plastic containers and then rinsed thoroughly with tap water. The acid treatment was meant to digest any organic matter present in the sand and the rinsing done to get rid o f excess acid until the pH was between 6.8 and 7.0. The sand, which was wet, was dried in the sun and then used to fill the top chamber o f Leonard jars. Centrally placed 43 University of Ghana http://ugspace.ug.edu.gh cotton wicks dipping into the sterile N-free nutrient solution irrigated the sand in the top chamber o f the jars. The isolates were grown in culture bottles containing 50ml YEM broth on a shaker for 5 days (Somasegaran and Hoben, 1985). The seeds were surfaced sterilised by immersion in 70% alcohol for 3 minutes and rinsed with 6 washes o f sterile distilled water. They were also immersed in in 0.1% mercuric chloride for 3 minutes and rinsed with 6 washes o f distilled waters and allowed to imbibe water by soaking in distilled water for 1 hour and then rinsed twice. Pre-germination was done by transferring the seeds aseptically onto 1% (w/v) water agar in petri dishes and incubated at 28°C for 3 days, after which they were planted four per ja r in the sterilised Leonard jars containing sterile N- free nutrient solution (Somasegaran and Hoben, 1985). After the seeds had fully germinated they were thinned to two plants per ja r and each plant inoculated with 1ml suspension o f YMA broth culture o f one o f the isolates grown in culture bottles on a shaker for 5 days. The two plants in the same jar were inoculated with the same isolate. Each ja r was replicated two times. There were two separate uninoculated controls, one supplied with nitrogen and the other without nitrogen. The inoculated plants and the uninoculated ones without nitrogen were supplied with N-free nutrient (Broughton and Dilworth, 1970) solution while the uninoculated N-control received N-free solution to which nitrogen had been added (Somasegaran and Hoben, 1985). The experiment was set up in the greenhouse and the jars arranged in completely randomised design. The plants were supplied with their respective nutrient solutions regularly throughout the period o f growth. They were harvested 35 days after planting and nodule number, nodule dry weight and shoot dry weight records taken. In the process the shoots were severed from their 44 University of Ghana http://ugspace.ug.edu.gh roots at the collar, put in labelled envelopes and oven-dried at 80°C for 72 hours after which their dry weights were taken. The mean shoot dry weight (x) was used to calculate the Effectiveness index E given by the following relation: E = xi - xTo x 100 xTn- xTo where j is the shoot dry weight o f inoculated strain,.To, that o f the uninoculated control and Tn that o f the nitrogen control. CRITERIA FOR GROUPING ISOLATES. Group Criterion Ineffective Isolate Isolate with effectiveness index less than 50% Moderately Effective Isolate Isolate with effectiveness index between 50%-80% Highly Effective Isolate Isolate with effectiveness index more than 80% 45 University of Ghana http://ugspace.ug.edu.gh 3.5 Inoculation of Soybean Genotypes with Bradyrhizobia Isolates. This experiment was conducted in the Bekwai soil series using three soybean types, namely, Bragg (anon-promiscuous genotype), TGX 1303 and Bengbie (which are promiscuous). A non- nodulating soybean genotype was included for estimating nitrogen fixed by the I5N isotope dilution method (Fried and Middelboe, 1977.) Plastic pots used for the experiment were each filled with 1.2 kg o f the sieved soil sample.. Seven bradyrhizobia isolates were used, five o f them being indigenous isolates obtained from the screening experiment while the other two (J2 and J23) were standard tropical isolates received from Thailand. All the isolates were first streaked onto YEM agar plates and incubated for 3 days after which they were transferred into sterile YEM broth in culture bottles and grown aseptically (Vincent, 1970) at 28°C on a wrist-action shaker for 5 days. The seeds were carefully selected by hand sorting to obtain viable, undamaged, clean seeds of reasonably uniform sizes and colour for planting so as to reduce as much as possible variability among the seeds. They were surface sterilised by immersion in 70% alcohol for 3 minutes and rinsed with 6 washes o f distilled water. They were also immersed in in 0.1% mercuric chloride for 3 minutes and rinsed with 6 washes o f distilled water and and then allowed to imbibe water by soaking in distilled water for 1 hour and then rinsed twice. Pre-germination was done by transferring the seeds aseptically onto 1% (w/v) water agar and incubated at 28°C for 3 days, 46 University of Ghana http://ugspace.ug.edu.gh after which they were planted four plants per pot. They were thinned out to two plants per pot after germination. A micropipette (dispensette) was used to dispense 1ml culture o f each isolate and inoculated onto each plant. The control plant for each genotype however did not receive any inoculation. Each treatment was replicated three times, and the pots arranged in a split-treatment design in the greenhouse. 15N- labelled ammonium sulphate fertilizer was dissolved in sterile distilled water and applied to all treatments at a rate equivalent to 10 kg N/ha. This was done in three split-applications at 1 week, 3 weeks and 5 weeks after planting. The plants were watered daily with sterile distilled water and harvested 7 weeks after planting. All the shoots were severed from the roots around the collar, put into labelled envelopes and oven-dried at 80°C for 72 hours after which their dry weights were taken. The roots were washed thoroughly to get rid o f adhering soil using a gentle stream o f water from a hose. Nodules per plant were counted and recorded, then carefully wrapped in aluminium foil, oven dried at 80°C for 48 hours, and their dry weights taken. 3.6 Soybean Response to Inoculation and Nitrogen Fertilizer Application. This experiment was conducted in Bekwai and Adenta soils. Three soybean types were used for the study; they were Bragg, TGx 1303 and Bengbie. In addition a non-nodulating soybean isoline was included as a reference crop. One isolate (isolate 38) was used to inoculate all treatments except the non-nodulating genotype, which was not inoculated. H alf o f the treatments received 15N-labelled ammonium Sulphate fertilizer at a rate equivalent to 10 kgN/ha. The rest received ammonium sulphate fertilizer enriched by I5N-atom excess at a rate equivalent tolOOkgN/ha. 47 University of Ghana http://ugspace.ug.edu.gh Table3.2 PHYSICOCHEMICAL PROPERTIES OF THE SOILS USED FOR THE INOCULA TION STUDIES. SOIL SERIES Bekwai Adenta CLASSIFICATION LOCAL Red Forest Ochrosols Savanna Ochrosols USDA Rhodic Paleudult Typic Paleustalf MATERIAL Phyllite Quartzite schist pH 5.6 4.6 ORGANIC CARBON (%) 2.86 0.84 TOTAL N (% ) 0.21 0.058 AVAILABLE 5.1 3.47 TOTAL P (mg/kg) 173 120 Isolate 38 was first streaked onto YEM agar plate and incubated for 3 days after which it was cultured aseptically at 28°C on a wrist-action shaker for 5 days in culture bottles containing YEM broth. The seeds were carefully hand-sorted to obtain clean viable seeds o f uniform size and colour for the study. They were surface-sterilised by immersion in 70% alcohol for three minutes and washed in 6 changes o f distilled water; they were also immersed in in 0 .1% mercuric chloride for 3 minutes and rinsed with 6 washes o f distilled water. They were made to imbibe water by soaking in distilled water for 1 hour and then rinsed twice with distilled water. They were then transferred aseptically onto 1% (w/v) agar plates and incubated at 30°C in an incubator for 3 days. Four of the pre-germinated seeds were planted in each pot and University of Ghana http://ugspace.ug.edu.gh subsequently thinned to two per pot after they had fully germinated. Each treatment was replicated three times, and the pots arranged in a split-treatment design in the greenhouse. A dispensette was used to dispense 1ml o f the culture o f bradyrhizobia isolates and used to inoculate each plant. The two different rates o f fertilizer were applied to their respective treatments by dissolving the required quantities o f l5N-labelled ammonium Sulphate fertilizer in sterile distilled water and applying them in 3 split-applications at lweek, 3 weeks and 5 weeks after planting. The plants were watered daily until they were harvested 7 weeks after planting. All the shoots were severed from their roots around the collar and put into labelled envelopes, oven dried at 80°c for 72 hours, after which their dry weights were taken. The roots, contained in a sieve were washed thoroughly to get rid o f the adhering soil, using a gentle stream o f water from a hose. The nodules per plant were counted and recorded, carefully wrapped in aluminium foil, oven dried at 80°c for 48 hours, and their dry weights taken. 3.7 1SN-Analysis After the determination o f the shoot dry weight, the plant shoots in the two previous experiments were milled and used for 15N-determination. A little quantity (550 mg) o f each o f the milled samples was weighed into labelled Kjeldahl flasks. A loopful o f selenium reaction mixture (catalyst) was added to each sample and mixed thoroughly. Eight mililitres o f concentrated sulphuric acid were added to each sample and shaken vigorously to ensure that the samples were properly mixed with the acid. The samples were digested using the digester for about 2 hours. About 5ml o f distilled water 49 University of Ghana http://ugspace.ug.edu.gh was then added to each sample in the flask followed by the addition o f two drops o f Tahiru indicator. Meanwhile 10ml o f 0.1N hydrochloric acid had been discharged into conical flasks and labelled the same way as those o f the Kjeldahl flasks. Two drops o f Tahiru indicator were also put into each flask containing the hydrochloric acid. The digest was distilled and the gas that evolved collected into the 0.1N HC1. A back titration o f 0.1N NaOH against the distillate was done and the volume o f the acid for the back titration recorded for the determination o f total nitrogen. The sample was vapourised using the evaporator until ju st about 2 ml or so was left. This was stored safely in a capped tube and kept in a refrigerator. The actual 15N readings were done using the Emission spectrometer (NOI-6e) (Fieldler and Proksch, 1975). 3.8 Counting of rhizobia The most probable number (MPN) method (Vincent 1970) also called the plant infection count was used to assess the rhizobial populations capable o f infecting soybean in the eight soils used for the screening experiment. The promiscuous soybean variety, Bengbie; was used for the enumeration. The seeds were carefully hand-sorted to obtain clean viable seeds o f uniform size and colour for the study. They were surface-sterilised by immersion in 70% alcohol for 3 minutes and washed in 6 changes of distilled water and in 1% mercuric chloride for 3 minutes and subsequently washed in six changes o f sterile distilled water. They were then allowed to imbibe water by soaking in distilled water for lhour and then rinsed twice (Somasegaran and Hoben, 1985). They were thereafter transferred aseptically onto 1% (w/v) water agar and incubated at 30°C until the radicles were 2cm long. The seedlings were planted in sterilised growth pouches, at two plants per pouch. The pouches used in the exercise were made o f transparent heat-resistant polythene 50 University of Ghana http://ugspace.ug.edu.gh (16 by 18cm) with paper wick liners (Somasegaran and Hoben, 1985). Each pouch contained N- free nutrient solution (Broughton and Dilworth, 1970). Tenfold dilutions o f each soil suspension with four replicates per dilution were used to inoculate the pouches at 1ml per pouch. When the plants were 5 days old the growth pouches were reorganised and randomised on wooden racks and kept in the greenhouse. The plants were observed periodically and the nutrient solution replenished as and when necessary. Signs o f nodulation were evident after 2 weeks. Twenty- eight days after inoculation, nodulation was assessed and the most probable numbers o f rhizobia calculated (Vincent, 1970). 