QR82.R45 F35 bJthrC.l G360936 University of Ghana http://ugspace.ug.edu.gh DIVERSITY AND SYMBIOTIC CHARACTERISTICS OF COWPEA BRAD YRHIZOBIUM STRAINS IN GHANAIAN SOILS BY JOSEPH OPOKU FENING A THESIS SUBMITTED TO THE DEPARTMENT OF SOIL SCIENCE, UNIVERSITY OF GHANA, FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (Ph.D.) IN SOIL SCIENCE. AUGUST 1999 University of Ghana http://ugspace.ug.edu.gh DEDICATION This work is dedicated to Mrs. Juliana Twum, my brother's wife, who supported and encouraged me during the period of my academic pursuits. In particular, when I decided to return to Legon for my Doctorate Programme, Mama invited me to share iheir East Legon home and ensured that I had my special diet regularly and promptly. This, of course, relieved me of the chores of campus life and contributed substantially to the completion of the programme on time. University of Ghana http://ugspace.ug.edu.gh DECLARATION I hereby declare that, except for references to works of other researchers, which have been duly cited, this work is the result of my own original research and that this thesis has neither in whole nor in part been presented to any other University for the award of a degree. ....... Joseph Opoku Fening (Student) / Prof S .K .l Danso (Major Supervisor) r. F.K KumagahD (Supervisor) (Supervisor) Dr. K.M. Bosompem (Supervisor) University of Ghana http://ugspace.ug.edu.gh ABSTRACT This study reports investigation of the biodiversity of bradyrhizobia isolates that nodulate cowpea in Ghanaian soils. As a prelude, some components of nitrogen fixation of cowpea in the various soils were examined through: (1) assessment of the natural nodulation of 45 cowpea cultivars in 20 soils sampled from 5 ecozones (coastal savanna, tain forest, semi deciduous forest, forest savanna transition and guinea savanna), (2) determination of the numbers of bradyrhizobial isolates in the soils and (3) determination of the response of cowpea to nitrogen fertilization. The results of the ability of 45 cowpea cultivars to nodulate naturally in different soil types showed large variability among the cultivars. Counts of the indigenous bradyrhizobia population in the soils showed that most of the soils in Ghana harbour large populations of bradyrhizobia (in the range of 0.6 x 10 to 31 x 103) capable of nodulating cowpea. Response of cowpea to nitrogen fertilizer differed in the different soils. In general all the cultivars showed significant responses to increasing levels of nitrogen, an indication that nitrogen fixation was not supplying the plants with all the external nitrogen required for maximum yield. A combination of morpho-physiological and molecular analysis was used to assess the diversity of the bradyrhizobia isolates. A total of 100 isolates were assessed. The results of the morpho- physiological analysis indicated that cowpea is nodulated by both fast and slow growing rhizobia. The results also showed that the isolates were versatile and could survive under different soil conditions particularly acidity and salt stress. A cross inoculation study of the isolates with nine legume species produced seven major groupings with 28 subgroups based on University of Ghana http://ugspace.ug.edu.gh iv distinct nodulation patterns. Results of the serology (ELISA) assay indicated that only a small fraction of the isolates reacted strongly with antisera of each other. The greater proportion showed no cross reactivity. Analysis of the 16S rRNA gene of the isolates by PCR-RFLP identified 20 different composite genotypes. Diversity among the genomic species identified was very high, reaching 80% diversity. The various methods used indicated large diversity among the isolates, but the groupings of the isolates by the various methods were inconsistent, due to the different levels of resolution by the various methods. Diversity of the isolates in symbiotic effectiveness showed that some of the isolates had high nitrogen fixing capabilities that were comparable to plants fertilized with inorganic fertilizer nitrogen. Some of the isolates even showed superiority in symbiotic effectiveness relative to the standard strain TAL 169, suggesting that the native isolates may be useful strains for cowpea inoculation. The Gus A marker gene technique was used to assess the competitive abilities of the effective and ineffective isolates. Competition between the isolates was examined at different population ratios. The results obtained indicated that competitive ability was not directly related to effectiveness of strains. Inoculation of cowpea with indigenous bradyrhizobia isolates increased the number of nodules, shoot dry weight and total nitrogen of plants. The method of inoculation was observed to influence these parameters The results indicated that response of cowpea to inoculation in the presence of native rhizobia in some soils is possible. University of Ghana http://ugspace.ug.edu.gh VACKNOWLEDGEMENT I wish to express my sincere gratitude to my supervisors, Professor S. K A Danso, Dr. F. K. Kumaga Dr. S. K Offei and Dr. K. M Bosompem, for their invaluable support, advice and research directions. I am extremely grateful to Professor Danso. Besides providing an excellent guidance, he took a special interest to support this work by obtaining an IAEA fellowship for me in Austria which enabled me to do all the molecular biology aspects of the work. As always, there are people without whose help this thesis could not have been written. In no particular order but with equal gratitude for their knowledge, support and expertise, I wish to thank the following: Mr. MS. Elegba, Mr. A Tonyinga, Mr J. Aggrey, Mr. Dogbe, Mr Osei Ampontuah, Miss Henrietta Mbeah and all staff of the Soil Science Department I also thank the Council for Scientific and Industrial Research (CSIR) and the Soil Research Institute (SRI) for granting me study leave to undertake this programme. The support and encouragement given to me by the Director of SRI, Mr RD Asiamah needs mentioning. Further thanks go to the National Agricultural Research Project (NARP) and the Ecological Laboratory of the University of Ghana, for providing funding and research materials, without which this work could not have been done. University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS Dedication .. .. .. - Declaration . .. •• Abstract .. .. .. .. Acknowledgement .. .. .. Table of contents . .. .. List of Figures.. .. .. List of Tables .. .. CHAPTER ONE 10 INTRODUCTION .. 1.1 Background 1.2 Problem Specification.. 1.3 Objectives of the Study CHAPTER TWO 2 0 LITERATURE REVIEW 2.1.1 The nodule bacteria 2.1.2 Rhizobium taxonomy 2.1.3 Abundance in soil 2 .2.1 Nodule development 2.2.2 Types of nodule University of Ghana http://ugspace.ug.edu.gh vii 2.2.3 Factors affecting legume nodulation .. 2.2.4 Longevity of nodule .. .. .. .. 2.3 Inoculation of legumes with rhizobia.. . 2.4 Diversity ofrhizobial isolates.. .. .. 2.4 1 Methods for analysing diversity of rhizobia .. 2.4.1.1 Cross inoculation .. .. 2.4.1.2 Cultural and metabolic characterisation 2.4.1.3 Serology .. .. .. 2.4.1 4 Molecular analysis .. .. .. 2.5 Symbiotic effectiveness .. .. .. 2.6 Competition for nodule occupancy .. .. 2.6 1 Molecular gene markers in competition studies CHAPTER THREE 3.0 MATERIALS AND METHODS .. 3.1 Soil sampling .. .. .. .. 3.1.1 Soil analysis .. .. .. .. 3.1.1.1 Soil pH ................................................. 3.1.1.2 Total phosphorus 3 . 113 Total nitrogen 3.2 Nodulation capabilities of cowpea . 3.2.1 Planting materials .. .. 3.2.2 Pot experiment .. .. 13 20 20 23 25 25 26 28 30 34 35 40 44 44 44 44 47 47 47 47 48 University of Ghana http://ugspace.ug.edu.gh viii 3.3 Enumeration of rhizobia . 3.4 Response of cowpea to nitrogen fertilization.. 3.4.1 Pot experiment -• •• 3.5 Isolation of rhizobia .. •• 3.5.1 Authentication of Isolates .. 3.5.2 Culture maintenance . . - •• 3.6 Physiological and metabolic characterisation of isolates 3.6.1 Growth rates and colony morphology.. .. . 3.6.2 Salt tolerance .. .. .. .. •• •• 3.6.3 Acid tolerance.. .. .. .. •• 3.6.4 Carbon utilizati on -. 3.7 Host range analysis . . .. .. 3.8 Serological characterisation .. .. .. 3.8.1 Preparation of rhizobia antigens .. .. 3.8.2 Formulation of antigens for immunization 3.8.3 Experimental animals.. .. 3.84 Coating of microtitre plates with antigen .. 3.8.5 ELISA Procedures .. .. .. 3.8.6 Evaluation of antigen for antibody production 3.8.7 Bleeding of mice and screening for antibody response 3.8.8 Selection of rabbits for immunization.. 3.8.9 Immunization of rabbits . 3 .8.10 Detection of antibody.. 48 49 49 50 50 50 51 51 51 51 52 52 53 53 53 53 54 54 55 55 55 56 56 University of Ghana http://ugspace.ug.edu.gh IX 3 9 Molecular characterisation . • 3.9.1 Sample preparation for DNA amplification .. 3.9.2 PCR amplification of the 16S rRNA gene . .. 3.9.3 Electrophoresis and imaging .. .. .. - 3.9.4 Restriction fragment analysis.. .. .. . .. 3.10 Effectiveness of isolates in fixing nitrogen .. .. 3.10.1 Relative effectiveness of isolates in fixing nitrogen . 3.11 Measurement of nodulation competitiveness by glucuronidase 3.11.1 Gus fusion donor strain . . . . . . 3.11.2 Marking rhizobia with gusA gene .. .. 3.11.3 Detection of gusA - marked Bradyrhizobium derivatives .. 3.11.4 Competition experiment .. .. .. .. 3.11.5 Staining of nodules .. .. .. .. . .. 3 11.6 Effect of placement and time of placement of inoculum on competitive ability of isolates.. .. 3.11.7 Speed of infection of host . . . . 3.12 Inoculation of cowpea with isolated indigenous cowpea bradyrhizobia isolates.. .. . .. 3.12.1 Soils used .. .. .. 3.12.2 Inoculation procedures 3 12.2.1 Seed inoculation .. .. „ 3 12.2.2 Soil inoculation 3.12.2.3 Plant growth .. 57 57 58 58 58 59 60 60 60 60 61 62 62 63 63 64 64 64 64 65 65 University of Ghana http://ugspace.ug.edu.gh XCHAPTER FOUR R E S U L T S .................................... .......................... 66 4.1 Examination of components of nitrogen fixation in cowpea.. 66 4 11 Nodulation potential of cowpea in Ghanaian soils .. .. 66 4.1.2 Estimation of indigenous bradyrhizobia numbers in the Soil 66 4 13 Response of cowpea to nitrogen fertilization .. 70 4.2 Diversity of indigenous cowpea bradyrhizobia isolates .. 77 4.2.1 Physiological and metabolic analysis.. .. .. .. 77 4.2.2 Host range analysis .. .. .. 77 4.2.3 Serology .. .. .. 81 4.2.3.1 Antibody responses in immunized mice .. 81 4.2 3.2 Serogrouping of cowpea bradyrhizobia isolates 84 4.2.4 Molecular analysis .. .. 84 4.3 Effectiveness of isolates in fixing nitrogen .. 89 4.3 1 Relative effectiveness of isolates in fixing nitrogen .. 98 4.4 Competition for nodule occupancy .. .. .. 98 4.4.1 Effect of placement and time of placement .. .. 108 4.4.2 Speed of formation of nodules by competing isolates .. 108 4.5 Inoculation of cowpea with indigenous HTa.dyrhizobium isolates . 116 University of Ghana http://ugspace.ug.edu.gh xi CHAPTER FIVE 5.0 DISCUSSION................................................... .......................... 122 5.1 Potential to improve nitrogen fixation of cowpea .. 122 5.2 Diversity of indigenous cowpea bradyrhizobia isolates 126 5.3 Symbiotic effectiveness .. .. 131 5.4 Competition for nodule occupancy . .. 132 5.5 Inoculation response of cowpea .. .. .. 135 CHAPTER SIX 6 0 CONCLUSION ...................................... 138 REFERENCES ............................................................. 143 APPENDIX ... 193 University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES FIGURE PAGE 4.1 Percent of cowpea which formed nodules in soils of the different Ecozones . .. .. .. 67 4.2 The means of cowpea bradyrhizobia populations in soils of the various ecological zones .. .. 69 4.3 Mean responses of cowpea to nitrogen application on various soils.. 71 4.4 Mean responses of cowpea varieties to nitrogen application on all soils 72 4.5 Response of cowpea varieties to nitrogen fertilization on Akuse soil series. 73 4.6 Response of cowpea varieties to nitrogen fertilization on Wacri soil series . .. .. .. 74 4 7 Response of cowpea varieties to nitrogen fertilization on Tafali soils 75 4 8 Response of cowpea varieties to nitrogen fertilization on Adenta soil Series .. 76 4.9 Percentage of plants of several legume species nodulated by cowpea bradyrhizobia isolates .. .. .. .. .. 82 4 10 Serum antibody response of BALB/C mice following immunization with cowpea rhizobia antigen extracts . . . 8 3 4.11 Relationship between cowpea bradyrhizobia isolates as determined by reaction of homologous and heterologous antisera 86 University of Ghana http://ugspace.ug.edu.gh xiii 4.12 4.13 4.14 4.15 4.16 4.17 4.18 4.19 4.20 4.21 Dendrogram showing relationship among genomic species of slow growing cowpea bradyrhizobia isolates as determined by RFLP analysis of 16S rDNA .. .. . - •• Dendrogram showing relationship among genomic species of fast growing cowpea bradyrhizobia isolates as determined by RFLP analysis of 16s rDNA .. - - •• - •• Percent number of cowpea bradyrhizobia isolates for groups of effectiveness Symbiotic effectiveness of cowpea bradyrhizobia isolates relative toTAL196 .. .. .. .. .. .. .. Linear regression of [Log (Px + P both)/Py + P both)] against Log (lx/1 y) for Gus-marked isolates, 2,10, 14 when competed against ineffective isolates 28, 29, 30, 72 and 9 4 ..................................... .......................... Effect of placement and time of placement of effective isolate (G2) and ineffective isolates (28 and 94) on shoot biomass .. .. .. Effect of placement and time of placement of effective isolate (G10) and ineffective isolates (28 and 94) on shoot biomass Effect of placement and time of placement of effective isolate (GI 4) and ineffective Isolates (28 and 94) on shoot biomass Effect of placement and time of placement of effective isolate (G2) and ineffective isolates (28 and 94) on shoot nitrogen .. Effect of placement and time of placement of effective isolate (G10) and ineffective isolates (28 and 94) on shoot nitrogen. .. 90 91 95 101 107 109 110 111 112 113 University of Ghana http://ugspace.ug.edu.gh xiv 4.22 4.23 4.24 4.25 Effect of placement and time of placement of effective isolate (GI 4) and ineffective isolates (28 and 94) on shoot nitrogen .. .. Time when nodules were first visible on cowpea roots inoculated with the competing isolates in single cultures .. .. .. .. Nodulation of cowpea plants inoculated with isolated native cowpea bradyrhizobia isolates .. .. .. .. Shoot yield of cowpea (Asontem) plants inoculated with native cowpea bradyrhizobia isolates . .. .. .. .. 114 115 117 118 University of Ghana http://ugspace.ug.edu.gh XV l is t o f t a b l e s TABLE PAGE 3.1 Classification of the various soils . - •• 45 3.2 Selected chemical properties of the soils.. .. .. •• ^6 4 .1 Most probable number estimates of cowpea bradyrhizobia isolates in the in the various soils .. .. .. •• •• 4.2 Physiological and metabolic characteristics of cowpea bradyrhizobia Isolates . . . . . .. 