51 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR 4.0 RESULTS 4.1 Nodulation potential of six soybean cultivars in eight Ghanaian soils. . The results o f studies carried out to assess the nodulation potential o f six soybean cultivars in eight soils without inoculation showed variable nodulation potential o f the cultivars in the soils studied (Fig 4.1). With the exception o f Bengbie which nodulated with naturally occuring rhizobia in about 63% o f the soils under study, the rest o f the cultivars nodulated in 50% o f the soils. On the whole, TGx 1519 gave the highest average number o f nodules per plant (Fig 4.1) which represented 36% o f the total number formed by all the six cultivars, with Davis recording the lowest nodulation, as low as 0.009% per plant on average. Considering soils, the highest nodulation occurred in Nyigbenya, with an average, o f 11 nodules per plant. Nodulation in Nyigbenya soil series alone represented 44% o f the total nodulation in the six soils under study. There was no nodulation in Anyinase, Bekwai and Nzima soil series, whiles only one cultivar, Bengbie, nodulated in the Toje soil series. The Adenta soil was the only one in which all the cultivars nodulated and thus also, the only one in which Davis formed nodules. 52 University of Ghana http://ugspace.ug.edu.gh Fig. 4.1 NODULATION OF SOYBEAN IN EIGHT GHANAIAN SOIL SERIES □ Bengbie □ TGX1303 □ Salantuya □ TGX1519 DD Bragg □ Davis Adenta Nyibenya Hatso Akuse Anyinase Toje Bekwai Nzima University of Ghana http://ugspace.ug.edu.gh 4.2 Estimation of population of indigenous soybean bradyrhizobia. The number o f naturally occurring rhizobia by the MPN count is presented in Table 4.1.The average population densities o f indigenous bradyrhizobia capable o f nodulating the six soybean cultivars in the eight soils indicated that soil had a great influence on the abundance o f soybean bradyrhizobia. While extremely few bradyrhizobia were detected in Nzima, Bekwai, Toje and Anyinase soils, the remaining four soils contained more than 2x103 bradyrhizobia cells per gram soil with the highest bradyrhizobia count o f 8x103/g soil occurring in the Adenta soil and the lowest, 3.2 x lO '/g soil, in Nzima soil. With the exception o f Toje, there appeared to be a relationship between the population o f soybean bradyrhizobia and the ecozones under which the soils developed. This is based on the fact that the other soils which contained less than 1000 bradyrhizobia (cell/g soil) were all not capable o f supporting nodulation in soybeans . A more interesting observation however was the fairly substantial bradyrhizobia count recorded in Anyinase, even though none o f the soybean genotypes nodulated in that soil during the screening studies. Table 4.1 Population of bradyrhizobia (cell/g soil) nodulating soybean. Soils Bradyrhizobia Count Adenta 6000 Nyigbenya 5600 Hatso 4000 Akuse 4000 Anyinase 700 Toje 700 Bekwai 45 Nzima 32 4.3 Selecting Effective Strains of Soybean Bradyrhizobia for Nitrogen Fixation Symbiotic effectiveness was determined using 60 isolates randomly selected from the screening experiment and the results presented in Tables 4.2 and 4.3. The results indicated variation 54 University of Ghana http://ugspace.ug.edu.gh among the isolates in terms o f nodule numbers formed on soybean as well as in symbiotic effectiveness. The estimated effectiveness values relative to the uninoculated but N fertilized control ranged from 0% to 144.6%. The isolates were categorised into effective, moderately effective and ineffective based on their effectiveness index with almost two-thirds (65%) o f the isolates being classified as ineffective (Fig 4.2). Table 4.2. Effectiveness grouping o f 60 soybean isolaes selected from the screening experiment. Effectiveness Groups Percentage o f Isolates Identification Nos. o flso ltes Highly Effective 15% 38,119,118,11,129,25,102,23, 39 Moderately Effective 20% 98,8,12,175,164,42,21,112,10 7,168,22,122, & 177 Ineffective 65% 57,174,79,47,75,40,10,153,10 5,128,41,169,114,143,1,5,167, 161,9,101,80,106,178,45,91,1 26,104,179,116,166,82,103,14 1,117,144,158,44, & 13 55 University of Ghana http://ugspace.ug.edu.gh T a b ic 4 .3 Effectiveness indices of 60 soybean rhizobia isolated from some Ghanaian soils. Isolate Effective index Shoot drv wt. of Nodule drv wt./plant sovbean/plantfe) (mg.) 38 144.6 3.14 252 119 97.8 2.28 172 118 96.7 2.26 220 11 93.5 2.2 122 129 91.3 2.16 84 25 89.1 2.12 225 102 84.8 2.04 241 23 83.7 2.02 152 39 81.5 1.98 210 98 77.2 1.9 85 8 75.0 1.86 151 12 75.0 1.86 103 177 73.9 1.84 206 175 73.9 1.84 81 164 72.3 1.8 136 42 64.1 1.66 156 21 58.7 1.56 105 112 57.6 1.54 90 56 University of Ghana http://ugspace.ug.edu.gh 1SU 1 107 168 22 57 174 79 47 75 40 10 153 122 105 128 41 169 114 143 1 5 Effective index Shoot dry wt. of Nodule dry wt./plant sovbean/nlantfa) (ms-) 54.2 72 64 52.2 1.44 103 51.1 1.42 103 45.7 1.32 70 43.5 1.28 105 41.3 1.24 64 41.3 1.24 100 34.8 1.12 36 32.6 1.08 63 31.5 1.06 62 29.3 1.02 85 28.3 1.0 77 27.2 0.98 86 26.1 0.96 64 25.0 0.94 72 23.9 0.92 93 22.8 0.9 64 20.7 0.86 42 18.5 0.82 48 18.5 0.82 35 17.5 0.8 51 57 University of Ghana http://ugspace.ug.edu.gh 161 9 101 80 106 178 45 91 126 104 179 116 166 82 103 141 117 144 158 44 13 Effective index Shoot drv wt. of Nodule drv wt./plant sovbean/Dlantfg) (mg.)64 14.1 0.74 77 12.0 0.7 65 12.0 0.7 41 12.0 0.7 35 10.9 0.68 69 10.9 0.68 52 9.8 0.66 74 7.6 0.62 86 7.6 0.62 31 6.5 0.6 62 6.5 0.6 22 5.4 0.58 17 5.4 0.58 20 4.3 0.56 15 4.3 0.56 15 2.2 0.52 19 1.1 0.5 36 0.0 0.48 18 0.0 0.48 11 0.0 0.48 15 0.0 0.48 10 58 University of Ghana http://ugspace.ug.edu.gh Fig 4.2 Sample of soybean plants used for effectiveness studies 1= Nitrogen control II, III and IV = Inoculated plants V = Uninoculated control 59 University of Ghana http://ugspace.ug.edu.gh Generally, effectiveness index ranking seemed to correspond well with shoot dry matter yield, however the same could not be said o f nodule dry matter. The most effective isolate (isolate 38) according to the ranking produced almost 10 times as much dry matter as the lowest ranked isolate (Isolate 13). 4.4 Cross Inoculation Groupings of Some Selected Legumes. The abilities o f indigenous soybean bradyrhizobia to form symbiotic relationships with two other commonly cultivated legumes in Ghana, cowpea and groundnut were examined to determine their level o f compatibility and promiscuity. The proportion o f the soybean isolates that nodulated these two legumes was high, with 88.3% nodulating cowpea and 80% groundnut Table 4.4). This shows a relatively higher nodulation in cowpea compared with groundnut.. Table 4.4__________________ Cross inoculation grouping of 60 soybean isolates with cowpea and groundnut. Number % B. japonicum isolates which nodulated soybean 60 100 B. japonicum isolates which nodulated Cowpea 53 88.3 B. japonicum isolates which nodulated Groundnut 48 80.0 60 University of Ghana http://ugspace.ug.edu.gh 4.5 Response of some selected soybean cultivars to inoculation. Nodule number and dry weight. With inoculation, all plants nodulated fairly well, with the highest number o f nodules (44 nodules per plant) produced on Tropical Glycine cross (TGx-1303), inoculated with isolate J23 (Table 4.5). However, there was no nodulation by the uninoculated plants. Nodule numbers seemed to have correlated well with nodule dry matter (r = 0.7), but the same could not be said of isolates J2 and J23 which in most cases produced many but rather tiny nodules. Also nodule dry matter seemed to correlate fairly well with % N-fixed (r = 0.7) as well as total N-fixed (r= 0.7) (Tables 4.5 and 4.6). Shoot dry matter: The shoot dry matter formed by Bragg, Bengbie and TGx inoculated with the seven selected soybean bradyrhizobia isolates is shown in Fig 4.3. Cultivar x isolate interaction was significant (P=0.05). Yield o f inoculated Bragg plants was significantly higher than Bengbie and TGx in all case except when inoculated with isolates J23 and 122. For Bengbie, yield was improved by inoculation with all the seven strains. Similarly, yield increased by inoculation o f TGx in all cases. For Bragg, inoculation with iso-38 gave the highest yield, significantly different from the other isolates, between 21 and 42% higher. Differences between yields when inoculated with the remaining isolates were not significant, although inoculation with Iso-38 was the only one among them that gave higher yield than the uninoculated control. 61 University of Ghana http://ugspace.ug.edu.gh Table 4.5 Number and drv matter of nodules formed by Bragg Bengbie and TGX Inoculated with seven soybean bradvrhizobial isolates in Bekwai soil. INOCULUM/VARIETY NODULE No/PLANT NODULE DRY MATTERfmg/plant) Isolate 38 Bragg 33.0 86.7 Bengbie 17.5 48.8 TGx 41.5 50.5 X 30.7 62.0 Isolate 119 Bragg 29.0 65.3 Bengbie 14.5 32.0 TGx 40.5 64.0 X 28.0 53.3 Isolate 129 Bragg 34.5 55.3 Bengbie 18.5 35.5 TGx 34.5 70.0 X 29.2 53.6 Isolate 102 Bragg 30.0 50.2 Bengbie 12.5 26.5 TGx 16.3 46.0 X 19.6 40.9 62 University of Ghana http://ugspace.ug.edu.gh INOCULUM/VARIETY NODULE No/PLANT NODULE DRY MATTER/mg/plant) Isolate J2 Bragg 39.0 32.0 Bengbie 26.5 49.8 TGx 35.5 77.0 X 33.7 52.9 Isolate J23 Bragg 36.5 33.0 Bengbie 36.5 40.5 TGx 44.0 55.0 X 39.0 42.8 Isolate 122 Bragg 17.5 41.5 Bengbie 15.0 40.5 TGx 16.0 55.0 X 16.2 45.7 Uninoculated control Bragg 0.0 0.0 Bengbie 0.0 0.0 TGx 0.0 0.0 x~ 0.0 0.0 LSD(0.05) 4.5 7.8 63 University of Ghana http://ugspace.ug.edu.gh SH OO T DR Y MA TT ER YI EL D /p la nt (g ) FIG 3 INOCULATION EFFECT OF SELECTED SOYBEAN ISOLATES ON SHOOT DRY MATTER WT. OF THREE SOYBEAN CULTIVARS. U iso-38 iso-119 iso-129 iso-102 iso-J2 iso-J23 iso-122 UC SOYBEAN ISOLATES □ Bragg 0 Bengbie □ TGX 1303 University of Ghana http://ugspace.ug.edu.gh For Bengbie, Iso-J23 gave the highest shoot dry matter yield, which was significantly higher than plants inoculated with five strains, 38, 119, 129, 102, & 122, but not significantly different from that o f Iso-J2. For TGx, inoculation with Iso-102 gave the highest dry matter yield, and this was significantly higher than for plants inoculated with three o f the strains, 129, J23 and 122, while the differences in yields were not significantly different when plants were inoculated with 38,119 and J2. On the whole, the best isolate for Bragg was 38, Bengbie J23, and for TGx 102, showing strain preference for each o f the three soybean genotypes. Total Nitrogen: Generally the trend for total N per plant was similar to that o f shoot