78 4.3 Diversity o f cowpea bradyrhizobia isolates by cross inoculation .. 80 4.4 Normalised absorbance values for antigen antibody reactions of cowpea Bradyrhizobia isolates .. .. .. ~ - .. ' 85 4.5 Restriction patterns of cowpea bradyrhizobia isolates revealed by RFLP analysis of PCR-amplified 16S rRNA genes 87 4.6 Distribution of cowpea bradyrhizobia isolates among 20 genomic species identified by RFLP analysis of PCR-amplified 16S rRNA genes .. 88 4.7 Symbiotic effectiveness of cowpea bradyrhizobia isolates .. .. .. 92 4.8 Correlation between effectiveness and some parameters of nitrogen fixation 96 4.9 Distribution of cowpea bradyrhizobia isolates in terms of effectiveness in the soils of the various ecological zones .. . . . . 99 4.10 Symbiotic effectiveness profile of cowpea bradyrhizobia isolates 10C University of Ghana http://ugspace.ug.edu.gh xv i 4.11 Percentage of nodule occupancy of GUS marked cowpea bradyrhizobia isolates co-inoculated with the corresponding parent isolates onto the same plants.. .. 102 4.12 Competitive characteristics of GUS-marked effective cowpea bradyrhizobia isolates against ineffective isolates .. .. .. .. . .. 104 4.13 Total nitrogen and percent nitrogen in shoots of inoculated and uninoculated cowpea plants .. . . . . . . .. .. 119 4.14 Comparison of means of total nitrogen and percent nitrogen by soil series .. .. 120 4.15 Comparison of means oftotal nitrogen and percent nitrogen by inocula .. 121 University of Ghana http://ugspace.ug.edu.gh 1CHAPTER ONE INTRODUCTION 1.1. Background Legumes belonging to the family Fabaceae form symbiotic associations with the soil bacteria of the genera Azorhizobium, Bradyrhizobium,Mesorhizobium, Rhizobium and Sinorhizobium. This symbiosis, often leads to the development of a new plant organ, the nodule, which provides the ecological niche required for the biological fixation of atmospheric nitrogen (Elkan and Bunn, 1991). This process is carried out by rhizobia that reside in the nodule and is represented as: N2 + 8H+ + 8e" = 2NH3 + H2 The ammonium generated is assimilated as the ion, NH4+and is subsequently converted into glutamate, which is further assimilated and utilised by the plant (Giller and Wilson, 1993). By virtue of this symbiotic association, the growth of the plant can be rendered to a large extent, independent of soil nitrogen (Pueppke, 1986; Long, 1989). The economic significance of the legume - Rhizobium association has led to intensive research to improve the efficiency of the symbiosis over the past one hundred years since it was discovered (Gordon et al., 1995) One very important finding made so far is that: Judging from the magnitude of biological nitrogen fixation in different cropping systems, it is considered agronomically significant, providing an alternative or at least a supplement to the use of energy — expensive, artificial nitrogenous fertilizers. University of Ghana http://ugspace.ug.edu.gh 2It has been estimated that approximately 120 million tonnes of atmospheric nitrogen is reduced by biological nitrogen fixation to ammonium each year (Freiberg et al., 1997). 1.2 Problem specification One major problem facing farmers in Africa is that either the inherent fertility of the soil is low, or the capacities of these soils to supply nitrogen decline rapidly once agricultural activities commence (Buresh, 1997). The annual rate of decline in soil nitrogen stocks has been estimated to vary from 22-112 Kg N ha"1 (Stoorvogel et a l, 1993). Although the application of fertilizer nitrogen is recognised as the convenient way for rapid correction of nitrogen deficiency in soils, its high cost limits its wide application by farmers (Peoples et al., 1995). Ghana’s case is a good example; during the past decade, domestic inflation and removal of fertilizer subsidies in Ghana, have contributed to a rapid increase in fertilizer prices to over 29,000% (Bump, 1994). Consequently, there has been a sharp fall in the purchase and use of fertilizer in Ghana (Bump, 1994), with the attendant lowering in crop yields. The use of legumes as renewable sources of nitrogen and soil organic matter amendment have long been a major component of many farming systems in Africa, with cowpea as the most prominent legume (Awonaike et a l, 1990). As a food legume, the impact of fixed nitrogen to the soil following harvest depends upon the balance between soil nitrogen in the crop and that which was fixed. This is determined by the difference between the amounts of nitrogen fixed and nitrogen removed with the seed. When effectively nodulated, cowpea can produce as much as 90% of its nitrogen requirements from biological nitrogen fixation for maximum yields (Eaglesham et al., 1977). Should this high rate of biological nitrogen fixation be attained, then University of Ghana http://ugspace.ug.edu.gh 3even when grown in soils deficient in available nitrogen, (as is the case in many tropical soils), cowpea should be able to give optimum yields without requiring nitrogen fertilizer. However, at the farm level grain yields of cowpea are often low and inconsistent (Summerfield et al., 1974, FAO, 1998). One of the possible hypotheses is that this is due to inadequate infectivity or efficacy of the indigenous rhizobia to supply all the nitrogen required for optimum yields (Singleton and Taveres, 1986). This will suggest a potential for improvement in cowpea yields through the use of microbial inoculants Although several studies have reported that nodulation of cowpea in tropical soils could not be improved by inoculation (Doku, 1969; Kang et al., 1977; Rhodes and Nangju, 1979; Awonaike et al., 1990), in some cases increased yields were obtained (Danso and Owiredu, 1988; Rajput, 1994). The need for cowpea inoculation is therefore still controversial. In order to overcome and settle this, some well-defined studies are necessary, and include studies on the genetic variability of the indigenous rhizobial strains that have to be evaluated thoroughly (Richardson et al., 1995; Mpepereki et al., 1997; Martins et al., 1997) Indigenous rhizobia that are able to nodulate cowpea have been described as a heterogenous group, characterised by a high degree of symbiotic promiscuity (Hada and Loynachan 1986; Singleton et al., 1992). Cowpea rhizobia were thought to be slow growing and alkaline producing (Fred et al., 1932). However, several fast-growing strains have now been isolated (Zablotowicz and Foct, 1981; Dakora and Vincent 1984; Mpepereki et al., 1997). These findings indicate that there is possibly more to be examined on the taxonomic relationships of rhizobia that fix nitrogen in symbiosis with cowpea (Jordan, 1984). Several reasons may contribute to the difference in results, one of which may be some reported cases of negative response of cowpea to inoculation (Ezedinma, 1963; Kang et al., 1977; Rhodes and Nangju, University of Ghana http://ugspace.ug.edu.gh 41979; Awonaike et a l, 1990). Additionally, most of the agriculturally important tropical legumes are members of the cowpea cross inoculation group, and therefore they are often assumed to be less selective than other non-tropical legumes in the choice of ihe rhizobial micro symbiont. Thus rhizobial diversity of tropical soils is not considered as important (Dobereiner 1978; Halliday, 1985). An array of different methods including host range analysis, serology, antibiotic resistance and biochemical analysis have been used to describe cowpea rhizobia (Eaglesham et a l, 1987; Ahmad et a l, 1981; Mpepereki et a l, 1997). Even though these methods are important and play significant roles in the characterisation of rhizobial strains, for species identification and taxonomic purposes molecular analysis has become an effective method (Graham et al., 1991; Ludwig and Schleifer, 1994; Young and Haukka, 1996; Sessitsch et a l, 1997, de Lajudie et al., 1998). Moreover, molecular biology techniques have become indispensable for the analysis of biodiversity (Laguerre et al., 1994; Richardson et a l, 1995; Laguerre et al., 1996; Sessitsch et al, 1997; Vinuesa et al, 1998). The advantage of molecular tools is that because they are more precise, they are able to reveal the existence of marked genetic differences within a group of organisms even when other methods are not sensitive enough to detect differences (Martinez- Romero, 1994). This study aims at finding out the genetic diversity of native cowpea (brady)rhizobia isolated from soils in different agroecological zones in Ghana. A combination of morpho-physiological analyses and molecular methods was used to characterise the various isolates and in addition their nitrogen fixation effectiveness as well as competitive abilities were determined This University of Ghana http://ugspace.ug.edu.gh investigation is providing fundamental information that could pave the way for the improvement of the cowpea-Rhiznhium symbiosis. 1.3. Objectives of the study (i) To assess the potential to improve nitrogen fixation of cowpea in Ghanaian soils. (ii) To isolate, identify and analyse biodiversity of the indigenous rhizobia that nodulate cowpea (iii) To determine the symbiotic properties of the isolates. 5 University of Ghana http://ugspace.ug.edu.gh 6CHAPTER TWO 2.0 LITERATURE REVIEW 2.1.1 The nodule bacteria Bacteria capable of nodulating legumes belong to the family Rhizobiaceae. They are Gram- negative, rod-shaped, aerobic and mobile saprophytes (Schlegel, 1996). Five separate genera of the rhizobia have been proposed to date. These are Rhizobium (Buchanan, 1926; Jordan, 1984), Bradyrhizobium (Jordan, 1982), Azorhizobium (Dreyfus et al., 1988), Sinorhizobiwn (Chen et al., 1988) and Mesorhizobium Chen et al., 1995). Some of the phenotypic characteristics used to differentiate between the different rhizobial genera are growth rate, flagella type, and carbohydrate metabolism. Some of these traits appear to have been propagated in the literature even when they later have proven not to give definite inter or intra generic differences (Giller and Wilson, 1993). Growth rate shows a gradation of generation times and the conventional terminology of fast or slow growing can only be interpreted according to experience. The concept of fast-growing species being acid-producing and slow-growing being alkaline-producing (Norris, 1965), is also not absolute, and may strongly be affected by the growth medium (Hernandez and Focht, 1984; Ahmad and Smith, 1985; Dreyfus et al., 1988). Phenotypic features such as host range or carbon substrate metabolised seem rarely to be a reliable guide to species identification within a genus. Perhaps the most direct way to assign a new rhizobial strain is to gain some information about its host University of Ghana http://ugspace.ug.edu.gh 7range and to carry out some form of analysis that compares the genomic structure of the new strain to that of other named species with a similar host range (Giller and Wilson, 1993). Nowadays, bacterial taxonomy is to a greater extent build on DNA-based methods. The 16S rRNA gene has been considered as a useful parameter for phylogenetic analysis as it is constant in its function, present in all bacteria and contains highly conserved as well as more variable regions (Woese, 1987; Schleifer and Ludwig, 1989). It also constitutes a significant component of the cellular mass and is readily recovered from all types of organisms, providing adequate sequence information to permit statistically significant comparisons. Furthermore, a ribosomal database has been established (Maidak et al., 1994). In addition, DNA:DNA relatedness has been considered as an important criterion as it has been shown that the 16S rRNA gene sequence similarity among bacteria can be high although the DNA relatedness may indicate different species (Oyaizu et al., 1992; van Beikum eta!., 1996). 2.1.2 Rhizobium taxono my The taxonomy of the nodule bacteria has undergone considerable revision in the past two decades and is still in a state of transition (Graham et al., 1991; Elkan, 1992). When nodule bacteria were first isolated, they were called Bacillus radicola (Beijerinck, 1888). The following year, Frank (1889), published the name Rhizobium leguminosarum, which is still in use today. The taxonomy of the nodule bacteria was reviewed by Fred et al. (1932), and was based on the nodulation host range Over time, overlapping host ranges have been reported (Wilson, 1944), and rhizobia were found to be diversed in symbiotic, physiological and other properties. Consequently, Jordan (1982), described two genera, Rhizobium and Bradyrhizobium Rhizobium University of Ghana http://ugspace.ug.edu.gh 8strains were said to be fast growing whereas Bradyrhizobium strains were considered as slow growing. Since 1984, three additional genera, Azorhizobium (Dreyius et a l, 1988), Sinorhizobium (de Lajudie et a l, 1994) and Mesorhizobium (Lindstrom et al., 1995) have been described and accepted. All Bradyrhizobium strains that nodulate soybean effectively were until recently known as B. japonicum (Jordan, 1984). Kuykendal et a/.(1992), proposed the name B.elkanii for group ii strains, a fairly clear subgroup of soybean bradyrhizobia. Another species, B.liaoningense, has recently been proposed (Xu et a l, 1995), which is distinct by the criterion of DNA-DNA hybridization, besides having an exceptionally slow growth rate. All other slow growing strains were assigned to Bradyrhizobium spp, the so called cowpea miscellany rhizobia or tropical bradyrhizobia. There is only one species, Azorhizobium caulinodans, in the genus Azorhizobium, although a second species has been recognised by DNA-DNA hybridization (Rinaudo et al., 1991). A.caulinodans nodulates both stems and roots of Sesbania rostrata (Dreyfus et a l, 1988). The DNA sequences of the small sub-unit ribosomal RNA genes suggest that the genus Rhizobium may comprise several species. The species R.leguminosarum, R. tropici, R. etli and R.galegae are clearly separated groups and their taxonomy is also supported by similarities and differences in other properties (Lindstrom, 1989; Segovia et a l, 1993; Martinez-Romero et a l, 1991). De Lajudie et al. (1994) have proposed that the branch that includes Rjneliloti and Rfredii should be transferred to the genus Sinorhizobium. This genus was originally proposed by Chen et a/.