dry matter (Table 4.6). With UC, Bengbie gave the lowest total N but this was not significantly different from TGx-1303. Total nitrogen produced by uninoculated Bragg was however significantly higher than that produced by Bengbie, but not TGx. There were no significant differences in total N among all varieties inoculated with 122 and 129, while with isolates J23, J2 and 102, Bengbie consistently produced highest and significantly different total N values from the other two cultivars. Bengbie responded greatly to inoculation, rising from the lowest when not inoculated to the highest 65 University of Ghana http://ugspace.ug.edu.gh when inoculated with J23 J2 and 102. TGx on the other hand seemed to exhibit a reversal o f that trend. The total N results seemed to be consistent with the dry matter weight data. In both cases, all the isolates, except isolates 38 and 102, did not significantly improve the perfomance o f Bragg (the non-promiscuous American genotype), while the same strains significantly improved the performance o f the 2 promiscuous types. Although Bragg produced the highest total N when uninoculated, with inoculation with seven strains, it produced significantly highest yield only with isolate 38, while the differences between Bengbie and TGx with this isolate were not significant. Comparing total N values o f the various isolates with the UC, TGx and Bengbie gave significantly different amounts o f total N with all isolates, the highest given by TGx inoculated with isolate 102, with higher total N value o f 80% over the uninoculated control. TGx gave the highest total N with isolates 102 and 119. Fixed Nitrogen: Percentage N fixed was generally low and ranged from zero for the uninoculated treatments to 52.3%, with an overall average o f 35.9% for the inoculated treatments (Table 4.6). Bragg appeared to be the best fixer; in four out o f the seven inoculation treatments (with strains 38,119,102 & 122), it gave the highest % N fixed, besides giving the highest % N fixed value o f 52.3% when inoculated with Iso-102. Percent nitrogen fixed by Bengbie was highest with strains J2 and J23, giving its highest % N fixed value o f 43.1% when inoculated with Iso-J23. TGx on the other hand recorded the lowest %N fixed values, with values between 24.3 and 33.9%. 66 University of Ghana http://ugspace.ug.edu.gh 4.6 Effect of nitrogen fertilization on nodulation, nitrogen fixation and yield of Bragg, Bengbie and TGx in Bekwai and Adenta soils. Nodulation: Generally and in most cases plants grown in Adenta soil produced significantly more nodules than for the Bekwai soil (Table 4.7). All plants were fairly well nodulated with the overall nodulation being 15% better in Adenta than in Bekwai. Similarly, nodule dry matter,was higher in Adenta than Bekwai in all cases. The nodules in Adenta were generally bigger than in Bekwai (data not presented). Also, apart from Bragg and TGx fertilized at 10 kgN/ha whose nodule dry matter values were not significantly different, nodule dry matter values were significantly different among thecultivars in the rest o f the cases. At 10 kg N/ha rate o f fertilizer application, nodule dry matter values were significantly higher than at 100 kg N /ha in both soils (Table 4.7). Shoot dry matter: In contrast to nodulation, shoot dry matter produced in Bekwai was significantly higher than in Adenta, with the highest produced by the non-nodulating (Non-nod) soybean plant at 100 kg N/ha fertilizer rate (Table 4.8). The increase in shoot dry matter o f the non-nodulating soybean with the higher rate o f fertilization was 185.7 % more than what pertained in Adenta. There were no significant differences in the shoot dry matter yield among the nodulating cultivars in Bekwai with the 67 University of Ghana http://ugspace.ug.edu.gh Table 4.6. Inocultion effects of seven soybean Bradyrhizobium isolates on three soybean cultivars, Bragg. Bengbie and TGx in Bekwai soil. INOCULUM / VARIETY Isolate 38 Bragg Bengbie TGx Mean Isolate 119 Bragg Bengbie TGx Mean Isolate 129 Bragg Bengbie TGx Mean * Continued on next TOTAL N/PLANT 83.0 73.1 75.1 77.1 66.4 61.5 74.6 67.5 64.1 67.5 74.6 67.5 page %N FIXED/PLANT 48.0 34.3 31.1 37.8 36.8 33.5 28.2 32.8 25.7 28.9 28.0 27.5 N FIXED (mg) 35.1 25.1 23.4 27.9 24.6 19.0 21.1 21.6 16.2 22.0 18.1 18.8 68 University of Ghana http://ugspace.ug.edu.gh INOCULUM / TOTAL N/PLANT %N FIXED/PLANT N FIXED (mg) VARIETY Isolate 102 Bragg 67.3 52.3 35.2 Bengbie 63.7 36.8 23.3 TGx 87.3 33.9 29.7 Isolate J2 Bragg 59.1 25.1 14.1 Bengbie 69.9 41.8 26.6 TGx 63.7 32.2 20.5 Mean 64.2 30.0 20.7 Isolate J23 Bragg 61.3 31.4 19.3 Bengbie 74.3 43.1 30.3 TGx 63.8 25.9 19.0 Mean Isolate 122 Bragg 60.2 34.3 20.6 Bengbie 59.6 26.4 13.6 TGx 62.3 24.3 15.1 Mean 60.7 28.3 16.4 * Continued on next page 69 University of Ghana http://ugspace.ug.edu.gh INOCULUM / TOTAL N/PLANT %N FIXED/PLANT N FIXED (mg) VARIETY Uninoc. control Bragg 59.8 Bengbie 48.4 TGx 55.9 Mean 54.7 LSD (0.05) 7.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.0 5.7 exception o f Bragg at 100 kg N/ha rate, which was significant. The trend in Adenta was similar to that o f Bekwai with only TGx producing significantly higher yield at the lower fertilizer rate over the higher rate. In both soils however the non-nods produced significantly higher yields than at 100 kg N/ha with more pronounced yield differences in Adenta. Among the cultivars there were no significant differences observed in yield in both soils at both rates o f fertilizer application except the non-nod, which was significantly lower than all the other cultivars in Adenta. Non-Nod gave significantly higher yield than TGX in Bekwai at 100 kg N/ha. 70 University of Ghana http://ugspace.ug.edu.gh Nitrogen fixation: Percent nitrogen fixed by plants in the Adenta soil (Table 4.6) was significantly higher than in Bekwai in all cases apart from Bragg fertilized at 100 kg N/ha rate. Percent N fixed was significantly higher at 10 kgN/ha rate than at 100 kg N /ha with the highest given by Bragg which registered over 2.5 times more %N fixation at 10 kg N /ha than at 100 kg N /ha in Adenta Table 4.7 Number and drv weightfmgVolant of nodules formed by Bragg, Bengbie and TGx in Bekwai and Adenta Soils Fertilized with 10 and 100 kg N/ha. Nitrogen Rate (kg/ha.) 10 kg/ha. 100 kg/ha. Bekwai Adenta X Bekwai Adenta X Bragg 25.5 33.0 29.0 7.5 7.5 7.5 (80) (91) (85.5) (26.5) (55) (40.8) Bengbie 22.0 34.5 28.0 8.5 12.5 10.5 (70) (87) (78.5) (20.5) (64) (42.3 TGx 34.5 25.0 30.0 12.5 15.0 14.0 (101) (103) (102) (21.5) (77) (49.3) X 27.0 (83.7) 31.0 (93.7) 9.5 (22.8) 12.0 (65.3) LSD (nodule numbers) = 5.5 LSD (nodule dry wt.) = 7.8 Numbers in brackets represent nodule dry weight (mg/plant) 71 University of Ghana http://ugspace.ug.edu.gh Table 4.8 Shoot Drv Matter Yield/plant (g) of Bragg. Bengbie and TGx in Bekwai and Adenta Soils Fertilized with 10 and 100 kg N/ha 10 kg/ha 100 kg/ha Bekwai Adenta X Bekwai Adenta _x Bragg 3.07 1.92 2.5 3.45 2.1 2.8 Bengbie 3.27 2.0 2.7 3.42 1.89 2.7 TGX 2.99 1.8 2.4 3.2 2.14 2.7 Non-nod 3.00 1.05 2.0 3.58 2.14 2.9 x" 3.10 1.7 3.4 2.1 LSD(5%): 0.31 Table 4.9 Percent Nitrogen Fixed by Bragg. Bengbie and TGx in Bekwai and Adenta Soils Fertilized with 10 lnd 100 kg /ha. Nitrogen Rate (kg/ha.) 10 kg/ha. 100 kg/ha. Bekwai Adenta X Bekwai Adenta X Bragg 48.2 64 56.1 22.4 24.4 23.4 Bengbie 33.3 56.9 45.1 14.8 30.2 22.5 TGx 29.7 54.8 42.3 13.0 34.9 24.0 X 37.1 58.6 16.7 29.8 LSD (0.05) 5.07 72 University of Ghana http://ugspace.ug.edu.gh Among the cultivars Bragg gave significantly higher percent nitrogen fixed values than Bengbie and TGx; the differences in % N fixed between Bengbie and TGx were not significant. Soil x cultivar x fertilizer interaction for amount o f N fixed was not significant. However the two-way interactions between fertilizer and soil, cultivar and soil as well as cultivar and fertilizer were all significant. Amount o f N fixed by plants grown in both soils at 10 kgN/ha was significantly higher, being about two times more than the amount fixed at 100 kg N/ha (Table 4.9). Percent nitrogen fixed in all the soybean cultivars was significantly higher in Adenta soil than in Bekwai Differences in total N accumulated in the plants were not significant for soil x fertilizer x cultivar interaction as well as cultivar x soil interaction. Cultivar x fertilizer and fertilizer x soil interactions were however significant. Total N accumulated in plants followed a similar trend as % N fixed (Table 4.10). Table 4.10 Amount of fixed and total N accumulated by Bragg, Bengbie and TGx in Bekwai and Adenta Soils Fertilized with 10 and 100 kg N/ha . Total Nitrogen/plantfmg) Fixed Nitrogen/plantfmg) Bekwai Adenta Bekwai Adenta Bragg 75.1 40.0 26.4 17.4 Bengbie 76.6 36.3 18.5 15.8 TGx 75.8 40.7 16.1 17.8 Non-nod 66.8 29.3 0 0 LSD(5%) 6.2 LSD(5%) 3.05 SxCxF=ns SxF=s SxC -=s CxF=s 73 University of Ghana http://ugspace.ug.edu.gh Table 4.11 Total N accumulated by Bragg. Bengbie and TGx in Bekwai and Adenta soils fertilized with 10 andlOOkgN/ha. Bekwai Adenta 10 kg N/ha 100 kg N/ha 10 kg N/ha) 100 kg N/ha Bragg 74.2 76.0 38.2 57.3 Bengbie 76.3 TGx 76.3 77.0 75.3 36.1 40.4 36.5 40.9 Non-Nod 63.1 70.6 20.5 38.1 X 74.5 LSD (0.05) = 8.7 74.7 33.8 43.2 S xCxF =ns SxC =ns S=Soil C=Cultivar F=Fertilizer SF= s C xF=s s=Significant ns = not significant Total nitrogen in plants was generally higher in Bekwai than in Adenta, (86.4 to 128.2% higher) with the highest difference in total N in the plants recorded by non-nodulating plants and the lowest by TGx. In both soils there were no significant differences among Bragg, Bengbie and TGx, which were all significantly higher than values for the non-nodulating plants (Tables 4.10 and 4.11). 74 University of Ghana http://ugspace.ug.edu.gh In Bekwai, total N values were not significantly different among the cultivars, which were significantly higher than the non-nodulating plants at 10 kg N/ha but not at 100 kg N/ha. In Adenta, total N values were also not significantly different among the cultivars which were all significantly higher (76.1-97% higher) than the total N values o f the non-nodulating plants. At, 100 kg N /ha however, Bragg gave significantly higher total N over the other cultivars, including the non-nod plants. There were no significant differences between Bengbie, TGx and Non-nod at the 100-kg N/ha rate. . Although soil x fertilizer x cultivar interaction was generally not significantly (P<0.05), (Table 4.10) total N values in Bragg and Non-nod were significantly higher at 100 kg N/ha than at 10 kg. Total N accumulated in the plants was significantly different in the two soils. Comparing total N at both fertilizer rates, there were significant significant differences between the soybean genotypes in Adenta but not in Bekwai. Nitrogen fixed was significantly different in the soils at 10 kg N/ha.but not significant at 100 kg N/ha. In both soils N-fixed at 10 kg N /ha was significantly different and more than double the values obtained at 100 kg N /ha (Tables 4.10 and 4.11). 75 University of Ghana http://ugspace.ug.edu.gh 5.0 CHAPTER FIVE DISCUSSION, SUMMARY AND RECOMMENDATION. 5.1 Introduction Studies were carried out to assess the nodulation potential o f six soybean cultivars in some uninoculated Ghanaian soil types, and to estimate the populations o f naturally occuring bradyrhizobia present in the soils, and their efficiency in nitrogen fixation. Experiments were in addition performed to assess the response o f soybean to bradyrhizobial inoculation and how this affects nitrogen fixation. 5.2. Nodulation potential of soybean in Ghanaian soils. Nodule number and mass have often been employed for indirect assessment o f nitrogen fixation (Weber, 1966; Westermann and Kolar, 1978). The results obtained from the studies showed that o f the eight soils, it was in only three o f these that no nodules were formed by any o f the soybean cultivars, and each cultivar was nodulated, at least in one o f the soils. What was perhaps interesting was the fact that Bragg a non- promiscuous American soybean genotype, from which several supernodulators have been derived, nodulated considerably well in these soils, an observation contrary to some well established reports in the literature that non-promiscuous American soybean genotypes do not normally nodulate in tropical soils (Pulver et al., 1978). However, nodulation o f another non- promiscuous American genotype, Davis, was poor, which o f course confirms the well- acclaimed view in the literature. 76 University of Ghana http://ugspace.ug.edu.gh The most probable number (MPN) count was carried out to estimate the population o f indigenous soybean bradyrhizobia present in the soils (Vincent, 1970), and also served as an indirect means o f predicting whether or not soybean would respond to inoculation (Thies et. al, 1991). The results obtained were indicative o f variability o f bradyrhizobia population in the soils. This is a confirmation o f Abaidoo’s (1997) observation that, bradyrhizobia population in African soils (by the MPN technique) was variable and ranged from 2 x 10° to 3.38 x 104 bradyrhizobia g '‘soil. Similar studies by Fening (1999) showed that average density o f indigenous bradyrhizobia capable o f nodulating cowpea varieties in Ghanaian soils varied within and between the localities for the study. The fact that ha lf o f the soils, hitherto not cropped to soybeans, contained large enough population to enable them to infect soybean is not unusual since Vincent (1980) reported o f the presence o f rhizobia even in virgin soils. This, perhaps indicates that either bradyrhizobia occur naturally as part o f the indigenous soil population or that other native legumes serve as hosts and thus inoculant sources o f bradyrhizobia for soybean. However the fact that Bekwai and Nzima soils contained less than 50 rhizobia per gram o f soil seems to confirm the view held by Cuttelan and Hungria (1994) that, where soybean has not been previously grown, there is generally a response to inoculation with bradyrhizobia especially for the non-promiscuous cultivars. 77 University of Ghana http://ugspace.ug.edu.gh The results from the screening experiment and MPN studies seemed to suggest some relationship between nodulation and bradyrhizobial numbers, from the viewpoint that soils which supported nodulation generally had higher bradyrhizobia counts, while those in which nodulation did not occur had low bradyrhizobia count. This indicates that nodulation and nitrogen fixation o f soybean can be improved significantly through inoculation with bradyrhizobia in different soils. Counts o f bradyrhizobia capable o f nodulating soybeans depicted that ha lf o f the soils studied contained reasonably large enough populations to enable them nodulate soybean well. This view is arrived at based on Danso’s (1992) assertion that population range o f 103 to 104 rhizobia per gram o f soil was by most standards adequate for nodulation to occur in food legumes. Interestingly, Anyinase which recorded zero nodulation during the screening studies had substantial bradyrhizobia count, giving the indication that in spite o f the fair amount of bradyrhizobia population present in Anyinase, conditions prevailing in the soil may not have been conducive for symbiotic compatibility between the legume and the microsymbiont. It is suggested that factors such as low pH and aluminium toxicity might be partly responsible for the zero nodulation score in Anyinase. Agronomic practices such as liming may greatly improve the nodulation potential in Anyinase. Bekwai and Nzima soils had very low bradyrhizobia counts, which may explain why there was no nodulation in these two soils, giving the indication that a yield response to inoculation could be obtained in these soils. 78 University of Ghana http://ugspace.ug.edu.gh 5.3 Cross inoculation. The cross inoculation group concept is based on the ability o f rhizobia to specifically nodulate a limited group o f legume host species (Fred et. al. 1932). It is based on this concept that Rhizobium species have been classified as promiscuous or specific. The concept was therefore applied in this study to determine the symbiotic specificity or otherwise o f native B. japonicum obtained from the screening experiment. The results showed that large number o f indigenous soybean bradyrhizobia had the potential o f nodulating cowpea and groundnut, two common legume genera cultivated in Ghana. The results suggest that these bradyrhizobia that nodulated soybean were not highly specific. The possibility that most o f thee bradyrhizobia belong to the Bradyrhizobium spp. rather than B. japonicum needs to be investigated. It would be interesting to do further work to establish, among other things, the relative effectiveness and competitive abilities o f these isolates on these hosts. 5.4 Symbiotic effectiveness. Symbiotic effectiveness is a primary factor for the determination o f incidence and magnitude o f legume response to inoculant production (Singleton and Travers, 1986, Thies et. al., 1991) The symbiotic effectiveness test was carried out to determine which o f the indigenous isolates were efficient in nitrogen fixation. Symbiotic effectiveness o f indigenous rhizobia is necessary for assessing need for inoculation, and also an important parameter for the selection o f the strains for inoculant production (Singleton and Travers, 1986; Thies et. al. 1991). 79 University of Ghana http://ugspace.ug.edu.gh The results indicated that only 15% o f the isolates were highly effective relative to the uninoculated control, 20% were moderately effective and 65% ineffective. This was not a drastic departure from the normal distribution pattern reported in a similar study by Singleton and Stockinger (1982). Also the results indicated that soybeans were nodulated by bradyrhizobia strains which were largely ineffective and thus in nitrogen fixation. However, the fact that some considerable number o f the isolates was effective means that the latter could, when selected, be o f immense use for inoculation, nitrogen fixation enhancement and inoculant production. Even though this requires further work and information, if this goal is achieved, it stands to benefit from comparative advantage in the sense that once the isolates are o f native origin, they are likely to adapt better to local conditions and may have a better competitive edge over imported inoculants. Further studies to assess what proportions of effective versus ineffective segments o f the isolates are specific for soybean, or which o f these belong to the cowpea miscellany and are only opportunistic nodulators o f soybean would be interesting. 5.5 Response of soybean to inoculation Nodulation is a primary requirement for nitrogen fixation in legumes, and particularly for a soil such as Bekwai where earlier screening studies had established no nodulation due to very low and rather inadequate bradyrhizobia count. The need to inoculate such soils cannot be over emphasized. Results obtained from the inoculation studies showed that all the inoculated plants nodulated and had fairly reasonably good nodule numbers whiles there was no nodulation in the uninoculated control, a first signal that perhaps inoculation had led to nitrogen fixation (Weber, 1966; Westermann and Kolar, 1978). 80 University of Ghana http://ugspace.ug.edu.gh Although, generally, inoculation with any o f the isolates produced significantly more shoot dry matter than the uninoculated control in all the three cultivars, the levels differed from cultivar to cultivar. In some instances, comparing the performance o f the uninoculated control with the isolates in one cultivar there were no significant differences. The implication is that, even though on the whole inoculation led to improvement in shoot dry matter, the levels were not the same among the cultivars. Several reports have shown that nitrogen fixed by a Rhizobium strain is strongly influenced by the host plant (Graham and Rosas 1977; Hardarson et. al. 1984), and that nitrogen fixation supporting traits often vary among different hosts. For example, while Bengbie produced the highest dry matter with four isolates, the same could not be said o f the rest o f the isolates even with the same cultivar. The other cultivars had trends quite different from Bengbie. This is an indication o f strain genotype interaction where the response for a strain was quite pronounced in some cultivars, others minimal, and yet others did not give significant responses. This suggests that, much as the bradyrhizobial isolate is considered to be very important in inoculation response, the plant genotype equally and significantly influences inoculation response, even though this consideration is often much overlooked (Pulver et. al. 1978; Awonaike et. al., 1990). The three cultivars again showed differences in total percent nitrogen fixed as well as in amounts o f nitrogen fixed. Again, the highest nodulation o f each of the three cultivars was by a different strain, emphasising differences in strain preference to their host plants and specificity differences; a phenomenon which might be o f prime importance in the screening and selection o f bradyrhizobial strains for soybean yield improvement. Another important consideration that emerged from the studies is that the two promiscuous genotypes seem to have responded to inoculation even better than the non-promiscuous 81 University of Ghana http://ugspace.ug.edu.gh genotype (Bragg). This seems to be in contrast to well established opinion that non-promiscuous genotypes have more propensity for responding to inoculation in the tropics because native bradyrhizobia are not capable o f infecting them; promiscuous genotypes in contrast are infected by native strains and hence may not readily respond to inoculation treatment (Rhodes and Nangju, 1979; Nangju, 1980). The results obtained are however in consonance with the observation made by Olufajo and Adu in 1992 that, significant responses could be obtained in promiscuous soybean genotypes on soils with low population o f indigenous bradyrhizobia.. The fact that Bengbie, (a locally cultivated soybean genotype) responded greatly to inoculation, rising from the lowest when not inoculated to highest when inoculated with J23, J2 and 102; does not seem to conform to the view held by Pulver et. al. (1982) that, there is no significant response to inoculation by local soybean cultivars because o f their incompatibility with B. japonicum strains and the presence o f more compatible indigenous bradyrhizobia in African soils. However, inoculation response in TGx was very low, probably confirming the observation made by Kueneman et.al. (1984) that the marginal response o f adapted soybean cultivars to B. japonicum inoculation was indicative o f the fact that Bradyrhizobium spp were capable of maintaining high soybean yields without inoculation. It has to be stated that the imported isolates from Thailand performed very well and were comparable to some o f the local isolates, probably because, these Thai strains are also o f tropical origin, and may thus easily adapt to the local conditions (Chowdhury, 1977; Awai, 1981). Perhaps this observation is a confirmation o f the observation made by Olufajo and Adu (1992). 