(1988), to emphasise differences between R.fredii and other rhizobia including R.meliIoti, but subsequent work has shown that these two species are rather similar (Jarvis et al., 1992), and the new definition o f Sinorhizobium includes both of them, as well as two new species S.teranga and S.saheli (de Lajudie et a l, 1994, Young and Haukka, 1996). The name Mesorhizobium has been suggested to refer to the growth rate of University of Ghana http://ugspace.ug.edu.gh 9strains, which are intermediate between typical growth rates imRhizobium and Bradyrhizobium. This new genus comprises M. loti (Jarvis etal., 1992), M. huakuii (Chen etal., 1991), M. cicer (Nour et al., 1995), M. thianshanense (Chen et al., 1995) and M. mediterraneum (Nour et al., 1995). The 16S rDNA sequence information ofi? galegae which nodulates Galega species does not allow it to fit into any of the above mentioned generic groups (Lindstrom, 1989). The sequence shows close similarity to Agrobacterium and therefore it has been proposed to be transferred to a different genus (Young and Haukka, 1996). 2.1.3 Abundance in soil The usual source of nodule bacteria is the soil. Nevertheless, many soils remain devoid of rhizobial strains capable of nodulating particular crops; for instance, soybean rhizobia do not occur naturally in Australia (Diatloff and Brockwell, 1976). For this reason a large number of surveys have been conducted to establish the levels of rhizobia in soil. Surveys of rhizobia encounter certain technical problems as no defined culture medium exits for their selective isolation and reliance has to be placed on legume infection. Most probable number (MPN) assays are conducted whereby the soil is progressively diluted with sterile water, saline or medium, and the various dilutions inoculated onto seedlings raised from surface-sterilised seeds (Somasegaran and Hoben, 1985). In a soil dilution containing at least one Rhizobium cell, the assumption is that the microorganisms can multiply in the rhizosphere and infect the plant, which is then examined for nodules Great variation occurs in the population size of field rhizobia The presence of an appropriate host and its rhizosphere are major determinants (Hiltbold et al., 1985; Rupela et al., 1987; University of Ghana http://ugspace.ug.edu.gh 10 Woomer et al., 1988). Other factors are soil acidity (Rice et al., 1977), seasonal effects (Rupela et a l , 1987), and soil texture (Woomer etal., 1988). Additionally, many biological factors could also affect the rhizobial population size in the soil including antagonist that produce antibacterial metabolites and effects such as predation by protozoa or attack by bacteriophages (Dansoefa/., 1975; Barnet, 1980; Roughley, 1985). The size of indigenous rhizobial populations could present a competition barrier to the establishment of inoculant strains, possibly leading to inoculation failures in some cases (Thies et al., 1991). Singleton and Tavares (1986) found that substantial nodule occupancy by an inoculant strain could only be predicted if the indigenous population was less than 100 cells g soil 1 Similarly, Weaver and Frederick (1974) found that the number of nodules formed on soybean increased with increasing amounts of Bradyrhizobium japonicum inoculant, but this increase was not observed in soils containing more than 1000 rhizobia g 1 Some of the surveys on indigenous cowpea rhizobial populations in soils in Africa have not been very encouraging, for example, numbers ranging from undetectable to about 100 cells g soil*1 have been recently reported in some Zimbabwean soils (Mpepereki and Makonese, 1995). Similar population counts were detected in some Egyptian soils (Moawad et a l, 1994) Contrary to these low population counts, higher counts ranging from 610 to 4500 g soil' 1 were detected in three Ghanaian soils (Danso and Owiredu, 1988). However, a comprehensive survey of the population sizes of cowpea rhizobia in Ghanaian soils is required in order to predict the success of rhizobial inoculation over a wider range of soils. University of Ghana http://ugspace.ug.edu.gh 11 2.2.1 Nodule development Root nodule formation can be divided into three stages: 1. pre-infection, 2. infection and nodule organogenesis, and 3. nodule function and maintenance (Vincent, 1980). The pre-infection starts when rhizobia are attracted by chemostaxis to the organic compounds excreted by root haiis (Turgeon and Bauer, 1985; Nap and Bisseling, 1990; Gerahty et al., 1992), followed by the attachment of the bacteria to the root hairs leading to root hair deformation and root hair curling (Bauer, 1981). Several hypotheses have been suggested to explain the mechanisms involved in the latter process. Hirsch (1992) proposed that the Nod factor, embedded in the rhizobial membrane by the lipid tail, causes root hair deformation and cortical cell division by the reaction of the N - glucosamine residue of the Nod factor with a sugar - binding site of the plant receptor, presumably a lectin. The strength of this interaction, regulating early events in nodulation, depends on several properties: the length of the glucosamine backbone and the presence or absence of various subtituents like sulphate (Dazzo and Hubbell, 1982; Chrispeels andRaikhel, 1991). Dazzo etal. (1984) suggested that the adhesion of the bacterial symbiont to the root surface is the critical step prior to successful infection and development There is substantial experimental evidence that support this hypothesis, for example, the work of Diaz et al. (1989) showed that the introduction of a pea lectin into white clover root, resulted in the production of a hairy clover root. However, other workers have not been able to demonstrate specific attachment (Smit et al., 1986; Smit et al., 1992). Another model explaining the attachment of rhizobial cells to root hairs is that the microbes bind weakly to a plant receptor with a calcium - binding protein on the bacterial surface known as rhicadesin (Smit et al., 1991; Swart et a l, 1994) Tighter adherence then occurs by means of bacterial extracellular cellulose fibrils (Smitef a/., 1986). University of Ghana http://ugspace.ug.edu.gh 12 Attachment of rhizobia to the surface of root hairs produce many deformations including the characteristic shepherd crook (Bauer, 1981). At the centre of the crook, disruption of the plant cell wall occurs which enables the rhizobia to enter the root hair (Bauer, 1981). As they do so, a new structure, the infection thread forms within the plant cell and encloses the rhizobia. The rhizobia themselves proliferate in these cells until they have almost filled them. These proliferating cells remain within the root endodermis and form the nodule (Newcomb, 1981). 2.2.2 Types of nodules The type of nodule that develops depends on the host plant, not on the rhizobial strain (Dart, 1977). There are two main nodule types; the determinate and the indeterminate. In general, temperate legumes such as Pisum, Vida, Trifolium, and Medicago develop indeterminate nodules, while tropical legumes such as Vigna, Phaseolus and Glycine develop determinate nodules (Corby et al., 1983). Both types are composed of similar tissues, all formed from the nodule meristem (van de Wiel et al., 1990). Indeterminate nodules are characterised by a persistent apical meristem whilst determinate nodules do not have a persistent meristem (Newcomb, 1976). Determinate nodules grow for a fixed period, all parts of the nodule essentially differentiating at the same time and have a finite life span. In contrast, indeterminate nodules have an apical meristem that continues to be active throughout the lifetime of the nodule, producing zones of new infection, and so giving rise to a gradient of differentiation progressing back towards the root. Nodules differ in shape and size, partly as a response to soil conditions and partly as a characteristic of the particular bacterial strain-plant variety interaction (Lynch and Wood, 1989). University of Ghana http://ugspace.ug.edu.gh 13 They may be spherical, cylindrical, flattened and often bidentate or with coralloid branching, or may have an entirely irregular shape. Their size may vary from that of a pinhead to over 1 cm. The larger nodules are never spherical but have shapes giving a high ratio of surface area to volume, possibly to ensure an adequate supply of nitrogen gas to the active nodule cells and an adequate means of disposal of carbon dioxide produced in the nodule. In general, a nodule will only contain a single strain of Rhizobium, although dual occupancy of nodules is well documented (Lindemann et al., 1974; Johnston and Beringer, 1976; May and Bohlool, 1983; Moawad etal., 1984; Sessitsch etal., 1997). Once the bacteria have filled a proliferating cell, they change their form into bacteriods. Only bacteriods produce the enzymes required for nitrogen fixation. However, the nodule bacteriods may be effective or ineffective in fixing nitrogen. Lack of bacteriod persistence appears to be the most common cause of nodules being ineffective (Lynch and Wood, 1989). The ineffective nodules are often short lived, and they are also much smaller than effective ones. Although, they are typically far more numerous than the effective nodules, the total volume of ineffective bacteriod tissue per plant is much smaller and the colour is less pink (Lynch and Wood, 1989), indicating a lower leghaemoglobin content Usually, the legume root can carry only a limited number of nodules per unit length, hence if root growth ceases early in the season, the root system can become saturated with nodules. Once this has happened no further bacteria of any other strain can produce additional nodules on the root. Therefore, nodules already present on the root can affect the numbers of new nodules produced. Initial nodulation is as rapid with effective and ineffective strains, but the effective nodules inhibit further nodulation as soon as nodule growth has started (Kosslak and Bohlool, 1984). University of Ghana http://ugspace.ug.edu.gh 14 2.2.3 Factors affecting legume nodulation The most obvious requirement for a legume to form an effective nitrogen fixing symbiosis is the ability of the legume to nodulate (Giller and Wilson, 1993). Different Rhizobium species have various degrees of specificity or host preference (QuispeL, 1983). Therefore, if legumes are introduced into soils in which the appropriate Rhizobium species is absent, no nodulation will occur (Rupela et al., 1987). Certainly, it cannot be assumed that a legume can nodulate under all conditions just because it is a legume (Giller and Wilson, 1993). Apart from the need for the presence of the appropriate rhizobial species, nodule formation may also fail as a result of any of the following incompatibilities: (i) bacterial defect, (ii) hereditary defect in the legume and (iii) specific interaction between the bacterial and the legume (Lynch and Wood, 1989). Hostile conditions such as high temperature, drought, acidity and nutrient deficiencies can at several different levels of the nodulation process prevent nodulation (Day et a l, 1978). Excessive soil temperatures can kill the majority of the bacteria in the surface layers of soil, although some rhizobia are able to survive at 70°C in dry soil (Marshall, 1964; Danso, 1977). The Survival of bacteria in soil at high temperature appears to be improved by the presence of clay particles and soil organic matter (Day et a l, 1978). Differences in the effect of temperature on the ability of various Rhizobium strains to nodulate have been reported (Kvien and Ham, 1985; Piha and Munns, 1987). Nodules formed by an effective strain at high temperatures (35 and 38°C) were observed to be ineffective (Hungria and Franco, 1993). Furthermore, deeper- placed nodules have been observed to be more active in nitrogen fixation when top soil temperatures are high (Piha and Munns 1987). Several studies have assessed the effects of temperature on the growth of various legumes (Dart and Day, 1971; Date, 1989; Schombergand University of Ghana http://ugspace.ug.edu.gh 15 Weaver, 1992). The work of Montanez et al. (1995) indicated that while the growth of the legume was not very sensitive to high temperatures, the symbiosis was seriously affected. Elevated temperatures may delay nodule initiation and development and interfere with nodule structure and functioning especially in temperate legumes, whereas in tropical legumes, nitrogen fixation efficiency is mainly affected Moderate (30 — 20°C) day — night temperatures give early nodulation and high nitrogen fixation rates. However, duration of active nitrogen fixation is shortened because of rapid degeneration of nodules (Graham, 1979). Low temperatures delay root hair infection, and decrease nodulation and nitrogenase activity (Waughman, 1977). Legumes are intolerant to shortage and excess of water. This is primarily due to the ultrasensitivity of the symbiosis to water stress (Sprent, 1984). Infection is restricted in dry soils, because of absence of normal root hairs; short stubby root hairs appear, instead, which are inadequate for infection by rhizobia (Lie, 1981). Water stress reduces both nitrogen fixation and respiration of nodules, and within certain limits this reduction is proportional to the degree of water loss of the nodules (Guerin et al., 1990). Excess water is particularly detrimental to nitrogen fixation. A thin layer of water on the nodule surface reduces nitrogen fixation considerably presumably due to the low diffusion of oxygen. Build up of carbon dioxide may occur under waterlogged conditions, which at high concentration inhibit nodule formation (Guerin et al., 1990). Another gas known to be produced in anaerobic soils is ethylene, which, at low concentrations, can also restrict nodulation (Eaglesham and Ayanaba, 1984). Legumes are generally more sensitive to salinity than bacteria (Singleton et al., 1982). This is perhaps not surprising in view of the feet that in the symbiotic state rhizobia live within cells University of Ghana http://ugspace.ug.edu.gh 16 which have much greater solute concentrations than those generally experienced in soils (Sprent, 1984). The process of root hair infection of legumes in particular is sensitive to saline stress, perhaps due to the common cessation of root hair growth in these conditions (Sprent, 1984). It may also be caused by the bacterial partner as different strains of rhizobia were found to show marked differences in their ability to infect and form nodules on pigeon pea under saline conditions (SubbaRao etal., 1990). Among the grain legumes, cowpea and groundnut are more tolerant of soil acidity than are soybean or common bean (Munns, 1978). Large differences in sensitivity to the toxic effects of acid soils have also been found between different species of tropical pasture legumes (Andrew, 1976; de Carvalho et al., 1982). Acid soils usually have some inherent adverse concentrations of certain elements coupled with related nutrient deficiencies. The principal effects of soil acidity may be resolved into hydrogen ion