82 University of Ghana http://ugspace.ug.edu.gh Finally, it might be worthy to mention here that, though there was some appreciable inoculation response which resulted in higher nitrogen fixation, the response levels seemed to be quite below expectation. Hence there might have been some environmental as well as unidentified factors, which may have hampered the realization o f the full potential o f the isolates. Temperature and antagonistic effects by some microorganisms might be culprit in this regard. Also according to Rennie (1982) N fixation o f soybean globally is about 50%, therefore lower N fixed in the study may also be attributed probably to the high presence o f ineffective rhizobia (65%) in the soils used for the studies, especially if the ineffective strains are highly competitive. 5.6 Response o f soybean to inoculation and nitrogen fertility. The advantage in using i5N is in its ability to distinguish between sources o f N, and thus makes it possible to distinguish the amount on fixed nitrogen in the plant from soil and fertilizer- derived N (Fried et. al., 1983). The response o f soybean to nitrogen fertilizer as measured was the net effect o f nitrogen uptake and nitrogen fixation over the growing period. The fact that nodulation and percent nitrogen fixed were generally better in Adenta than in Bekwai at both rates o f fertilizer application was not unexpected because o f the presence o f substantially more numbers o f soybean bradyrhizobia in Adenta soil than in Bekwai. Nodulation was also better at 10 kg N/ha than at 100 kg N /ha o f fertilizer application, which is in conformity with reports that high rates o f inorganic N fertilizer inhibit or have a depressing effect on nodulation and subsequently nitrogen fixation (Hardarson et. al., 1984; Rennie and 83 University of Ghana http://ugspace.ug.edu.gh Kemp, 1983). In fact Carroll and Gresshoff (1983) explained that high external concentrations of nitrogen inhibit root infection by rhizobia. N accumulation in Adenta was largely from fixation and not from soil, thus significant differences in total N and yield reflected variability in nitrogen fixing abilities o f the soybean genotypes. However, for Bekwai, fixation was low, compared to uptake from soil and thus differences in nitrogen fixation had less variable effect. At both rates o f nitrogen application total nitrogen in Bekwai was higher than in the Adenta soil suggesting that perhaps soil chemical properties could have had a role to play in nitrogen accumulation and nitrogen fixation in the soybean plants. Adenta is known to be poor in terms of soil fertility (Table 3.2). Even though nodulation and percent nitrogen fixed were generally higher in Adenta, the actual amount o f nitrogen fixed as well as total nitrogen accumulated in the soybean plants in Bekwai soils was higher than Adenta. This implies that BNF may not have been optimal enough to supply all the nitrogen needed in Adenta soil, in addition to the possibility o f chemical fertilizer being more limiting in Adenta. On other hand, there could have been factors inherent in Bekwai that might have enhanced plant growth and hence total nitrogen fixed. Soil nitrogen, organic matter and phosphorus support dry matter yield, which are more favoured by Bekwai against Adenta (Table 3.2). Observation o f variability in cultivar response to inoculation, fertilization and perhaps soil effects is an indication that inoculation, fertilizer and soil response are influenced by plant genotype. 84 University of Ghana http://ugspace.ug.edu.gh 5.7 SUMMARY Enumeration o f bradyrhizobia capable o f nodulating soybeans showed that ha lf o f the soils studied contained large enough populations to enable them nodulate sobeans well. This was confirmed by the screening test on soybean cultivars, where both promiscuous and non- promiscuous soybean genotypes were nodulated reasonably well by native soybean bradyrhizobial strains in these four soils. Randomly selected native bradyrhizobial isolates from the screening studies used to cross nodulate groundnut and cowpea showed that large number o f the native bradyrhizobia capable of nodulating soybean also had the potential for nodulating cowpea and groundnut, two commonly cultivated legumes in Ghana. Effectiveness test performed on 60 randomly selected native bradyrhizobia isolates showed that 15% of the isolates were highly effective, 20% moderately effective and 65% ineffective. The inoculation studies gave an indication o f improvement in nodulation, nitrogen fixation and improved shoot dry matter yield by the native and a few imported standard soybean bradyrhizobia strains from Thailand; with the native isolates performing as well as their imported counterparts. The promiscuous soybean genotypes seemed to have responded to inoculation even better than the non-promiscuous genotypes. Nodulation and %N fixed were generally better in Adenta soil series than in Bekwai series at the two rates o f fertilizer application, probably due to the substantially higher numbers o f soybean 85 University of Ghana http://ugspace.ug.edu.gh bradyrhizobia in Adenta soil than in Bekwai. Nodulation was also better at 10kg N/ha than at 100kg N/ha o f fertilizer application in conformity with well documented reports (Hardarson et. al., 1984). Total N in plants grown in Bekwai was however significantly higher than in the Adenta soil, probably due to the fact that Bekwai is more fertile than Adenta. 86 University of Ghana http://ugspace.ug.edu.gh 5.8 RECOMMENDATION The studies gave a general overview o f the presence and behaviour o f soybean bradyrhizobia present in some Ghanaian soils, and there is the need to do follow up studies to characterise bradyrhizobia into cowpea bradyrhizobia and soybean bradyrhizobia (Bradyrhizobium japonicum ), and to study their performance in nodulation and nitrogen fixation in soybean, cowpea and other related legumes. Further work needs to be done to ascertain among other things, the effectiveness and competitive ability o f bradyrhizobia strains before they could be used for inoculant production. Also further studies to assess what proportions o f effective versus ineffective segments o f the isolates are specific for soybean or which o f these belong to the cowpea miscellany and are only opportunistic nodulators o f soybean would be worth looking at. The levels o f inoculation response and nitrogen fixation were quite lower than expected, due probably to environmental factors (such as temperature) and other soil factors including antagonistic effects o f other soil microorganisms. There is also the possibility that the preponderance o f ineffective strains in the soil may have impeded nodulation by the inoculant strains which calls for competition studies as mentioned ealier. The other factors also need to be investigated to further enhance the nitrogen fixing ability o f soybean bradyrhizobia. 87 University of Ghana http://ugspace.ug.edu.gh Finally, it is my expectation that the results and further work would provide the basis and impetus for inoculant production in Ghana and the West Africa sub-region as a whole to help alleviate the protein malnourishment prevalent in the aforementioned areas through the consumption o f soybean and other soya-products. University of Ghana http://ugspace.ug.edu.gh REFERENCES Abaidoo, R.C. 1997. Diversity within indigenous bradyrhizobia populations that nodulate soybeans in Africa. (DoctorateDissertation Univ. o f Hawaii, Hawaii.) Alexander, M. 1965. Most-probable-number method for microbial populations. In : Methods o f Soil Analysis, Part 2. Chemical and Microbiological Properties, ed. Black, C.A., Evans, D.D., White, J.L., Ensminger, L.E. & Clark, F.E. pp. 1467-1472. Am. Soc. o f Agron., Inc., Madison, Wisconson: Alexander, M. 1971. Introduction to Soil Microbiology. John Willey & sons. Alexander, M. 1977. Introduction to Soil Microbiology. John Willey & sons. Alios, H.F., and Batholomew, W.V. 1959. Replacement o f symbiotic fixation by available N. Soil Sci. 87: 61-66. Amarger, N., Mariotti, A., Mariotti, F., Dorr, J.C., Bourguignon, C., and Lagacheri, B. 1979. Estimate o f symbiotically-fixed nitrogen in field-grown soybeans using radiations in the I5N natural abundance. Plant Soil 52: 269-280. Anderson, A.J., 1956. Effect o f fertilizer treatments on pasture growth. Proc. 7th Int Grassl. Congr. 323-333. Annan V. 1998. Inoculation response o f two soybean varieties in two Ghanaian soils. (B.Sc. Dissertation. Univ. o f Ghana, Legon). Anon, 1975. Root activity patterns o f some tree crops. Results o f a Five-year Co-ordinated Research Programme of Joint FAO/IAEA Division o f Atomic Energy in Food Agriculture. STI/DOC/10/170. IAEA, Vienna. University of Ghana http://ugspace.ug.edu.gh Anon, 1982.' International. Institute o f Tropical Africa Annual Report for 1981. 99. 76-80. Ibadan, Nigeria. Anon. 1993 Validation o f the publication o f new names and new combinations previously effectively published outside the IJSB. Int. J. Syst. Bacteriol. 43: 398-399. Anon. 1996. The Rhizobium ecology network o f East and Southern Africa (RENEASA) Phase II Activities: Legume inoculation response and farmer perceptions o f nitrogen fixation and legume inoculants. (Prepared by P.L. Woomer and N.K. Karanja). Technical report o f the Rhizobium Ecology Network o f East and Southern Africa. Rockefeller Foundation Grantl 994-0020-0042 Awai, U. 1981. Inoculation o f soybean in Trinidad. Trop. Agric. (Trinidad) 58: 313-319. Awonaike, K.O., Kumarasinghe, K.S., and Danso, S.K.A. 1990. Nitrogen fixation and yield o f cowpea (Vigna unguiculata) as influenced by cultivar and Bradyrhizobium strain. Field Crops Res., 24: 163-171. Awuku, K.A., Brese, G.L., Fosu, G.K and Baidu, S.O. 1991 Agricultural and Environmental Studies for S.S.S.: Soybean. Min. o f Education, Accra, pp. 112. Ayanaba, A. 1977. Towards a better use o f inoculants in the humid tropics. In A. Ayanaba and P.J.Dart (ed.) Biological Nitrogen Fixation in Farming Systems o f the Tropics pp 181- 187. John Wiley & Sons. New York, Ayanaba, A. and Nangju, D,, 1973. Nodulation and nitrogen fixation in six grain legumes. In: Proceedings o f the First IITA Grain Legume Improvement Workshop. Pp. 198-204. International Institute o f Tropical Agriculture. Barnet, Y. M. 1980. The effect o f rhizophages on population o f Rhizobium trifolii in the root zone o f clover plants. Canadian Journal o f Microbiology, 26: 527-576. 90 University of Ghana http://ugspace.ug.edu.gh Batholomew, W.V. 1977. Soil nitrogen changes in farming systems in the humid tropics. In Biological Nitrogen Fixation in Farming Systems o f the Tropics. Pp. 27-42. A. Ayanaba and P.J. Dart (ed.). John Wiley & Sons, New York. Bergersen, F. J.T. Brockwell,J. Gibson, A. H. and Schwinghamer, E. A. 1971. Studies on natural populations and mutant o f Rhizobium in the improvement o f legume inoculants. Plant Soil Special Volume 3-16. Bauer, W. D. 1981. Infection o f legumes by rhizobia. Ann. Rev. PI. Physiol. 32: 407-449 Boonkerd, N., and Weaver, R.W. 1982. Survival o f cowpea rhizobia in soil as affected by soil temp, and moisture. Applied and Environmental Microbiology 43: 585-589. Borkert C.M. and Sfredo G.J. 1994. Fertilizing tropical soils for soybean. In: Tropical Soybean Improvement and Production; Brazilian Agricultural Research Enterprise. F.O.A. o f the United Nations. Rome. Bowen, G.D., and Kennedy, M.M. 1959. Effect o f high soil temperature on Rhizobium spp. Qld. J. Agric. Sci. 16: 177-197. Broadbent, F.E., Nakashima, T.and Chang, G.Y. 1982. Estimation o f nitrogen fixation by isotope dilution in field and greenhouse experiments. Agron. J. 74: 625-628. Brockwell, J., Gault, R.R., Chase, D.L., Hely, F.W., Zorin and Corbin, E.J. 1980. An appraisal o f practical alternatives to legume seed inoculation: Field experiments on seed bed inoculation with solid and liquid inoculants. Aust. J. Agric. Res. 31: 47-60 Bromfield, E.S.P., and Ayanaba, A. 1980. The efficacy o f soybean inoculation on acid soil in tropical Africa. Plant and Soil 54: 95-106. 91 University of Ghana http://ugspace.ug.edu.gh Bromfield, E.S.P and Barran, L. R. 1990. Promiscuous nodulation o f Phaseolus vulgaris, Macroptilum atropurpureum and Leucaena leucocephala by indigenous Rhizobium meliloti. Canadian Journal o f Microbiology. 36: 369-372. Broughton W.J. and Dilworth M. 1970 N-free nutrient solution. In: Methods in Legume- Rhizobium Technology. Bumb, B. L. 1994. World nitrogen supply and demand: An overview. In P.E. Bacon (ed.). Nitrogen Fertilization and the Environment. Marcel Dekker Inc., New York, USA. Bums, R. C., and Hardy, R. W. F. 1975. Nitrogen Fixation in Bacteria and Higher Plants. Springer-Verlag, New York. Burton, J.C. 1972. Nodulation and symbiotic N fixation. In Hanson CH ed.. Alfalfa Sci. and Tech. ASA. Monograph No. 15. Am. Soc. o f Agron. J.66: 229-232. Burton, J.C., and Allen, ON . 1949. Inoculation o f crimson clover with mixtures o f rhizobia strains. Soil Sci. Soc. Am. Proc. 14: 191-195. Calwell, B. E. and Vest, G. 1970. Effects o f Rhizobium japonicum strains on soybean yields. Crop Sci. 10: 19-21. Carroll, B J . and Gresshoff, P.M. 1983. Nitrate inhibition o f nodulation and nitrogen fixation in white clover.2. Planzenphysiol.l 10: 77-88. Cattelan A.J., and Hungria M. 1994. Nitrogen nutrition and inoculation. In: Tropical Soybean Improvement and Production; Brazilian Agricultural Research Enterprise. F.O.A. o f the United Nations. Rome Chaiwanakupt, P,. Siripaibool, C., and Snitwonsgse, P. 1991. Evaluation o f the appropriate non-N2-fixing crops to quantify nitrogen fixation by soybean using the l5N isotope 92 University of Ghana http://ugspace.ug.edu.gh dilution method. In: Proceedings Symposium on Stable Isotopes in Plant Nutrition, Soil Fertility and Environmental Studies, pp 89-99. STI/PUB/845, IAEA, Vienna. Chalk, P.M. 1985. Estimation o f N2 fixation by isotope dilution: An appraisal o f techniques involving !5N soil enrichment and their application. Soil Biol. Biochem. 17: 389-410. Chen, M., and Alexander, M. 1971. Resistance o f soil microorganisms to starvation. Soil Biol. Biochem. 4: 283-288. Chowdhury, M.S. 1977. Response o f soybean to Rhizobium inoculation in Morogoro, Tanzania. In: Biological Nitrogen Fixation in Farming Systems o f the Tropics, pp. 27-42. A. Ayanaba, P.J. Dart (ed.). John Wiley & Sons, New York. Dakora, F.D. 1977. Effect o f inoculum size on inoculation and nitrogen content o f five selected grain legumes in two Ghanaian soils. (B.Sc. Dissertation. Univ. o f Ghana, Legon.) Danso, S.K.A.1977. The ecology o f Rhizobium and recent advances in the study o f the ecology o f Rhizobium. In Ayanaba A. and Dart P.J. (Editors), Biological Nitrogen Fixation in Farming Systems o f the Humid Tropics.pp. 115-125. John Wiley & Sons, Chichester, pp 115-125. Danso, S.K.A. 1985. Methods o f estimating biological nitrogen fixation. In: Ssali H and Keya S.O. (eds). Biological Nitrogen Fixation in Africa pp. 213-244.MIRCEN, Nairobi. Danso, S.K.A. 1986 Review: Estimation o f N2-fixation by isotope dilution: An appraisal of techniques involving N enrichment and their application-comments. Soil Biol. Biochem. 18:243-244. Danso, S.K.A. 1988, Nodulation of soybean in an acid soil: The influence o f Bradyrhizobium inoculation and seed pelleting with lime and rock phosphate. Soil Biol. Biochem. 20: 259-260 93 University of Ghana http://ugspace.ug.edu.gh Danso, S,K.A.,1991 Natural and artificial methods o f 15N labelling o f soil to estimate biological nitrogen fixation: Review o f symposium papers. In: Proceedings Symposium on Stable Isotopes in Plant Nutrition, Soil Fertility and Environmental Studies, pp. 89-99. STI/PUB/845, IAEA, Vienna. Danso, S.K.A. 1992. Biological nitrogen fixation in tropical agrosystems: Twenty years of biological nitrogen fixation reseach in Africa. In Biological Nitrogen Fixation and Sustainability o f Tropical Agriculture pp. 3-13. A Wiley-Sayce Co-Publication. Danso, S.K.A. 1995. Assessment of biological nitrogen fixation. In Fertilizer Res. 42:33-41 Danso, S.K.A., and Alexander, M. 1974. Survival o f two Rhizobium strains in soil. Soil Science o f America Proceedings 38: 86-89. Danso, S.K.A., Bowen, G.D., and Sanginga, N.1992. Biological nitrogen fixation in trees in agro-ecosystems. Plant Soil 141: 177-182 Danso, S.K.A., Hardarson, G., and Zapata, F.1986. Assessment o f dinitrogen fixation potentials o f forage legumes with ISN technique. In: Proc. o f a Workshop on Potentials o f Forage Legumes in Farming Systems o f Sub-Saharan Africa Eds. I Haque, S Jutzi and PJH Neate (eds.). Pp. 26-57 ILCA, Addis Ababa, Ethiopia. Danso, S.K.A., Hardarson, G. and Zapata, F. 1993. Misconception and practical problems in the use o f 15N soil enrichment techniques for estimating N2 fixation. Plant Soil 152: 25-52. Danso, S.K.A., Keya, S. O., and Alexander, M. 1975. Protozoa and the decline o f Rhizobium populations added to soil. Canadian Journal o f Microbiology, 21: 884-895. 94 University of Ghana http://ugspace.ug.edu.gh Danso, S.K.A., and Owiredu, J.D. 1988. Competitiveness o f introduced and indigenous cowpea Bradyrhizobium strains for nodule formation on cowpeas ( Vigna unguiculata[L] Walp) in three soils. Soil Biol. Biochem. 20: 305-10. Dart, P.J., and Mercer, F.V. 1965. The effect o f growth temperature, level o f ammonium nitrate, and light intensity on the growth and nodulation o f cowpea ( V sinensis Endl. Ex. Hassk.) Aust. J. Agric. 13: 129-142. Dart, P.J., and Wilson, D.C. 1970. Nodulation and nitrogen fixation by V. sinensis and Vicia atropurpurea: The influence o f condition, form, and site o f application o f combined N. Aust. J. Agric. Res. 21: 45-56. Date, R.A. 1975 Inoculation o f tropical pasture legumes. In: J.M Vincent, A.S. Whitney and J. Bose (Eds.) Exploiting the Legume-Rhizobium Symbiosis in Tropical Agriculture, pp. 293-311. Univ. Hawaii, Honolulu. Dart, P.J. 1977. Infection and development o f leguminous nodules. In: Hardy, R. W. F., Silver, W. S.(eds.). A Treatise on Dinitrogen Fixation. Vol. 3. Wiley Press. New York pp. 367- 472. Dazzo, F.B., and Brill, W.J. 1978. Regulation by fixed nitrogen o f host-symbiont recognition in the Rhizobium-clover symbiosis. Plant Physiol. 62: 18-21. Dazzo, F.B., Truchet, G., Sherwood, J. E., Hrabark, E.M., Abe, M., and Pankratz, S.H. 1984. Specific phases o f root hair attachment in the Rhizobium trifolii clover symbiosis. Appl. Environ. Microbiol. 48: 1140-1150. de Lajudie, P., Willems, A., Pot, B., Dewettinctk, D., Maestrojunan, G., Collins, M. D., Dreyfus, B., Kersters, K., and Gillis, M. 1994. Polyphasic taxonomy o f rhizobia, 95 University of Ghana http://ugspace.ug.edu.gh emendation o f the genus Sinorrhizobium and description o f sinorrhizobum melliloti comb. nov. Sinorrhizobum saheli sp.nov. Sinorrhizobum teranga sp. nov. Int. J. Systematic Bacteriology 44: 715-733. Deibert, E.J., Bijeriego, M., and Olson, R. 1979. Utilization o f fertilizer by nodulating and non- nodulating soybean isolines. Agron. Journal 71: 717-723. Denison, R.F., Weisz, P.R., and Sinclair, T.R. 1983. Analysis o f acetylene reduction rates of soybean nodules at low acetylene concentrations. Plant physiol. 73: 648-651. Dennis, E.A. 1975. Nodulation and nitrogen fixation in legumes in Ghana. In Biological Nitrogen Fixation in Farming Systems o f the Tropics, pp. 27-42. A. Ayanaba, P.J. Dart (ed.). John Wiley & Sons, New York. de Haen, H. 1994. Preface o f Tropical Soybean Improvement and Production.; Brazilian Agricultural Research Enterprise. F.O.A. o f the United Nations. Rome. Dilworth, M. 1966. Acetylene reduction by nitrogen-fixing preparations from Clostridium pasteurianum. Biochem. Biophys. Acta. 127: 285-294. Dixon, R. 1966. Rhizobia with particular reference to relations with the host plants. A. Rev. Microbiol. 23: 137-158. Doku, E.V. 1969. Host specificity among five species in the cowpea-cross inoculation group. Plant Soil 30: 126-128. Dowuona, G, N. N. 1985. Correlation o f the Ghanaian system o f soil classification with other international systems. Dissertation. Univ. o f Ghana, Legon). Dreyfus, B., Garcia, J.l. and Gills, M. 1988. Characterization o f Azorhizobium caulinodans gen. nov., sp. nov., a stem-nodulation nitrogen-fixing bacterium isolated from Sesbania rostrata. Int. J. o f Syst. Bacteriol. 48: 89-98. 96 University of Ghana http://ugspace.ug.edu.gh Duhoux. E., and Dommcrgues, Y. 1985. The use o f nitrogen fixing trees in forestry and soil restoration in the tropics. In Biological Nitrogen Fixation in Africa Ssali H. and Keya S.O. (Eds).pp. 384-400.MIRGEN, Nairobi. Dunham, D.H., and Baldwin, I.L. 1931. Double infection o f leguminous plants with good and poor strains o f rhizobia. Soil Sci. 32: 235-249. Duong, T.P., Djep, C. N., Khiem, N. H., Toi, N.V., and Nhan, L.T.K. 1984. Rhizobium inoculant for soybean Glycine Max.( L) Merril). In: Mekong Delta. I. Response o f soybean to chemical nitrogen fertilizer and Rhizobium inoculant. Plant Soil. 79,241- 247. Duque, F.F., Neves, M.C.P., Franco, R.L., and Boddey, R.M. 1985. The response o f field grown Phaseolus vulgaris to Rhizobium inoculation and the quantification o f N2 fixation using 15N. Plant Soil 88: 333-342. F.A.O. 1978. Production Year Book .50: 99-105 F.A.O. 1984. Production Year Book Fening, J.O. 1997. Diversity and symbiotic characteristics o f cowpea Bradyrhizobium strains in Ghanaian soils. (Doctorate Dissertation, University o f Ghana, Legon.) Fielder, R. and Proksch, G. 1975. The determination o f I5N by mass-spectrometry in biochemical analysis. Anal. Chim. Acta. 78. Foulds, W. 1971. The effect o f drought on tree species o f Rhizobium. Plant Soil 35. 665-667 Foy, C.D., Chaney. R.L., and White, M.C. 1978. The physiology o f metal toxicity in Plants Ann. Review. Plant Physiol. 29: 511-566. Franco, A.A., and Vincent, J.M. 1976. Competition among rhizobial strains for the colonization and nodulation o f two tropical legumes. Plant Soil 45: 27-48. 