concentration, deficiencies of calcium, phosphorus and molybdenum, and excessive quantities of aluminium and manganese. The presence of available aluminium in acid soils inhibits nodulation directly and indirectly by stunting root growth, and also inhibiting calcium uptake (Bell and Edwards, 1987). Nodule number decreases with decreasing calcium availability and with increasing aluminium level in the soil. Aluminium is a potent stress to the growth of fiee-living rhizobia. Fast-growing rhizobia appear to be less tolerant to aluminium than slow-growing rhizobia (Munns and Keyser, 1981). Aluminium not only prevents some plants from nodulating, but also delays and depresses nodulation. Effects of high pH on rhizobial growth, nodulation and legume growth have been reported to some extent in the literature. Yadav and Vyas (1971), in two surveys of 23 rhizobial isolates University of Ghana http://ugspace.ug.edu.gh 17 from eight legume species, reported that all grew well at pH values up to 10. By contrast, none of the 17 strains of Bradyrhizobium japonicum tested showed significant growth at pH 8.5 (DiatlofC 1970). However, beneficial effects on root hair infection and nodulation on alfafa were reported on high extremes of soil pH (Lakshi-Kumari et al., 1974), whereas pH above 6.0 reduced nodulation in lupins (Tang and Robson, 1993). Several of the nutrients essential for the growth of plants or bacteria play specific roles in the nodulation process and acute deficiency can hinder nodulation (O’Hara et al., 1988). Nutrients such as phosphorus and sulphur are required for nodule metabolism. When legumes dependent on symbiotic nitrogen fixation receive an inadequate supply of phosphorus, they also suffer from nitrogen deficiency. Under these conditions, nitrogen deficiency symptoms are dominant and can be alleviated by the application of phosphorus fertilizers (Dadson and Acquaah, 1984). Nitrogen has the most prominent influence on nodulation of legumes (Horst, 1986). This influence can be stimulating or depressing, depending on the level of available soil nitrogen and the legume. Except for the stimulatory effect of "starter" doses where nitrogen deficiency or hunger occurs in young seedlings, high levels of inorganic nitrogen are generally inhibitory to biological nitrogen fixation (Giller and Wilson, 1993). The effect of nitrogen on the different stages of the nodulation process has been examined, and all the various steps, from the induction of rhizobial nodulation genes, through root hair curling, penetration and infection thread formation, are inhibited to a greater or lesser extent by the presence of inorganic nitrogen (Carrol and Mathews, 1990). The result is that, the actual number of nodules formed is reduced, leading eventually to complete suppression of nodulation if concentrations exceed a certain threshold value This may vary from plant species to species (Harper and Gibson, 1984) and cultivar to cultivar (Hardarson et a l, 1984; Senaratne et a l, 1987). At intermediate University of Ghana http://ugspace.ug.edu.gh 18 concentrations the effect of nitrogen is manifested in the developing nodules being smaller, such that the nodule mass per plant is reduced while the total number of nodules remains almost unaltered (Streeter, 1988). Another effect of nitrogen is the actual inhibition of fixation in active nodules. This has been demonstrated both in greenhouse (Streeter, 1985) and in field-grown plants (Eardly et al., 1984). The requirement for molybdenum, the metal component of nitrogenase, explains the occurrence of nitrogen deficiency symptoms in legumes growing on soils low in available molybdenum (Parker and Hams, 1977). Cobalt is also required for the synthesis of leghaemoglobin. Therefore, in legumes, there is a close correlation between cobalt supply, nitrogen fixation and leghaemoglobin content of nodules (Chatel et al., 1978). Exploitation of the potential to increase nitrogen fixation in a legume requires a good knowledge of its nodulation capabilities. Studies on the genetics of legume nodulation have indicated that some legume genotypes have lost the ability to form effective nodules, whilst others have lost the ability to nodulate completely (Gibson, 1988; Vance et al., 1988). The presence of large genotypic variability for nodulation in cowpea has long been known (Zari et a l, 1978). Variations in nodulation within a species may be due to cultivar effects and duration of growth (Awonaike et al., 1990; Armstrong et al., 1994; Bell et al., 1994). Large genotypic variability for nitrogen fixation traits like nodule number, nodule mass and acetylene reduction activity per plant has been known since the early eighties for groundnut and pigeon pea (Nambiar et al., 1988) soybean (Wacek and Brill, 1976), cowpea (Zari et al., 1978), common bean (Graham and Rosas, 1977). These studies have shown that low as well as high nodulating lines occur among cultivars and that even non-nodulating plants occur among normal cultivars or races. University of Ghana http://ugspace.ug.edu.gh 19 Late maturing cultivars have been found to fix more nitrogen than early maturing cultivars (Rennie et al., 1982). However, within maturing groups only small differences in nitrogen fixation were observed (Paterson and La Rue, 1983). Rhizobium strains also differ in their ability to form nodules and to support nitrogen fixation and yield of legumes (Bezdicek et al., 1978; Rennie and Dubertz, 1984). The interaction between cultivar and strain has been reported to significantly influence plant diy weight, nitrogen yield, percent nitrogen derived from the atmosphere, and amount of nitrogen fixed in soybean (Senaratne et al., 1987; Rennie and Dubetz, 1984) Nitrogen fixation in some nodulated legume cultivars can be maximised by inoculating with effective Rhizobium strains. The need to inoculate is a question that is established in several ways (Allen and Allen, 1961; Roughley and Brockwell, 1987; Thies et al., 1991c). Field experiments have been designed to determine the need for inoculation (Bell and Nutman, 1971; Brockwell, 1971; Date, 1977; Thies et al., 1991b), but take several months to complete. Bonishy (1979), using dilutions of soil samples to inoculate clover seedlings growing aseptically in test tubes demonstrated a laboratory means for characterising simultaneously the size and nitrogen fixing capacity of soil-bome populations of rhizobia. Brockwell et al. (1988), developed this method into an expeditious assay which could be combined with a serial dilution plant infection technique for the enumeration of rhizobia. A related procedure (Thies et al., 1991c), makes it possible to forecast the likely success of introducing inoculant rhizobia into the soil, by considering indices of size of resident rhizobial population and the nitrogen status of the soil. A unique proposal for predicting the need for inoculation on a regional basis using a Geographical Information System has also been advanced by Thies et al. (1994). University of Ghana http://ugspace.ug.edu.gh 20 2.2.4 Longevity of nodule The amount of nitrogen fixed by a leguminous plant depends largely on the longevity of the nodules formed. Four factors affect nodule longevity or persistence: (1) the physiological condition of the plant, (2) the moisture content of the soil, (3) parasites in the nodule, and (4) the bacterial strain inhabiting the nodule (Lynch and Wood, 1989). The effect of the physiological condition applies particularly to annual plants whose nodules tend to die at flowering and seed set (Lynch and Wood, 1989). This is presumably because at this time the flowers and developing seeds are drawing on the carbohydrate reserves of the plant very heavily, and the young seeds may also be drawing on the nitrogen compounds in the nodules and leaves. Perennial legumes differ appreciably in the longevity of their nodules. Leguminous shrubs and trees may carry nodules for several years. Nodules seem to remain on the roots of many leguminous plants if soil is kept moist (Albrecht et al., 1984). The first effect at the onset of drought is for the plant to shed its nodules (Lynch and Wood, 1989), though unfortunately no systematic work has been done on the moisture deficiency, or the water tension in soil, at which shedding is severe. 2.3. Inoculation of legumes with rhizobia An important aspect of the successful exploitation of the Rhizobium - legume symbiosis has been the opportunity of introducing effective rhizobial strains into the soil. This is usually carried out at seed planting time with the aim of ensuring adequate nitrogen fixing rhizobia. Successful inoculation depends on the selection of the appropriate rhizobial strains and their provision in high enough numbers to colonise the developing root system. Inoculation is usually adopted for two main reasons. The first instance is when a legume is grown and ihe appropriate rhizobia are absent or present in numbers too low to form adequate nodules. A different and University of Ghana http://ugspace.ug.edu.gh 21 more difficult problem exits for many legume crops grown in the tropics. Here, the soil often contains abundant rhizobia capable of nodulating the crop but the indigenous rhizobia are often of low nitrogen fixing effectiveness (Nambiar and Dart, 1982). The most important point to consider is whether inoculation is needed Information regarding the nodulation of the legume species to be grown must be available, other important aspects are. the presence or absence of compatible rhizobia in the soil, their efficiency in fixing nitrogen and the previous cropping history of the soil. Where native rhizobia are ineffective in fixing nitrogen and the numbers fluctuate during the year, knowledge of their population shifts as well as their saprophytic competence is also important when legumes are to be inoculated (Obaton, 1975). Although information on the nodulation responses of legumes in tropical soils is scanty, a moderate amount of information on nodulation of cowpea is available (Giller and Wilson, 1993) Inoculation of cowpea in most of the humid tropics is considered to be unnecessary, due to the occurrence of large populations of highly competitive indigenous cowpea bradyrhizobia (Sellschop, 1962; Ezedinma, 1963; Doku, 1969; Ayanaba and Nangju, 1973), though in some cases inoculation has increased yields (Rotini, 1972; Danso and Owiredu, 1988; Rajput, 1994) Application of inoculants to the seed surface prior to sowing is the traditional and most commonly means of inoculation, although viability of the rhizobial inoculant strain is subject to the hazards of drying (Salema et a l, 1982), fertilizer contact (Kremer et al., 1982), seed coat toxicity (Materon and Weaver, 1984), incompatible pesticides and mineral additives (Gault and Brockwell, 1980) and inimical soil factors (Kremer and Peterson, 1983). Proposals to extend the life expectancy of rhizobia on seed, including curing inoculants before use (Burton, 1976; Materon and Weaver, 1985), and suspending cultures in alginate gel rather than sucrose before University of Ghana http://ugspace.ug.edu.gh 22 their application to the seed (Rawsthome and Summerfield, 1984), have been adopted. There are some situations where seed application of rhizobia may be an inefficient means of inoculation. For example, for seeds dressed with a pesticide incompatible with rhizobia, for inoculation of broad acre sowing of crop legumes with high seeding rates, and for seeds such as peanut which are too fragile for seed-surface inoculation (Brockwell, 1982). Preparations and procedures for inoculant application directly into the seed bed have been developed, either as solid inoculant (Barkdoll et al., 1983; Hegde and Brahmaprakash, 1992) or liquid inoculant (Hely et al., 1980). These methods have proved to be more effective in most cases than conventional seed inoculation for initiating nodulation and nitrogen fixation (Danso et al., 1990; Rice and Olson, 1992; Rice et al., 1998), but are often more labourious. The normal criteria for rhizobial strain selection for inoculant production have been summarised by Keyser et al. (1992). Where there are large indigenous populations of rhizobia with poor effectiveness, it is necessary to add a further criterion to strain selection, that of competitiveness (Keyser et al., 1992). The latter characteristic is very difficult to define and may vary from soil to soil. There is no correlation between effectiveness and ability to compete with other strains. Beattie et al., (1989), stated that competitiveness reflects the behaviour of a particular strain in a particular soil with a particular plant and in a particular season. If any other factor is altered the competitiveness may be altered. Simply increasing the number of inoculant rhizobia is not always a guarantee of success as a good competitive strain, however, ineffective, may be able to overcome a numerical disadvantage of as high as 1:1000 as observed by Lynch and Wood, (1989). At least part of the competition phenomenon is related to the pre-infection stage of rapid rhizobial growth in the rhizosphere (Keyser et al., 1992). University of Ghana http://ugspace.ug.edu.gh 23 Very useful information can be gained from laboratory and greenhouse trials, but the eventual performance of an inoculant must be evaluated from field trials in a variety of soils. Some insurance against poor rhizobial performance in specific situations can be gained by using inoculants that comprise a mixture of strains. Here the precaution needed is that the strains are not mutually antagonistic 2.4. Diversity of rhizobial isolates Successful management of symbiotic associations between plants and their bacterial endosymbionts requires that specific strains of the bacteria can be readily identified. (Richardson et al., 1995; Mpepereke et al., 1997; Di Cello et al., 1997). Any microbial utilisation in agriculture requires an evaluation of the environmental risks that are associated with the introduction of indigenous or non indigenous microorganisms into the rhizosphere (Di Cello et al., 1997). It also requires assessment of the most suitable conditions for the effective and successful establishment of the inoculum in the rhizosphere of the host plant (de Leij et al., 1994; 1995) Analysis of the structure of the microbial population therefore has practical importance; the results can he used to assess the fate of released strains and their impact on resident microbial communities. African soils may harbour a large diversity of rhizobial populations, however, only little information is available. Cowpea rhizobia indigenous to Nigerian soils are probably the only group that has been studied (Ahmad et al., 1981; Eaglesham et al., 1987; Sinclair and Eaglesham, 1984) Assessing the diversity of West African cowpea bradyrhizobia based on physiological and biochemical characteristics, Eaglesham et al. (1987), found some traits University of Ghana http://ugspace.ug.edu.gh 24 common to all or most of the isolates, some related to geographical origin and some colony morphology. The indigenous cowpea rhizobia strains in Zimbabwean soils showed considerable cultural and physiological diversity that included unique types belonging to several, as yet undefined species (Mpepereki et al., 1997). Recent studies on fast growing rhizobia of Sesbania and Acacia species obtained from soils in Senegal, has led to the description of two fast growing species, Sinorhizobium saheli and S. teranga (de Lajudie et al., 1994). Slow growing rhizobia nodulating soybean in Zimbabwean soils have also been reported by Davis and Mpepereki, (1994). In Ghana, the diversity of rhizobial populations of different legumes has not been examined. Several classical and/or molecular techniques are available for the identification and analysis of the biodiversity of bacterial strains of a natural population. They include intrinsic antibiotic resistance (Muller et al., 1988), serology (Ayanaba et al., 1986), biolog automated analysis (Klinger et al., 1992), multilocus enzyme electrophoresis techniques (Wise et a l, 1996), PCR ribotyping (Kostman et al., 1992), and the random amplified polymorphic DNA (RAPD) method (Fani et al., 1993). Analysis of the diversity of the cowpea rhizobia in this thesis was carried out by a combination of classical and molecular methods which had previously been successfully applied to studies of rhizobial populations isolated from different environments (Sessitsch et a l, 1997; Santamana et a l, 1997; Tyler et a l, 1997; Zewdu e ta l, 1998; Vinuesa e ta l, 1998). University of Ghana http://ugspace.ug.edu.gh 25 2.4.1. Methods for analysing diversity of rhizobia 2.4.1.1 Cross inoculation The cross inoculation group concept is based on the ability of Rhizobium strains to specifically nodulate a group of legume host species (Fred et al., 1932). Based on this concept, rhizobial strains have long been described as specific for strains apparently restricted in their host range or promiscuous for strains with a very broad host range (Burton, 1972). However, examination of a wide range of species has shown that many legumes are nodulated by rhizobia outside their own groups (Thies e ta l, 1991). Consequently, the integrity of the cross inoculation concept as a system for determining relatedness among rhizobia strains has been questioned (Wilson, 1944; Bromfield and Barran, 1990), and is now in general disrepute. Many Rhizobium strains have also been found to be so promiscuous that their host ranges do not even consist of closely related legumes, but may include legume plants that are so distantly related as to be placed in different sub-families within the leguminosaese. For example, the last growing Rhizobium strain NGR234 has been shown to elicit nodules on over 37 genera of legumes including members of different sub families such as Lablab purpureus and Leucaena leucocephala (Trinick, 1980). The cross inoculation concept like the other methods for the characterisation of Rhizobium strains pays no attention to nitrogen fixation abilities. It is common for instance, to find strains of rhizobia that can elicit nodules on say ten different legume host species and yet in association with perhaps five of those host plants fix nitrogen only weakly or not at all (Wilson et al., 1987). In view of these negative features of the cross inoculation concept, its continued usage in systematics has been justified on the basis of convenience and agronomic significance (Graham et al., 1991). The concept actually has some University of Ghana http://ugspace.ug.edu.gh 26 practical use for selecting rhizobial strains which have the potential to be used as inoculants for particular legume crops (Mpepereki et al., 1996). Using the cross inoculation classification system, Habish and Khairi (1968), showed that most grain legumes including cowpea could be grouped accordingly, even though the cowpea group showed some inconsistencies. Thies et al., (1991), also found that only 18% of cowpea-derived isolates formed effective nodules on peanut, although peanut nodulates heavily in most African soils. Similar results were obtained by Mpepereki et al., (1996), who concluded that the indigenous cowpea rhizobia of Zimbabwean soils had relatively narrow host ranges. These reports contradict the statement that cowpea rhizobia indigenous to African soils are promiscuous, and nodulates a wide range of legumes (Singleton etal., 1992). These bring to the fore that very little is actually known about the symbiotic characteristic of the cowpea rhizobia indigenous to African soils. The cross inoculation concept was therefore used in this study to (i) determine the extent of symbiotic specificity of indigenous cowpea rhizobia isolated from a range of physiographic environments in Ghana and (ii) characterise the indigenous rhizobia. 2 .4 .1.2 Cultural and metabolic characterisation Cultural and metabolic characteristics have been described as useful guides for the recognition of rhizobial groups at the species level (Vincent, 1970). Rhizobial strains may be recognised as such by a combination of a large number of traits such as growth characteristics, carbon source utilisation, stimulation by sugars or vitamins, limits of pH, temperature tolerance and production of hydrogen sulphide (Vincent, 1970). This variety of tests by which the root nodule bacteria could be characterised was suggested by Lange (1961), as a way to resolving the taxonomic University of Ghana http://ugspace.ug.edu.gh 27 difficulties within the genus Rhizobium. Meanwhile, many researchers consider the above mentioned methodology as impractical (Graham and Parker, 1964). However, cultural and metabolic parameters are used for a phenotypic characterisation that is frequently earned out in combination with an analysis of the genotype. A central dogma in rhizobiology is that only two different types of rhizobia exist, the so-called slow-growing strains that nodulate tropical legumes and the fast-growing strains that nodulate temperate legumes (Jordan, 1984). Evidence has however, accumulated through a variety of cultural and physiological tests which show that some tropical legumes are nodulated by both fast and slow-growing rhizobial strains (Lim and Ng, 1977, 1979; Pankhurst, 1977; Keyser et al., 1982; Lawrie, 1983; Broughton et al., 1984; Padmanabhan et al., 1990; Mpepereki et al., 1997). The results of these reports point to the possible existence of several unique but unidentified Rhizobium species that nodulate tropical legumes. Currently, no data exist on the cultural and physiological characteristics of the indigenous rhizobial populations of Ghanaian soils. However, it is important to describe the cultural and physiological characteristics of the native rhizobia since it serves as a guide to species identification (Vincent, 1970). It also provides information about the performance of rhizobial strains under stress conditions, which gives additional criteria for the selection of rhizobial strains for inoculant production. University of Ghana http://ugspace.ug.edu.gh 28 2.4.1.3. Serology Serological analysis characterises rhizobia according to their reactions with antisera produced against strains having some agronomic or particular interest. The most common serological methods currently used are agglutination (Means et al., 1964; Date and Decker, 1965, Wollum, 1987), flourescent antibody techniques (Bohlool, 1987) and various forms of the enzyme-linked immunosorbent assay (Asanuma et al., 1985; Ayanaba et al., 1986; Fuhrmann and Wollum, 1985; Kishinevsky and Jones, 1987). Polyclonal antisera have been used in most cases, although monoclonal antibodies have been used successfully with soybean bradyrhizobia (Vellez et al., 1988). Serological studies of indigenous rhizobia have revealed considerable diversity within and among rhizobial from different geographysical locations. In some instances it has been possible to correlate the presence of particular serogroups within a restricted region to soil properties such as pH (Damirgi et al., 1967; Ham et al., 1971) or total nitrogen content (Bezdicek, 1972). One common goal of serological characterisation of bacterial strains is to identify groups that have practical importance to the management of a particular symbiosis. Yet a/though many studies have documented serological diversity within rhizobial populations, relatively few have assessed the value of the resulting groupings in predicting symbiotic performance. One problem with using serology to characterise rhizobia is the presence of strains that do not react with all antisera tested and the frequency of non-reactive strains is often significant (Fuhrmann, 1990; Mpepereki and Wollum, 1991). Furthermore, serological analyses commonly reveal strains that cross - react with antisera derived from two or more reference strains. The best documented example of this is the suite of soybean bradyrhizobia that constitute serocluster 123 (serogroups University of Ghana http://ugspace.ug.edu.gh 29 123, 127 and 129) (Schmidt et a l, 1986). Although related serologically, the serogroups comprising this serocluster are known to exhibit physiological and symbiotic diversity (Gibson etal., 1971; Hickey etal., 1987; Sadowsky e ta l, 1987). Numerous studies have reported that the incidence of serogroups of Bradyrizobium japonicum in root nodules of soybean can be affected by the plant genotype (Caldwell and Vest, 1968; Caldwell and Hartwig, 1970; Kvien et a l, 1981; Cregan and Keyser, 1986). However, the results of Fuhrmann (1989), suggested that the influence of plant genotype is minimal. The incidence of different serogroups have also been attributed to the sampling location or soil type (Ham et al., 1971; Keyser et a l, 1984; Kamicker and Brill, 1986.). Caldwell and Hartwig (1970), examined the effect of the location as well of the plant genotype on the serological distribution of B. japonicum in field sampled nodules. The study revealed high significant effects of similar magnitude for both variables. An analysis of cowpea miscellany rhizobia isolated from three West African sites showed that only 50% of the 53 isolates examined were serologically typed; those with dry colony morphology were found to be serologically reactive and diverse, whereas wet strains were found to be unreactive (Ahmad etal., 1981). Similarly, in a study of rhizobial isolates from groundnut grown in Sudan, high cross reactivity of surface antigens were observed and strains with no cross-reactivity were found to have different colony morphologies (Hadad and Loynachan, 1986). University of Ghana http://ugspace.ug.edu.gh 30 2.4.I.4. Molecular analysis The polymerase cfiain reaction (PCR) The polymerase chain reaction was developed about a decade ago (Mullis and Faloona, 1987). Yet PCR-based techniques have been shown to be one of the most effective means of differentiating complex genomes (Williams et al., 1990; Welsh and McClelland, 1990; Caetano- Anolles et al., 1991). Various PCR-based techniques have been used to differentiate the genomes of a wide range of diverse organisms including Rhizobium (Harnson et al., 1992; Richardson et al., 1995; Sessitsch et al., 1997). PCR is basically a biochemical amplification process where a single target DNA segment can be amplified a million-fold or more in several hours (Mullis and Faloona, 1987). The main feature of PCR is that, if the sequences of DNA flanking an unknown region of a DNA molecule are known, the unknown DNA can selectively be copied repeatedly to generate large quantities of DNA copies for further analysis (Wilson and Walker, 1995). Analysis of the amplification product is usually done by standard agarose or polyacrylamide gel electrophoresis. A standard PCR protocol employs DNA primers, usually 10 - 20 bases in length, which have been synthesized complimentary to specific, known segments of the target DNA The primers are complimentary to positions of opposite strands of the target DNA The primers are added to the test sample in a buffered solution containing a balanced mix of the four deoxynucleotides, magnesium chloride and a heat resistant DNA polymerase. A thermocycler is used to provide a strict regime of temperature cycling, and each cycle consists of a denaturation, an annealing and an extension step For denaturation, the temperature is raised to 94 - 95°C in order to melt the target DNA and subsequently lowered to 30 - 55°C to allow the primer to anneal with the University of Ghana http://ugspace.ug.edu.gh 31 corresponding sequence of the template. The annealing temperature depends on the melting point of the primer — template complex. Finally, a temperature of usually 72 C is used for the DNA synthesis of the new DNA molecule. The PCR consists typically 30 to 35 cycles. The utilisation of PCR in conjunction with either arbitrary or directed primers to differentiate individual strains of Rhizobium has proved to be highly useful for ecological studies. Potential applications ofPCR-based methods include: (i) the authentication of specific inoculant strains, (ii) the determination of nodule occupancy in competition trials where mixed inocula are used (iii) the analysis of the recovery and persistence of inoculant strains under field conditions (iv) the identification of predominant nodulating strains from particular sites, and (v) the assessment of the genetic diversity and relatedness of rhizobial field populations (Richardson et al., 1995). The PCR technique has several distinct advantages over more conventional techniques that have been used for Rhizobium strain identification. These advantages comprise a limited requirement to extensively purify and culture large amounts of rhizobial isolates prior to their identification, the precision with which individual strains can be identified, the rapidity of the procedure, and the large number of isolates that may be handled at one time (Richardson et al., 1995). More importantly, the procedure does not necessarily require a detailed prior knowledge of the individual strains, and there is no need to specifically mark target strains. Therefore, PCR-based fingerprinting techniques allow useful information pertaining to any particular Rhizobium strain or isolate, or the rhizobial population itself, to be readily obtained (Richardson et al., 1995). However, one of the limitations of this methodology are that its huge amplification capacity makes the system very vulnerable to errors from contamination; even a trace of foreign DNA, such as may be present in dust particles, will be amplified to significant