97 University of Ghana http://ugspace.ug.edu.gh Fred, E.B., Baldwin, I.L., and McCoy, E. 1932. Root nodule bacteria and leguminous plants. University o f Wis. Press, Madison. Fried, M. 1978. In second review meeting I.N.P.U.T.S. Project (S. Ahmed and HPM. Grunasena eds,). pp. 217-224. Fried, M., and Broeshart, H. 1975. An independent measurement o f the amountof nitrogen fixed by a legume crop. Plant Soil 43, 707-711. Fried M; Danso S.K.A. and Zapata F. 1983. The methodology o f measurement o f N2 fixation by non-legumes as inferred from field experiments with legumes. C anJ. Microbiol. 29: 1053-1062. Fried, M., and Middelboe, V. 1977. Measurements o f amount o f nitrogen fixed by legume crop. Plant Soil 43: 713-715. Frobisher, M., Hinsdill, R.D., Crabtree, K.T., and Clyde, R.G. 1974. Fundamentals o f Microbiology, pp. 665-668. W.B.Saunders Company. West Washington Square. Gaur, Y.D. and Lowther, W.L. 1982. Competitiveness and the persistence o f introduced rhizobia on oversown Clover: Influence o f strain, inoculation rate and lime pelleting. Soil Biol. & Biochem. 14, 99-102. Gerahty, N., Caetony-Annolle’s, G., Joshi, P.A.and Gresshoff, P. M. 1992. Anatomical analysis of nodules and additional autoregulatory control point. PI. Sc. 85: 1-7. Gibson, A.H. 1974. Consideration o f legume as a symbiotic association. In Indian Nat. Sci. Acad. Proc. 40B. pp 741-767. Gibson, A.H., and Nutman, P.S. 1960. Studies on the physiology o f nodule formation VII. A reappraisal o f the effect o f preplanting. Ann. Bot. 24: 420-433. 98 University of Ghana http://ugspace.ug.edu.gh Giller, K .E., and Wilson, K.J. 1993. Nitrogen fixing organisms in the tropical cropping systems. CAB International. Redwood Press Melksham, Wiltshire pp. 30-36. Giltner, W., and Langworthy, H.V., 1916. Some factors influencing the longevity o f soil solution. Agr. J. Res. 5: 927-942. Graham P.H. and Rosas, J.C. 1977 Growth and development o f intermediate bush and climbing cultivars o f Phaseolus vulgaris. L inoculated with Rhizobium. Journal o f Agricultural Science. 88: 503-505. Graham P. H., Sadowsky, M., Keyser, H.H., Barnet, Y.M., Bradley, R.S., Cowper, J.E., de Ley, D.J., Jarvis, B.D.W., Roslycky, E.B., Strydom, B.W., and Young, J.P.W. 1991. Proposed minimal standards for the description o f new genera and species o f root and stem nodulating bacteria. Int. J. Syst. Bacterol. 41: 582-587. Ham, G.E. Cardwell, V.B., and Johnson, H.W. 1971. Evaluation o f Rhizobium japonicum inoculants in soils containing naturalized populations of rhizobia. Agron. J. 63: 301-303. Haque, I., Amara, D.S., and Kamara, C.S. 1980. Effects o f inoculation and nitrogen fertilizer on soybean in Sierra Leone. Soil Science and Plant Analysis. II (1): 11-24 Hardarson, G., Danso, S.K.A., and Zapata, F. 1987. Biological nitrogen fixation in field crops. In:Handbook o f Plant Science Agriculture. B.R. Christie(Ed.). pp.165-192. CRC Press Inc., Boca Raton, FL. Hardarson, G., Golbs, M., and Danso S.K.A. 1989. Nitrogen function by soybeans (Glycine max(L) Merill) as affected by nodulation patterns. Soil Biol. Biochem. 21: 783-787. Hardarson, G., Zapata, F., and Danso, S.K.A. 1984 Field evaluation o f symbiotic nitrogen fixation by rhizobial strains using 15N methodology. Plant and Soil 82:369-375. 99 University of Ghana http://ugspace.ug.edu.gh Hardley, H.H., and Hymowitz, T. 1973. Speciation and cytogenenetics. Pp 97-116. In B.E.Caldwell (ed.) Soybeans: Improvement, Production, and Uses. American Society of Agronomy, Inc., Madison, WI. Hardy, R.W.F. Holsten, R.D., and Burns, R.C. 1973. Applications o f the acetylene-ethylene assay for measurement o f N fixation. Soil Biol. Biochem. 5: 47-48. Hardy, R.W.F., Holsten, R.D., Jackson, E.K., and Burns, R.C. 1968. The acetylene-ethylene assay fo rN 2 fixation: Laboratory and field evaluation. Plant Physiol. 43: 1185-1207. Hartel, P.G., and Alexander, M. 1983 Growth and survival o f cowpea rhizobia in acid, Al.-rich soils. Journal o f Soil Sci. Soc. o f Am. 47: 502-506. Hartel, P.G., and Alexander, M. 1984. Temperature and desiccation tolerance o f cowpea rhizobia. Can. J. o f Microbiol. 30, 820-823. Haydock, K. P. et. al. 1980. Plant Soil 57: 353-362. Hellriegel, H., and Wilfarth, M. 1888. Unterschungen uber die Stickstoffnahrung der Gramineen und Leguminosen. Beilageheft su der Zeschr. ver Rubenzucker-Industrie Deutschen Reichs. Hirsch, A. M., 1992. Developmental biology o f legume nodulation. New Physiol. 122: 211- 237. Proc. Soil Sc. Am. 10: 202-205. Hofer, A. W. 1945. Nitrogen fixation by mixed cultures of Rhizobium. Holding, A.J., and Lowe, J.F. 1971. Some effects o f acidity and heavy metals on the Rhizobium-leguminous plant association. Plant and Soil Special Vol. 117-127. Holland, A.A. 1970. Competition between soil and seed-borne Rhizobium trifolii in nodulation o f introduced Trifolium subterraneum. PI. Soil 32: 293-302. 100 University of Ghana http://ugspace.ug.edu.gh Holland, A.A., and Parker, C.A. 1966. Studies on microbial antagonism in the establishment of clover pasture II. The effect o f saprophytic soil fungi upon Rhizobium trifolii and the growth o f subterranean clover. PL Soil 25: 329-340. Hungria, G., and Franco, A. A. 1993. Effect o f soil temperature on nodulation and N function by Phaseolus vulgaris L. Plant Soil 149: 95-102 Hattugh, M. J., and Luow, H. A. 1966. The antagonistic rhizoplane bacteria on clover-legume symbiosis. Phytophylactica 1: 205-208. Hymowitz, T. 1970. The domestication o f soybean. Econ. Bot. 24: 408-421. Johnson, H.W., Means, U.M., and Weber, C.R. 1964. Competition for nodule sites between strains o f Rhizobium japonicum applied as inoculum and strains in the soil. Agron. J. 57: 179-185. Jordan, D.C. 1982. Transfer o f Rhizobium japonicum gen. nov., a genus o f slow-growing, root nodule bacteria from leguminous plants. Int. J. Syst. Bacteriol. 32: 136-139 Kang, B.T. 1975. Effect o f inoculation and nitrogen fertilizer on soybean in Western Nigeria. Experimental Agric. 11: 23-31. Keeney, D. 1982. Nitrogen management for maximum efficiency and minimum pollution. In Nitrogen in Agricultural Soils. Ed. F. J. Stevenson. Agronomy Monograph 22, pp 605- 649. ASA, Madison, WI Kueneman E.A. 1994. Tropical Soybean Improvement and Production.; Brazilian Agricultural Research Enterprise. F.O.A. of the United Nations. Rome. Kueneman E.A., Root, W.R., Dashiell, K.E., and Hohenberg. J. 1984. Breeding soybean for the tropics capable o f nodulating effectively with indigenous Rhizobium spp. Plant and Soil 82: 387-396. 101 University of Ghana http://ugspace.ug.edu.gh Kumarasinghe, K.S. Danso, S.K.A., and Zapata, F. 1992. Field evaluation o f N2 fixation and partitioning in climbing bean (P. vulgaris L.) using I5N. Biol. Fertil. Soils 13: 142-146. Labandera, C.A., and Vincent J.M. 1975. Competition between an introduced strain and native Uruguayan strains o f Rhizobium trifolii. Plant Soil 42: 327-247. Ledgard, S.F., Simpson J.R, Freney J.R. and Bergersen F. J. 1985 Field evaluation o f 15N technique for estimating nitrogen fixation in legume-grass associations. Aust. J. Agric. Res. 36: 247-258. Legg, J.O., and Sloger, C. 1975. A tracer method for determining symbiotic nitrogen fixation in field studies. In: Proc. o f the Second Int. Conf. on Stable Isotopes. Eds. ER Leonard. L.T. 1923. Nodule production kinship between the soybean and the cowpea. Soil Sci. 15 277-283.; Leonard, L.T. 1930. A failure o f Austrian winter peas apparently due to nodule bacteria J. Am. Soc. Agron. 22: 277-280. Lindstrom, K , van Berkum, P., Gillis, M., Martinez, E., Novikova, N., and Jamis, B. 1995. Report from the roundtable on Rhizobium taxonomy. In: I.A. Tikhonovich, N. A. Provorov, V.I. Romanov, and W.E. Newton (ed), Nitrogen Fixation: Fundamentals and Applications. Pp. 807-810. Lynch, J.M., and Wood, M. 1989. Interactions between plant roots and microorganisms. In: Soils Conditions and Plant Growth. Alam Wild (eds.) pp 526-563. Bath Press London. Marshall, K.C. 1956. A lysogenic strain o f Rhizobium trifolii. Nature (London) 177: 92. Marshall, K.C. and Roberts, F.J. 1963. Influence o f fine particle materials on survival o f Rhizobium trifolii in sandy soil. Nature (London) 1968:410-411. 102 University of Ghana http://ugspace.ug.edu.gh Minchin. F.R., Sheehy, J.E.and Muller, M. 1983. A major error in the acetylene reduction assay: Decrease in nodular nitrogenase activity under assay conditions. J Exp Bot 34: 641-649. Minchin, F.R., Sheehy, J.E.and Muller, M. 1986. Further errors in the acetylene reduction assay: Effects o f plant disturbance J. Exp Bot. 37: 1581-1591. Mpepereki, S., Wolhum A. G. and Makonese, F. 1996. Diversity in symbiotic specificity of cowpea rhizobia indigenous to Zimbabwean soils. Plant and Soil, 186: 167-171. Mulongoy, K., and Ayanaba, A. 1986. Dynamics o f population sizes o f cowpea and soybean rhizobia at three locations in West Africa. MIRCEN J. 2: 301-308. Mulongoy, K., Ayanaba, A., and Pulver, F. 1981. Exploiting the diversity in the cowpea- rhizobia symbiosis for increased cowpea production. In GIAM VI Global Impacts o f Applied Microbiol. Emejuaiwe S.O., Ogumbi O. Sanni S.O. (Ed )pp 119-125. London : Academic Press. Munns, D.N. 1968. Nodulation o f Medicago sativa in solution culture III. Effects o f nitrate on root hairs and infection. Plant Soil 28: 33-47. Munns, D.N. and Keyser, H.H. 1981. Response o f Rhizobium strains to acid and Al. stress. Soil Biol and Biochem. 13 115-118. Na Lampong, A. 1976. Inter-relationship between soybean varieties and indigenous Rhizobium strains in Northeast Thailand. Pp. 198-199. In Proc. of a Conf. for Asia and Oceania. Chang Mai, Thailand. INSTOY Publication Series No. 10. Nangju, D. 1980. Soybean responses to indigenous rhizobia as influence by cultivar origin. Agronomy J. 72: 403-406 Nap, J., and Bisseling, T. 1990. Developmental biology o f a plant-prokaryotg,symbiosis o f the 103 University of Ghana http://ugspace.ug.edu.gh root nodule. Science, 205: 948-954. Nambiar, P.T.C., Rupela. O.P., and Kuar Rao, J.V.D.K. 1988. Nodulation and N fixation in Groundnut (Arachis hypogea L.) Chickpea (Cicer arietinum) and pigeon pea (Cajanus cajan L. Mill) In: Subba Rao N.S. (ed.) Biological N fixation, Recent Developments pp. 53-70: Oxford and IBH Publishers, New Delhi. Nye, P.H.and G reenland, D.J. 1960. The soil under shifting cultivation. Technical Communication 51: Commonwealth Agric. Bureau. Bux. England. Obaton, M. 1975. Effectiveness, saprophytic and competitive ability. Three properties of Rhizobium essential for increasing the yield o f inoculated legumes. In A. Ayanaba, P.J. Dart (Ed.) Biological Nitrogen Fixation in Farming Systems o f the Tropics. Pp.127-133. John Wiley and Sons. New York. Okereke, G.U., and Eaglesham, A.R.J. 1992. Nodulation and nitrogen fixation by 79 “promiscuous” soybean genotypes in a soil in East Nigeria. Agrononomie Africaine V (2): 123-136. Association Argentina de la Soja, Buenos Aires, Argentina. Olufajo, O.O., and Adu J.K. 1992. Response o f soybean to inoculation with Bradyrhizobium japonicum in the Northern Guinea savanna o f Nigeria. In Biological Nitrogen Fixation and Sustainability o f Tropical Agriculture. A Wiley-Sayce Co-Publication. Owiredu, J. D. 1980. The use o f streptomycin-resistant inoculants in the study o f competition among strains o f rhizobia.