levels and may give University of Ghana http://ugspace.ug.edu.gh 32 misleading results (Wilson and Walker, 1995). Also at low population densities, PCR signals are difficult to interpret, because free DNA can also be amplified. The application is therefore mostly qualitative although attempts are being made to quantify the PCR signals (Simonet et a l, 1990; Hill e ta l, 1991; Wimpece ta l, 1991). The development of the PCR has led to a battery of new fingerprinting methods. For instance, it had been shown that DNA primers corresponding to repetitive extragenic palindromic (REP) (Stem et al., 1984) and enterobacterial repetitive intergenic consensus (ERIC) (Hulton et al., 1991) sequences, coupled with the polymerase chain reaction technique, can be used to fingerprint the genomes of a variety of gram-negative soil bacteria (Versalovic et a l, 1991; de Bruijn, 1992; Sessitsch et al., 1997). The REP and ERIC sequences contain highly conserved central inverted repeals, do not show significant homology to each other, and are normally found in intergenic transcribed, but not translated, regions (Versalovic et a l, 1991; Lupski and Weinstock, 1992). REP and ERIC PCR have been demonstrated to be a powerful tool for community analysis at the strain level (Judd et a l, 1993; Laguerre et al., 1996; Sessitsch et a l, 1997). In addition, PCR-based methods using short oligonucleotide primers, that bind to random sequences of the genome, have proven to be a valuable means to generate strain-specific fingerprints of Rhizobium (Dooley et al., 1993; Kay et al., 1994; Selenska-Pobell et al., 1995). Based on a reiterated Rhizobium n if promoter consensus element, Richardson et al., (1995) developed the RPOl-directed primer that also has been demonstrated to be suitable to fingerprint rhizobial genomes (Richardson e ta l, 1995; Sessitsch etal., 1997). Various methods employ PCR-amplified DNA as a substrate for a restriction analysis leading to a specific restriction fragment length polymorphism (RFLP). Nitrogen fixation (nif) and University of Ghana http://ugspace.ug.edu.gh 33 nodulation (nod) genes have been analysed for the genetic characterisation of rhizobial strains (Laguerre et al., 1996). However, the ribosomal operon has been looked at most frequently by PCR-RFLP analysis (Nour et a l, 1994; Massol-Deya et al., 1995; Sessitsch et al., 1997). The sequence of the small sub-unit of ribosomal, the 16S rRNA gene, is a highly suitable phylogenetic marker (Woese, 1987; Schleifer and Ludwig, 1989), and genetic variation within this molecule give useful information on microbial taxonomy. Therefore RFLP analysis of PCR-amplified 16S rRNA genes has been used frequently for Rhizobium species identification (Laguerre et a l, 1994; Sessitsch et a l, 1997). Greater discrimination can be obtained by analysis of the intergenic spacer (IGS) between the 16S and the 23 S rRNA genes and has been applied to examine chromosomally encoded genetic variations at the strain level (Barry et a l, 1991; Sessitsch etal., 1997). Differentiation of rhizobial isolates indigenous to tropical soils using molecular methods is yet to be exploited. However, the realisation that genetic characterisation underlies the diverse expressions of life, led to the use of the PCR-based methods in this study in order to enable a holistic understanding of the relationship among cowpea rhizobia. 2.5. Symbiotic effectiveness Determination of the population size of indigenous rhizobia and their diversity do not provide information on symbiotic effectiveness. However, it is important to know to what extent the native rhizobial strains nodulate cowpea effectively, because a low rate of nitrogen fixation can occur with normal nodule appearance (Eaglesham, 1985). Singleton and Tavares (1986), found that within a soil sample, the range of effectiveness of indigenous rhizobial strains differed significantly. This indicates that considerable diversity in the relative effectiveness of the University of Ghana http://ugspace.ug.edu.gh 34 indigenous rhizobia forming symbiotic association with cowpea may exist. Such population diversity may be reflected in differences in the sizes of indigenous rhizobial populations (Singleton and Tavares, 1986; Thies et a l, 1991). The results of screening 23 cowpea rhizobia in two field sites in Nigeria showed that only 30% of the isolates were effective (Ahmad et al., 1981) Ferreira and Marques (1992), found great variation in effectiveness among 170 strains isolated from native clover, with a predominance of strains with medium and high effectiveness. Similar findings were reported by Fredericks et al,. (1990), with clover isolates obtained from Ethiopian soils. In contrast to these results, Gibson et al., (1975), observed only small differences in effectiveness among Rhizobium trifolii populations from eight regions in Australia. Lie and Goktan (1984) as well as Lie etal. (1987) also verified that the symbiotic variation among the European Rhizobium population nodulating pea was relatively small. Differences in effectiveness among cultivars of legume species may occur. This hypothesis was supported by Robinson (1969), who reported that rhizobia isolated from Trifolium pratense were ineffective on T. subterraneum Similar findings were reported by Ferreira and Marques (1992). Several environmental factors that may affect the symbiotic effectiveness of a rhizobial strain have been suggested. Holding and King (1963) found that the mean effectiveness of Rhizobium trifolii was significantly correlated with the base saturation, pH, as well as the exchangeable calcium and exchangeable magnesium content of a soil. Similar results were obtained by Hagedom (1978), who correlated the poor effectiveness of Rhizobium trifolii populations with soil base status and phosphorus levels. These results however contradict with that of Ferreira and Marques (1992). They showed that soil type and plant origin did not influence general University of Ghana http://ugspace.ug.edu.gh 35 effectiveness of natural populations of Rhizobium leguminosarum bv. trifolii. Brockwell and Katznelson (1976) also found that soil type did not affect the general effectiveness of natural populations Rhizobia are well adapted to life in the free-living state in soil and can survive for over 30 years even in the complete absence of legumes (Martesson and Witter, 1990). However, with time, genomic changes may occur (Weaver and Wright, 1987; Gibson et a l, 1975). Gibson et a l (1990) observed that the symbiotic effectiveness of Rhizobium leguminosarum bv trifolii re-isolated from the field after several years differed from the parent culture. The Rhizobium genome carries a high number of repeated sequences (Flores et a l, 1988; Martinez-Romero and Palacios, 1990; Brom e ta l, 1991), which cause recombination and lead to rearrangements and deletions (Hahn and Hennecke, 1987). 2.6. Competition for nodule occupancy Competition in broad terms refers to interactions between two or more organisms struggling in order to gain advantage over limited resources such as nutrients, water, light and space, that are present in the environment in an amount insufficient to meet the biological demand (Alexander, 1971). In the case of rhizobia, competition is most commonly used to refer to struggle for supremacy in nodule occupancy. Rhizobial strains differ greatly in competitiveness, a feet that underlies the observed differences in percentage of nodules formed by individual strains present in the same root environment of legumes. A number of reviews are available on rhizobial competition (Date and Brockwell, 1978; Amarger, 1984; Dowling and Broughton, 1986; Triplett, 1990; Bottomley, 1992) Each summarises the profuse literature which illustrates the fact that different rhizobial strains may show different abilities to compete for nodule occupancy. Using three streptomycin-resistant cowpea bradyrhizobial strains, Danso and University of Ghana http://ugspace.ug.edu.gh 36 Owiredu (1988) showed that the three strains differed in their inherent competitive abilities, which was revealed by great differences in nodule occupancy when the strains were equally represented in mixed inocula. Relative success in achieving nodule occupancy is affected by environmental factors, host plant species and cultivar, initial population size and distribution in the soil and by competition from other organisms (Bottomley, 1992). Soil is the reservoir of Rhizobium strains and the intrinsic make up of the soil can affect the outcome of competition (May and Bohlool, 1983; Moawad and Bohlool, 1984). Rhizobium strains do not respond equally to the application of nitrogen. McNeil (1982), found that one strain of B.japonicum out-competed another for nodulation sites in the presence of nitrate. Correspondingly, this strain would have an advantage in soils with relatively high nitrogen levels. A study on competition of two serogroups of R trifolii in a tropical soil demonstrated that when the soil was limed (to increase available phosphates) the dominant serogroup was replaced by a minor one (Almendras and Bottomley, 1985). The addition of phosphate alone had little effect on the outcome of competition between the two strains. However, the addition of phosphate and lime restored the dominance of the original serogroup (Almendras and Bottomley, 1985). This suggests that phosphorus limitation is exacerbated by low pH and the combination of pH and phosphorus levels can have a strong influence on competition. The effect of soil pH on rhizobial competition is well documented. Dughri and Bottomley (1983) were able to alter the outcome of competition between indigenous rhizobia in the soil by changing the pH. When Russel and Jones (1975) adjusted soil pH from acid to neutral by liming, they inadvertently favoured nodule formation by the ineffective strain. Soil temperature can also alter the outcome of competition between strains. In mixed inoculants containing Vigna rhizobia as well as B. japonicum applied to a promiscuously nodulating University of Ghana http://ugspace.ug.edu.gh 37 cultivar of Glycine max, the Vigna rhizobia were found to be more competitive at higher temperature (36°C) whereas B japonicum competed better at lower temperature (24- 30°C)(Roughley et a l, 1980) Drought conditions, which are often found in conjunction with salt stress, also affect Rhizobium competition (Joshi et a l, 1981; Osa-Afiana and Alexander, 1982). Bradyrhizobium strains isolated from arid areas were found to be more tolerant of desiccation than strains isolated from cooler, wetter regions (Hartel and Alexander, 1984). Rhizobial strains differ in their motility in soil and it has been suggested that motile strains may be able to occupy lateral roots resulting in late stage nodulation and increased higher nitrogen fixation (Wadisirisuk et al., 1989). Rhizobia must multiply in the rhizosphere and attach to the host plant in order to initiate nodulation The macrosymbiont therefore plays an important role in competition (Hardarson et al, 1982; Cregan and Keyser, 1989). In a study by Moawad et a l (1984), competition among three serogroups of B japonicum was examined in the rhizosphere and in non-rhizosphere soil. They observed that the Bradyrhizobium populations increased gradually from 104g * to 10^g * in the rhizosphere soil during the first weeks after planting Glycine max, while the numbers in fallow or non-rhizosphere soil remained at 104g_1. Furthermore, Moawad et al., (1984) reported that there were no significant differences between the abundance of the three serogroups, and serogroup 123, which formed 60 -100% ofthe nodules, and thus showed no obvious dominance in the rhizosphere. Other authors have shown that the proportion of nodules formed by a strain could be correlated with its relative representation on the root surface (Marques et a l, 1974; Franco and Vincent 1976). University of Ghana http://ugspace.ug.edu.gh 38 The soil population density of indigenous rhizobia is a major factor in determining competition for nodule occupancy. In field trials Weaver and Frederick (1974), demonstrated that, nodule occupancy of greater than 50% by an inoculant strain was achieved by applying it at a rate of at least 1000 times greater than the estimated number of indigenous rhizobia. Several authors have actually demonstrated that there is little chance of inoculated strains forming many nodules if there is a significant indigenous population of compatible rhizobia (Carter et al., 1995). The negative response of cowpea to rhizobial inoculation in tropical soils for instance, has been attributed to the occurrence of large populations of highly competitive indigenous cowpea rhizobia (Sellschop, 1962; Kang etal., 1977). Numerous efforts have been made to bias the outcome of competition between rhizobial strains. These include the improvement of the inoculant formulation (Zdor and Pueppeke, 1990; Zablotowick et al., 1991), the application of extremely high inoculation rates (McLoughlin et al., 1990a and b) and variation in the inoculum placement (McDermott and Graham, 1989). Breeding programmes with this as a major objective have also been carried out For example, experiments have been directed towards the selection of highly effective combinations of host plant cultivar and bacterial strains (Alwi et al., 1989) or the development of host lines with restricted nodulation and thus are able to bypass the native soil rhizobia (Cregan and Keyser, 1986; Montealegre and Kipe-Nolt, 1994). The other approach involves screening for plants that are nodulated by the most effective strains present in a natural soil population (Kueneman et al., 1984; Herridge and Rose, 1994). University of Ghana http://ugspace.ug.edu.gh 39 Bacteria are almost always haploid; they possess only one single set of genes, but they also form zygotes. These are never the result of fusion of whole cells. Usually a part of the genetic material of a donor cell is transferred to a recipient (acceptor) cell, so that a partial zygote (Merozygote) is produced. The subsequent chromosomal and cell division results in a cell that contains only the recombined chromosome (Schlegel, 1996). Three types of transfer of genetic character are known in bacteria: conjugation, transduction and transformation. In the course of all three processes, DNA is transferred from a donor bacterium to a recipient bacterium. The process differs only in the manner in which the DNA is transported. The transfer process is immediately followed by DNA recombination in the recipient cell (Schlegel, 1996). Transfer of genetic material between Rhizobium strains in the legume rhizosphere has been observed to occur most frequently by conjugation (Broughton etal., 1987; Sullivan et al., 1995; Bo Normander et al., 1998), although transformation by circular plasmid DNA and bacteriophage-mediated transduction also occurs (Kiss and Kalman, 1982; Finan et al., 1984; Martin and Long, 1984). Most fast-growing strains of Rhizobium harbour symbiotic genes in large plasmids (Hombrecher et al., 1981; O’Connel e ta l, 1984), and some of these plasmids are self transmissible (Johnston et a l, 1978). In addition, Rhizobium plasmids are capable of genetic recombination, producing novel plasmids (Djordjevic et a l, 1983). Thus, genetic exchange among rhizobia and genomic instability of Rhizobium (Flores et al., 1988; Brom et al., 1991) can lead to altered competitiveness and nodulation properties. University of Ghana http://ugspace.ug.edu.gh 40 2.6.1. Molecular gene markers in competition studies Low symbiotic nitrogen fixation in plants is in many cases a result of competition between effective and ineffective rhizobia for nodule occupancy (May andBohlool, 1983). In such cases, solving the rhizobial competition problem is essential in order to improve the symbiotic interaction between bacteria and plants. Apparently, the lack of suitable methodology to properly identify rhizobial strains has been the greatest barrier (Wilson, 1995). Evaluation of the competitive ability of rhizobial strains has been done by employing intrinsic (Broughton et a l, 1987) or induced (Bushby, 1981; Danso and Owiredu, 1988) antibiotic resistances as identifying markers. Other markers used include the enzyme linked immunosorbent assay (ELISA) (Beiger et al., 1979). Analysis of plasmid profiles has also been used in rhizobial competition studies (Broughton et al., 1987; Pepper e ta l, 1989; Shishido and Pepper, 1990). Techniques also have been developed by which a specific marker gene can be introduced into the genome of the organism to be studied. The marker gene codes for an enzyme that gives rise to a coloured product following incubation with a histochemical substrate. The marker gene thus allows the visual detection of the marked organism. Such a marker gene in current use in ecological studies of rhizobia is the gus A gene encoding the enzyme B -glucuronidase (GUS) (Jefferson et a l, 1987; Wilson et al., 1991; Wilson et al, 1995). GUS is a hydrolase that cleaves a wide range of substrates - almost any aglycone conjugated to D - glucuronic acid in the configuration. Frequently used substrates are X-gluc and Magenta-gluc giving rise to a blue or red colour. Nodules occupied by gw.s/4-marked rhizobia are detected by virtue of a simple colour change (Wilson, 1995). The greatest advantage of GUS is the nearly complete lack of endogenous activity in plants and most agriculturally important bacteria. The assay has so far been used with bradyrhizobia that nodulate cowpea (Wilson et al., 1991) and Rhizobium sp. inoculated onto University of Ghana http://ugspace.ug.edu.gh 41 siratro and pigeon pea (Wilson et al., 1992; Wilson, 1995). Furthermore, the gusA marker gene has been used for competition studies of R. etli (Streit et al., 1995) and R. tropici (Sessitsch et al., 1997). Another marker gene that has been used for the detection of bacterial strains and that can be used in combination with gusA, is the celB gene (Sessitsch et al., 1996). It encodes the enzyme IJ-glucosidase with a high galactosidase activity that is thermostable and thermoactive and has a half time of 85 hours at 100°C (Voorhorst et al., 1995). Assays for the detection of celB activity within a nodule or on plant sample are simple. The washed legume root is incubated in phosphate buffer at 70°C in order to destroy endogenous enzymes. The roots are then incubated in the presence of a chromogenic substrate for celB product such as X-gal (5- bromo-4-chloro-3-6-D-galactoside) giving rise to a blue colour (Sessitsch etal., 1996) To date only a limited amount of data has been collected on the impact of inserted genes on the ecological fitness of the host organism (Doyle et al., 1995). Initially it was widely assumed that genetically engineered organisms (GEMs) would be impaired in fitness compared to their parental strains, due to the additional metabolic load imposed by expression of the inserted DNA In practice, this has proven not to be the case, with a number of studies demonstrating equal survival of GEMs and their parents. Sessitsch et al. (1997) measured the competitiveness index of various gusA-maiked derivatives ofi? tropici strain CIAT899 In that study, no effect on the competitive ability due to the presence of the marker gene was found indicating that the gusA cassette used is a suitable marker for rhizobial competition studies. Likewise, a strain of Erwinia carotovora, engineered to contain a chromosomal kanamycin resistance gene, showed equivalent survival capabilities as its parental strain in soil (Orvos et al., 1990). Pseudomonas aeroginosa and P putida growth rates were unaffected by introduced plasmids, although University of Ghana http://ugspace.ug.edu.gh 42 survival capabilities may have declined slightly (Yeung et a l, 1989). However in some other examples, fitness was found to be compromised. For example, strains of P. fluorescens marked with a Bacillus-endotoxin gene had slightly decreased growth and survival capabilities compared to the parental strain (van Elsas et al., 1991). Further complications derive from the observation that effects on fitness may be dependent on the nature of the host strain rather than the nature of the foreign DNA (Devanas et a l, 1986) and that the host genome may even evolve to become adapted to the introduced DNA such that loss of the DNA subsequently reduces fitness (Bouma and Lenski, 1988). There are few examples where fitness parameters other than growth or survival have been measured. Lam et al., (1990) analysed over 1200 mutants of P. putida containing a promoterless lacZ gene on a transposon Tn5 derivative, for their ability to colonize roots and found isolates with both increased and decreased colonisation ability. The majority of the isolates, however, showed a colonisation ability that differed little from the wild type strain. A few studies have looked at the effect on competition of marking rhizobia with the intact transposon Tn5 element. Sharma et a l (1991) found that the competitive ability as well as the nitrogen fixation ability of two strains of chickpea rhizobia tagged with Tn5 was not affected. Similar findings were reported by Sessitsch et a l (1997) where R tropici derivatives carrying gusA minitransposons showed symbiotic traits as well as growth characteristics similar to their parent strain. Aspects of the \egame-Rhizobium symbiosis relevant to this study have been reviewed in the foregoing literature analysis. While it is quite clear that the legame-Rhizobium symbiosis has and continues to receive research attention, information from tropical Africa is scanty. For the purposes of improving nitrogen fixation and yield of cowpea to enhance its contribution in University of Ghana http://ugspace.ug.edu.gh 43 farming systems in Ghana, research on the population structure of the native rhizobia that nodulate cowpea is needed This will not only help to reveal the numerous yet undiscovered strains of rhizobia, but would lay firm foundations on which agronomic strategies can be developed. University of Ghana http://ugspace.ug.edu.gh 44 CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 Soil sampling Soil samples representing 20 soil series (Table 3.1) were collected (from the 0-15 cm layer) from the five ecological zones of Ghana (Fig 3 .1). Each soil type was air-dried, undecomposed plant materials were removed by hand from the dried soil, and the soil aggregates were gently crushed in a mortar to pass through a 2mm sieve. Subsamples were used to analyse for soil physiochemical properties (Table 3.2). 3.1.1 Soil analysis 3.1.1.1 Soil pH Soil pH was determined in both distilled water and in 0.01M CaCl2 Soil pH was measured in 1:2 soil solution ratio using a Pracitronic pH meter Twenty grammes of soil was weighed into a beaker and 40ml of distilled water or 0.01 M CaCfe added to give a soil to solution ratio of 1:2 . The mixture was stirred several times for 30 min and left for about 1 hr to allow most of the suspended clay to settle. The pH meter electrodes were then immersed into the partly settled suspension and the reading on the pH meter recorded. There were three replicates of each soil type for the pH measurement. University of Ghana http://ugspace.ug.edu.gh H CA 2 t-1 ,w n | 8La? a < o'CflO cr ‘2o a ucu Ea g: n3 3 5S3 Pi GO C«JS pj <; 5 305 Oo3* o JT < I s o o IT.o K* £ f 2 -3 § B cdc I Jo nQ £ 5 ' e Cfl Cd < 2 < 5 3 S ' 3 CD s 5* 3 a. crCD CDa. < | & | £ S ’ £3 cc nus s Q X f I no. tt>o 3 §• % s*3 ^ 3 *■ i . H CO r °CT*O B £ 3 E" £fD cn § s soo e* go dd 3 •A O 3 a oo i« 5* 8O CO ?o 3 rt 2 »- 3 o J? 3 a 1/1eg ►flo Oo 3* oo 3“*iot/3 o On3" OcoO "HO o Cr‘o TlQ O X »■oq o o ►Ba 5ai#-► ox 5 QO ST* HICDT> So o 0*O oa. o >-a *-aB. BL o m c c a- o- c c O go 9* s*o o ' *V£ E2.CD CDC ea. Q-c^ C s s£ E 8 8 cr & cr- sr CO Cflo o no_ 5"< no hd 3* 3 “>< v; I t 3 TO C 3 S 1 3 •a 3*v<< s H H CD CD3 5 C* ^ 3 Q - Q * C t CD T 3 T 3 O O Cfl Cfl & ? o oo_ c< 5 3 a.n 3 . 3CL o 3 cr o' 3-CD TO 3 3 c < 5 3 cCflO QQ Q* m © I s s s g gt3Q C 6 O E.o c H m;•a o ZP P3 ^ " aCfl E Hv: ■a o S ’B.ae E 5‘ fe oo_ c"< 5 3 o oo c ~ e c S. c 3 S. et> q>0 o 3 3 o* 5» 1 I 3- "o <2 3 n 2 «' fT «°3a.n OQ3« COO3* H Hi5T U) ir1 O n ►r ecn a ► n t-1 ►ce Cfl n »► HM i o B S io S- ^o 3 Oi^ >r^ - o COO a. ►-* • CDa* University of Ghana http://ugspace.ug.edu.gh •Bswfcu <20) ♦Tamale (18) ♦Yapei (17, 19) D ♦Ejura (13, 14, 15,16) •Akumadan (9) Tafo (12) ♦ Kade (10, 11|* / #Avieme (4) *AKuse (2. LeaonI 200 KEY A Coastal Savanna Zone B Rainforest '^onc C Semi-dee duous Forest Zone D Forest-Savanna Transitional Zone E Guinea Savanna Zone K ilom eters Fig. 3.1 Map of Ghana showing the ecological zones and sites where the soils (showing by numbers) were sampled University of Ghana http://ugspace.ug.edu.gh Lima 6.4 0.99 0.12 61 Nyankpala 6.2 0.69 0.05 129 Siare 6.6 0.51 0.08 166 Tafali 6.2 0.58 0 06 138 a s**t? CD a S'5Cfl cf. 3 3 . z N 3B) C/5o (t o(A O' O ' Ln o c LA L/1 L/1 O' O' k> O J SO O ' O ' •tk. Ul so ■a 53 5o L/lvO O s | 00 to L/lo o LA LA U> VO 00O ooo> ^ o00 — VO KJ to as ^ — ONN) N>-U OO 0 00 1n 6 §■3 ? Ho 8# © O — i - U O ' o o o o .u r J — —ro vo L*J —-fc. K) U)00 L/lOO s*o0Qtta -oUJ 0 0 \ ' j ^ 0 ' v l * k u i [ d 000 w oo O ' K»o Ho £ ■TO 3 "Oui-atro 3CA 3OQ oa Table 3 .2 Some chem ical characteristics of the soils University of Ghana http://ugspace.ug.edu.gh 3.1.1.2 Totyl phosphorus Total phosphorus was determined by digesting 2 g soil with 30 ipls of 60% perchloric/nitric acid mixture for 40 mins. The soij sample was pre-digested with the nitric acid for 20 mins and the sample allowed to cool before adding the perchloric acid, which was allowed to digest for 30 min. The digest was filtered into a 250ml volumetric flask and made to volume. Phosphorus in the digest was measured with the Phillips PU 8620 spectrophotometer using the molybdate ascorbic acid iflethod of Watanabe and Qlson (1962) 3.1.1.3 Total nitrogen A tablet of selenium catalyst was added onto 0 5 g of soil in a 250 ml Kjeldahl flask. This was followed by digestion with 5 ml concentrated H2SO4 until the digest became clear. The contents of the flask were transferred into a 100 ml volumetric flask and brought to volume with distilled water An aliquot of 5 ml of the digest was taken into a Markham distillation apparatus. Five milliliters of NaOH was added to the aliquot and fhe mixture distilled- The distillate was collected in 5 ml of 2% boric acid to which 3 drops of mixed methyl red and methylene blue indicator had been added This was then titrated against 0.01M HC1. Total nitrogen was calculated by the method of Bremner (1960), 3.2. Nodulation capabilities of cowpea 3.2.1 Planting materials Seeds of 45 cowpea cultivars were obtained from the Plant Genetic Resources Centre of the Council for Scientific and Industrial Research (CSIR), Bunso, Ghana. All the cultivars were of determinate growth habit and mature between 60 to 80 days 17 University of Ghana http://ugspace.ug.edu.gh 48 3.2.2 J*ot experiment The abilities of the 45 cowpea cultivars to nodulate in the various soils were evaluated in black polythene seedling bags (15 x 23 cm) containing 1.5 Kg of each soil type. The bags wpre perforated at the bottom to allow free drainage of excess water. Four surface sterilised seeds of each of the 45 cultivars were planted in each bag which were thinned to two plants per bag after germination. There were three replicates for each soil for each cultivar. The plants were grown in the greenhouse and watered regularly. The plants were harvested 5 weeks after planting and assessed for nodulation. At harvest the seedling bag was removed from the soil-root matrix and a gentle stream of water from a hose was used to wash off the soil to expose the nodules. Nodulation was scored as positive when a plant bore at least a single nodule. 3.3. Enumeration of rhizobia Rhizobial populations capable of nodulating cowpea in all the 20 soils were enumerated by the most probable number (MPN) method (Vincent, 1970) using plastic growth pouches (17cm x 15cm sizes) (Weaver and Frederick, 1972), Cowpea var. Asontem which gave the highest nodulation score was the host used to enumerate rhizobia. Clean seeds free of damage and of uniform size and good viability (99%) were surface sterillised in 70% alcohol for 4 minutes and 0.1% mercuric chloride for 3 minutes. The seeds were then rinsed in several changes of sterile distilled water (Somasegaran and Hoben, 1994). Seeds were germinated on 1% water agar until cotyledons were about 2 cm long. Seedlings were planted two per pouch. The pouches contained N- free nutrient solution (Somasegaran and Hoben, 1994). Ten-fold dilutions of each soil sample with four replicates per dilution were used to inoculate the pouches, at 1 ml per pouch. The pouches were randomly arranged on wooden racks and kept in the greenhouse with temperature of 30/23 C day/night and natural light of approximately 12 h photoperiod. The University of Ghana http://ugspace.ug.edu.gh 48 3.2.2 pot experiment The abilities of the 45 cowpea cultivars to nodulate in the various soils were evaluated in black polythene seedling bags (15 x 23 cm) containing 1.5 Kg of each soil type. The bags wpre perforated at the bottom to allow free drainage of excess water. Four surface sterilised seeds of each of the 45 cultivars were planted in each bag which were thinned to two plants per bag after germination. There were three replicates for each soil for each cultivar. The plants were grown in the greenhouse and watered regularly The plants were harvested 5 weeks after planting and assessed for nodulation. At harvest the seedling bag was removed from the soil-root matrix and a gentle stream of water from a hose was used to wash off the soil to expose the nodules. Nodulation was scored as positive when a plant bore at least a single nodule. 