(M. Sc.Dissertation. Univ. o f Ghana, Legon). Pankhurst, C.E. 1981. Effects o f plant nutrition supply on nodule effectiveness and Rhizobium strain competition for nodulation o f Lotus pendunculatus. Plant Soil 60: 325-339 104 University of Ghana http://ugspace.ug.edu.gh Pascale. P.P., Pate, J.S. and Dart, P.J. 1961. Nodulation studies in legumes IV. The influence o f inoculum strain and time o f application o f ammonium nitrate on symbiotic response.Plant Soil 15: 329-346. Pate, J. S. and Dart, P.J. 1961 Nodulation studies in legumes IV. The influence o f inoculum strain and time o f application o f ammonium nitrate on symbiotic response. Plant Soil 15: 329-346. Pattterson, T.G., and LaRue, T.A. 1983. Nitrogen fixation by soybeans: Seasonal and cultivar effects and comparison o f estimates. Crop Sci. 23: 488-492. Piha, M.I., and Munns, D.N. 1987. Sensitivity o f the common bean (P. vulgaris L.) symbiosis to high soil temperature. Plant Soil 98: 183-194. Pulver. E.L., Brockman, F., Nangju, D., and Wien, H.C. 1978. IITA’s programme on N2 fixation. In: Isotopes in Biological Dinitrogen Fixation pp. 269-284. Vienna: IAEA. Pulver, E.L., Brockman, F., Nangju, D., and Wien, H.C. 1982. Nodulation o f soybean cultivars with Rhizobium spp. and their response to inoculation with R. japonicum. Crop Sci. 22: 1065-1070. Pulver, E.L., Kueneman, E.A., and Ranga Rao, V. 1985. Identification o f promiscuous nodulating soybean efficient inN 2 fixation. Crop Sci. 25: 660-663. Rachie, K. O. and Roberts, L. M. 1974. Grain Legumes o f the Lowland Tropics. Academic Press Inc. Ranga Rao, V., Thottappilly, G., and Ayanaba, A. 1982. Studies on the persistence o f introduced strains o f Rhizobium japonicum in soil during fallow and the effects on soybean growth and yield. In Biological Nitrogen Fixation Technology for Tropical 105 University of Ghana http://ugspace.ug.edu.gh Agriculture. Graham, PH & Harris S.C. (ed.) pp. 309-315. Workshop at CIAT, March 9- 13, 1981. Cali, Colombia: CIAT. Rennie, R.J. 1982. Quantifying N2 fixation in soybeans by 15N isotope dilution: The question of the non fixing control plant. Can.J. Bot. 60: 856-861. Rennie, R.J. 1984. Comparison o f methods o f enriching a soil with 15N to estimate dinitrogen fixation by isotope dilution. Agron. J. 78: 158-163. Rennie, R.J., Dubetz, S., Bole, J.B., and Muendel, H. 1982. Dinitrogen fixation measured by I5N in two Canadian soybean cultivars. Agron. J.71: 719-723. Rennie, R.J., and Kemp, G.A. 1983. Nitrogen-fixation in field beans quantified by 1SN isotope dilution. II. Effect o f cultivars o f beans. Agron. J. 1975: 645-649. Rennie, R.J., and Rennie, D.A. 1983. Techniques for quantifying N2 fixation in association with non-legumes under field and greenhouse conditions. Can. J. Microbiol. 29 1022-1035. Rhodes, E.R., and Nangju, D. 1979. Effects o f pelleting cowpea and soybean seed with fertilizer dusts. Exp. Agric. 15: 27-32. Robson, A.D., O ’Hara, G.W., and Abott, L.K. 1981. Involvement o f phosphorus in nitrogen fixation by subterranean clover (Trifolium subterranean L.) Aust.J. Plant Physiol. 8: 427-436. Roughley, R.J. 1970. The preparation and use of legume seed inoculants. Plant and Soil 32, 675-701 Roughley, R.J., 1985. Effect o f soil environmental factors on rhizobia. In: Shibles R., (ed.), World Soybean Research Conference III. Proceedings. West V iew Press. Boulder, Cororado, pp. 903-910. 106 University of Ghana http://ugspace.ug.edu.gh Roughley, R.J. Bromfield, E.S.P., Pulver, E.L., and Day, J.M. 1980. Competition between species o f Rhizobium for nodulation o f Glycine max. Soil Biol. Biochem. 12: 467-470. Ruschel, A.P., Vose, P.M., Matsui, E., Victoria. R.L., and Saito, S.M. 1982. Field evaluation of N2-fixation and N-utilization by Phaseolus bean varieties determined by 15N isotope dilution. Plant and Soil 65: 397-407. Ruschel, A.P., Vose, P.M., Matsui, E., Victoria, R.L., and Salati, E. 1979. Comparison of isotope techniques and non-nodulating isolines to study the effect o f ammonium fertilizer on dinitrogen fixation in soybean, Glycine max. Plant and Soil 53: 513-525. Sanyal, S.K., Chan, P.Y., and de Datta, S.K. 1990. P sorption description behaviour o f some acidic soils in South and Southeast Asia. Paper presented at the 6th Philippine Chemistry Congress, Cebu city, Philippine, 24-26. Schmidt, E.L., Bankole, R.O., and Bohlool, B.B. 1968. Fluorescent-antibody approach to study o f rhizobia in soil. J.Bacteriol. 95: 1987-1992. Scott, W. 0 . and Aldrich, S. R. 1983. Modem Soybean Production. S and A Publication, Champaign I L.pp 209. Sears, O.H., and Carroll, W.R. 1927. Cross inoculation with cowpea and soybean nodule bacteria. Soil Sci 24, 413-419. Sellschop, J.P.F. 1962. Cowpeas, Vigna unguiculata (L.)Walp. Field Crop Abstracts. 15: 259- 266. Shanmugasundaram, S. 1989. Global cooporation for improvement o f soybean research and development. In:Proceedings, World Soybean Res Conf. IV Ed. AAJ Shearer, G., and Kohl, H. 1986. N2-fixation in field settings: Estimations based on natural !5N abundance. Aust. J. Plant Physiol. 13: 699-757. 107 University of Ghana http://ugspace.ug.edu.gh Sinclair, T.R., Muchow, R.C., Ludlow, M.M., Leach, G.J., Lawn, R.J., and Foale, M.A. 1987. Field and model analysis o f the effect o f water deficits on carbon and nitrogen accumulation by soybean, cowpea and black gram. Field Crops Res. 17: 121-1400. Singleton, P.W., and Stockinger F. 1982. Compensation against ineffective nodulation in soybean, Crop Science 23: pp 69 Singleton, P.W., and Travers, J. W. 1986. Inoculation response o f legumes in relation to the number and effectiveness o f indigenous Rhizobium populations. Appl. Environ. Microbiol. 51:1013-1018. Somasegaran P., and Hoben H.J. 1985. Methods in Legume-Rhizobium Technology. Hawaii Institute o f Tropical Agriculture and Human Resources. University o f Hawaii. Sprent, J.I. 1979. The biology o f N fixing organisms. McGraw-Hill, London, 196. Sprent, J.I., Becana, M., and Sutherland, J.M. 1988. Optimising nitrogen fixation in legume crops and trees. In N fixation: A Hundred Years After Bothe H de BruijinFJ and Newton WE 725-733. Gustav Fisher, Stuttgart, Germany. Streeter, J.G. 1979. Allantoin and allantoic acid in tissues and stem exudate from field-grown soybean plant. Plant Physiol 63: 478-480. Summerfield, R.J., Dart, P.J., Huxely, P.A., Eaglesham, A.R.J., Minchin, F.R., and Day, J.M. 1977. Nitrogen nutrition o f cowpea ( Vigna unguiculata) In: Effects o f Applied N and Symbiotic N fixation on Growth and Seed Yield. Exp. Agric. 13: 129-142 Thies, J.E., Singleton, P.W., and Bohlool, B.B. 1991. Influence o f the size o f indigenous rhizobial populations on establishment and symbiotic performance o f introduced rhizobia on field-grown legumes. Appl. and Environ. Microbiol. 57: 19-28. 108 University of Ghana http://ugspace.ug.edu.gh Tisdale, S., and Nelson, W. 1956. Soil fertility and fertilizer use. Macmillan Publishing Co. Inc. New York pp 125-127. Tisdale, S., and Nelson, W. 1975. Soil fertility and fertilizer use. Macmillan Publishing Co. Inc. New York. Turgeon, B. G., and Bauer W.D. 1985. Ultrastrusture o f infection-thread development during the infection o f soybean by Rhizobium japonicum. Planta 163: 328-342. Tuzimura, K.I., Watanabe, and Shih, K.F. 1963. Difference in the rhizosphere effect on Rhizobium trifolii and Rhizobium meliloti between soils. Ecology o f root-nodule bacteria in soil. Soil Sc. Fertil. 28: 1713. Vance, C.P. 1983. Rhizobium infection and nodulation: A beneficial plant disease. Ann. Rev. Microbiol. 399-424. Van Rensburgh, H.J., Strijdom, B.W., and Kriel, M.M., 1976. Necessity for seed inoculation of soybeans in South Africa. Phytophylactica 8, 91-96. Van Schreven, D.A. 1970. Some factors affecting growth and survival o f spp. in soil-peat cultures PI. Soil 32: 113-130. Van Schreven, D.A. 1971 .The resistance o f effectiveness o f R. trifolii to low pH. PI. Soil. 37: 49-55 Vessey, J.K. 1994. Measurement o f nitrogenase activity in legume root nodules. In: Defence of the Acetylene Reduction Assay. Plant and Soil. 158: 151-162. Vincent, J.M. 1970. A manual for the Practical Study o f Root- Nodule Bacteria. IBP Handbook No. 15 pp. 46 Blackwell Scientific Publications, Oxford. Vincent, J.M. 1977. Quality control o f inoculants. In: The Biology o f nitrogen fixation pp 263- 341. A Quipsel (Ed) North Holland Publishing Co. Amsterdam. 109 University of Ghana http://ugspace.ug.edu.gh Vincent, J.M., and Waters, L.M. 1954. The root-nodule bacteria as factors in clover establishment in the red basaltic soils o f the Lismore district, New South Wales II. Survival and success o f inocula in laboratory trials. Aust J. o f Agron. Res. 5: 61-76. Vincent J.M. 1980. Factors controlling the legvme-Rhizobium symbiosis. In: W.E and Orne-Johnson, W.H. (eds.) Nitrogen fixation. 2: 101-129. University Park Press, Baltimore. Vose, P.B., and Victoria, R.L. 1986. Re-examination o f the limitations o f I5N isotope dilution technique for measurement o f dinitrogen fixation. Soil Sci.Soc. Am. J. 677: 23-41. Wadisirisuk, P., Danso, S.K.A., Hardarson, G., and Bowen, G.D. 1989. Influence of Bradyrhizobium japonicum location and movement on nodulation and N fixation in soybean. Appl. Environ. Microbiol. 55: 1711-1716. Wagner, G.H., and Zapata, J. 1982. Field evaluation o f reference crops in the study o fN fixation by legumes using isotope techniques. Agron.J 74: 607-612. Weaver, R.W., and Frederick, L.R. 1974. Effect o f inoculum rate on competitive nodulation of Glycine max L. Merril II. Greenhouse studies. Agron. J. 66 229-232. Weaver, R.W., and Frederick, L.R. 1972. A new technique for most-probable-number counts of rhizobia. Plant and Soil 36: 2199-2222. Weber, C.R. 1966 . Nodulating and non-nodulating soybean isolines. The response to applied nitrogen and modified conditions. Agron. J 58: 43-46. Weingartner, K.E. 1981. Bioavailability o f Calcium and Zinc in Soy Products. Urbana, University o f Illinois (Doctorate dissertation). West, C.P., and Wedin, W.F. 1985 Dinitrogen fixation in alfafa-orchard grass pastures. Agron. J. 77: 89-94 110 University of Ghana http://ugspace.ug.edu.gh Westermann, D.T., and Kolar, J.J. 1978. Symbiotic N2 (C2H4) fixation by bean. Crop. Sci. 18: 986-990. Williams, W.A., Jones, M.B., and Delwiche, C.C. 1977. Clover N-fixation measurement by total-N difference and l5N A-values in lysimeters. Agron. J. 69: 1023-1024 Wilson, K.J., 1944. Over five hundred reasons for abandoning the cross-inoculation groups of legumes. Soil Science 58: 61-69. Witty. J.F., and Minchin, F.R. 1988. Measurements o f N fixation by the acetylene reduction assay: myths and mysteries. In Beck DP and Materon LA (eds.) N fixation by legumes in Mediterranean Agriculture, pp. 331-334 Dordrecht: Martinus N ijhoff Publishers Witty, J.F., Minchin, F.R. Skot, L. and Sheehy, J.E. 1986. N fixation and Oxygen in legume root nodules. In Miflin BJ (ed) Oxford Surveys o f Plant Molecular and Cell Biology, Vol 3 Oxford Univ. Press, Oxford,pp. 275-314. Woomer, P., Singleton, P.W., and Bohlool, B.B 1988. Ecological indicators o f native rhizobia in tropical soils. Applied and Environmental Microbiology. 54: 1112-1116. Zengbe, M. 1980. Presence et distribution de Rhizobium cowpea dans les sols de Cote d’Tvoire. In Rosswall T (ed) Nitrogen Cycling in West Africa Ecosystems. Proc. of SCOPE/UNEP/UNESCO/IITA Workshop, Ibadan, Nigeria, Dec. 1978. Stockholm, Sweden; Sundt Offset AB 52R. I l l University of Ghana http://ugspace.ug.edu.gh