3.3. Enumeration of rhizobia Rhizobial populations capable of nodulating cowpea in all the 20 soils were enumerated by the most probable number (MPN) method (Vincent, 1970) using plastic growth pouches (17cm x 15cm sizes) (Weaver and Frederick, 1972). Cowpea var. Asontem which gave the highest nodulation score was the host used to enumerate rhizobia Clean seeds free of damage and of uniform size and good viability (99%) were surface sterillised in 70% alcohol for 4 minutes and 0.1% mercuric chloride for 3 minutes. The seeds were then rinsed in several changes of sterile distilled water (Somasegaian and Hoben, 1994). Seeds were germinated on 1% water agar until cotyledons were about 2 cm long Seedlings were planted two per pouch. The pouches contained N- free nutrient solution (Somasegaran and Hoben, 1994). Ten-fold dilutions of each soil sample with four replicates per dilution were used to inoculate the pouches, at 1 ml per pouch. The pouches were randomly arranged on wooden racks and kept in the greenhouse with temperature of 30/23 C day/night and natural light of approximately 12 h photoperiod. The University of Ghana http://ugspace.ug.edu.gh 4$ 3.2.2 fo t experiment The abilities of the 45 cowpea cultivars to nodulate in the various soils were evaluated in black polythene seedling bags (15 x 23 cm) containing 1.5 Kg of each soil type. The bags wpre perforated at the bottom to allow free drainage of excess water. Four surface sterilised seeds of each of the 45 cultivars were planted in each bag which were thinned to two plants per bag after germination. There were three replicates for each soil for each cultivar. The plants were grown in the greenhouse and watered regularly. The plants were harvested 5 weeks after planting and assessed for nodulation. At harvest the seedling bag was removed from the soil-root matrix and a gentle stream of water from a hose was used to wash off the soil to expose the nodules. Nodulation was scored as positive when a plant bore at least a single nodule. 3.3. Enumeration of rhizobia Rhizobial populations capable of nodulating cowpea in all the 20 soils were enumerated by the most probable number (MPN) method (Vincent, 1970) using plastic growth pouches (17cm x 15cm sizes) (Weaver and Frederick, 1972). Cowpea var. Asontem which gave the highest nodulation score was the host used to enumerate rhizobia. Clean seeds free of damage and of uniform size and good viability (99%) were surface sterillised in 70% alcohol for 4 minutes and 0 1% mercuric chloride for 3 minutes. The seeds were then rinsed in several changes of sterile distilled water (Somasegaran and Hoben, 1994). Seeds were germinated on 1% water agar until cotyledons were about 2 cm long. Seedlings were planted two per pouch. The pouches contained N- free nutrient solution (Somasegaran and Hoben, 1994). Ten-fold dilutions of each soil sample with four replicates per dilution were used to inoculate the pouches, at 1 ml per pouch The pouches were randomly arranged on wooden racks and kept in the greenhouse with temperature of 30/23 C day/night and natural light of approximately 12 h photoperiod. The University of Ghana http://ugspace.ug.edu.gh 49 plants were supplied with sufficient N- free nutrient solution when required. Nodulation was assessed after 28 days of inoculation and the most probable numbers of rhizobia were calculated (Vincent, 1970). 3.4. Response of cowpea to nitrogen fertilization 3.4.1 Pot experiment Response of cowpea to nitrogen fertilization was evaluated in four cowpea cultivars (Asontem, Benga, Sanji and Soronko) that showed variable nodulation abilities from the nodulation experiment. The experiment was conducted in plastic pots(l 8cm high, 15cm wide at the top and 12cm at the base), using soils collected from Adenta, Akuse, Tafale and Wacri soil series, found to contain variable rhizobial populations Each pot was filled with 5 kg of soil. Four seeds were planted in each pot and later thinned to two after germination. Treatments were (i) no fertilizer as control, and (ii) six levels of nitrogen fertilizer of 40, 80, 120, 160, 200 and 240 mg/kg (equivalent to 40, 80, 120, 160, 200 and 240 kg/ha). The fertilizer was applied in two splits at 4 days after germination (DAG) and at the onset of flowering, at 28 DAG. The experiment was arranged as a randomised complete block design with three replications. The plants were watered daily until harvest. Plants were harvested 35 days after planting (DAP) by cutting the stem at soil level. Roots were carefully washed from the soil in running tap water Nodules were then separated from roots and counted. The vegetative parts were dried in the oven at 60°C until constant weight was obtained. 3.5. Isolation of rhizobia Representative nodule samples were taken from each of the MPN assays performed. The nodules were surface sterilised wiih 70% alcohol for 3 min and then with 0.1% mercuric University of Ghana http://ugspace.ug.edu.gh 49 plants were supplied with sufficient N- free nutrient solution when required. Nodulation was assessed after 28 days of inoculation and the most probable numbers of rhizobia were calculated (Vincent, 1970). 3.4. Response of cowpea to nitrogen fertilization 3.4.1 Pot experiment Response of cowpea to nitrogen fertilization was evaluated in four cowpea cultivars (Asontem, Benga, Sanji and Soronko) that showed variable nodulation abilities from the nodulation experiment. The experiment was conducted in plastic pots(18cm high, 15cm wide at the top and 12cm at the base), using soils collected from Adenta, Akuse, Tafale and Wacri soil series, found to contain variable rhizobial populations. Each pot was filled with 5 kg of soil. Four seeds were planted in each pot and later thinned to two after germination. Treatments were (i) no fertilizer as control, and (ii) six levels of nitrogen fertilizer of 40, 80, 120, 160, 200 and 240 mg/kg (equivalent to 40, 80,120,160, 200 and 240 kg/ha). The fertilizer was applied in two splits at 4 days after germination (DAG) and at the onset of flowering, at 28 DAG. The experiment was arranged as a randomised complete block design with three replications. The plants were watered daily until harvest Plants were harvested 35 days after planting (DAP) by cutting the stem at soil level. Roots were carefully washed from the soil in running tap water. Nodules were then separated from roots and counted. The vegetative parts were dried in the oven at 60°C until constant weight was obtained 3.S. Isolation of rhizobia Representative nodule samples were taken from each of the MPN assays performed. The nodules were surface sterilised with 70% alcohol for 3 min and then with 0.1% mercuric University of Ghana http://ugspace.ug.edu.gh 50 chloride for another 3 min and rinsed with several washes in sterile distilled water (Somasegaran and Hoben, 1994). The nodules were each crushed in a drop of sterile distilled water in a petri dish with a sterile glass rod. A loopful of the suspension was then streaked on yeast extract mannitol (YEM) agar plates and incubated at 28°C. 3.5.1 Authentication of isolates The isolates were evaluated as pure cultures that could form nodules on cowpea Surface sterilised cowpea seeds were pre-germinated on 1% (w/v) water agar and planted in growth pouches containing N- free nutrient solution. The pouches were inoculated with 1ml 5 days YEM broth culture of each isolate. Uninoculated pouches served as controls. The pouches were placed on racks and kept at the green house The plants were harvested afrer 28 days and the roots were observed for the presence of nodules. One hundred out of the total of 300 isolates obtained were randomly selected for further studies and characterisation. 3.5.2 Culture maintenance Bradyihizobia isolates were maintained on slopes of YEM and stored at 4°C. All the isolates were re-plated on YEM and checked for purity at least every three months. ^•6. Physiological and metabolic characterisation of isolates 3.6.1 Growth rates and colony morphology The abilities of the isolates to change the pH of their growth medium were scored on YEM agar plates amended with 0.25mg l'l bromthymol blue. Each isolate was cultured in YEM broth cultures grown to early log phase (population density of approximately 108 cells ml-1) and University of Ghana http://ugspace.ug.edu.gh 51 streaked on duplicate YEM plates. Estimation of population density of cells was by the Miles and Misra drop count method (Collins and Lyne, 1985). Streaked plates were incubated at 28®C for 5 days and examined daily to determine the time to first appearance of colonies. Colony appearance was scored as dry where the surface was smooth and firm and wet for those which were watery or slimy. 3.6.2 Salt tolerance Tolerance of the isolates to NaCl was determined by checking for growth on YEM agar plates containing 1, 2, 3, and 5% NaCl (w/v). Each isolate was streaked on duplicate plates and incubated at 28°C for 5 days. 3.6.3 Acid tolerance Growth was determined at pH 3.5, 4 5, 5.5, in the acid range and pH 7.5, 8.5, and 9.5 in the alkaline range. The pH of tubes containing 10 ml of YEM broth with KH2PO4 omitted (Zablotowicz and Focht, 1981), was adjusted after sterillization by the addition of HC1 or NaOH. Tubes were inoculated with 100^1 aliquots of each isolate and incubated at 28°C for 5 days before scoring for growth. 3.6.4 Carbon utilization The isolates were tested for their abilities to grow when provided with different carbohydrates as the sole carbon source. The test was earned out in a basal medium with mannitol omitted (Zablotowicz and Focht, 1981). The carbohydrates were added to give 10% (w/v) solution. The carbohydrates were sterilised separately by Millipore membrane filtration and were then added University of Ghana http://ugspace.ug.edu.gh 52 to the sterilised liquefied mediurp just before plates wpre poured. Each isolate was streaked on duplicate plates and after 5 days incubation at 28°C, growth was scored. The following carbohydrate sources were tested: L- arabinose, D- glucose, D- galactose, fructose, lactose, maltose, mannitol and sucrose. 3.7. Host range analysis The ability of the isolates to nodulate eight leguminous crops (Arachis hypogea (L), Calopogonium phaseoloides (L), Clotalaria Spp, Glycine max (L) Merrill, Leucaena leucocephala (L) de Wit, Mimosa ratusa and Voandzeia subterranean (L) Verde) randomly selected from the cowpea subfamily Papillionoidea and subfamily Mimosoideae were tested These legumes are commonly found growing in Ghana. Seeds of the legumes were surface sterilised with 0.1% mercuric chloride (Somasegaran and Hoben, 1994). The seeds were pre­ germinated on 1% (w/v) water agar and planted in growth pouches containing sterile N- free nutrient solution. Each growth pouch was inoculated with 1ml suspension of late log phase YEM broth culture of one of the isolates. Uninoculated pouches served as controls. The pouches were arranged randomly on wooden racks and plants grown in the greenhouse. After 35 days of growth, the plants were scored for nodulation. 3.8. Serological characterisation 3.8.1 Preparation of rhizobia antigens Fourteen of the isolates were randomly selected to test for the sero-relatedness of the isolates The isolates were grown in YEM broth until late log phase. The cultures were centrifuged at 15,000 rpm for 10 min to obtain pellets. The pellets were resuspended and washed three times in sterilised 0 85% NaCl and boiled for 30 min. University of Ghana http://ugspace.ug.edu.gh 53 3.8.2 Formulation of antigens for immunization. Antigens for immunization were prepared with a 1:1 ratio of antigen suspension and Freund's incomplete adjuvant (Nambiar and Anjaiah, 1985). The antigen-adjuvant mixture was prepared by repeated drawing in and expelling in a glass syringe. The right consistency was reached when a drop of the mixture did not disperse when dropped in water (Somasegaran and Hoben, 1985). The antigen adjuvant mixture was used for only the first immunization. Subsequent boosters were done with 1:1 ratio mixture of sterile 0.85% NaCl and antigen suspension. 3.8.3 Experimental animals Inbred BALB/c mice, aged between 12 and 14 weeks and 56 weeks old New Zealand rabbits were used for the production of antibodies. The mice were obtained from the Animals Breeding Unit of the Noguchi Memorial Institute for Medical Research and the rabbits from the Animal Husbandary Division, Ministry of Agriculture, Nungua- Accra. 3.8.4 Coating of microtitre plates with antigen Rhizobial antigens were diluted in carbonate-bicarbonate buffer, pH 9.6 (coating buffer) (Voller et al, 1977) Flat bottom 96-well polysterene microtitre plates (Sumilon type C), were coated with 100 jil per well of antigen coating buffer. The plates were shaken for 1 min and incubated overnight at 37°C to enable adsorption of antigen onto polysterene wells. 3.8.5 ELISA procedure Screening of antigens for antibody production, screening of rabbits for background antibodies, estimation of sera and conjugate titres and the detection of antibody response of the rabbits to antigen immunization were all assayed by the indirect ELISA procedure (Voller et a l, 1977). University of Ghana http://ugspace.ug.edu.gh 54 In this assay, micro-ELISA plates coated with antigens were rinsed twice with washing buffer (PBS Tween 80, pH 7.4), flipped empty and banged to remove excess unbound antigen. The plates were incubated with titrated test sera diluted in washing buffer for lhr at room temperature. The plates were flipped empty, banged and rinsed twice with washing buffer to remove excess unbound antibody. The plates were then incubated with 100 jil of goat anti­ mouse or goat anti-rabbit horseradish peroxidase (HRPO) conjugate for 30 min at room temperature. The plates were then washed three times, each for 10 min. with washing buffer to remove excess unbound conjugate. The presence of bound conjugate was revealed or visualised by the addition of substrate ABTS and hydrogen peroxide pH 4.0, and incubated at room temperature for 30 min. The colourless substrate solution changed to green in wells with bound enzyme conjugates. The optical densities (OD) were read at 415 nm wavelength using MTP-32 ELISA reader 3.8.6 Evaluation of antigen for antibody production Inbred BALB/c mice were immunized with the prepared antigens to test the production of antibodies by the antigens. The mice were injected intraperitoneally with 200 pi each of antigen and Freund's incomplete adjuvant mixture. The mice were given booster immunization with antigens only at 2 and 3 weeks. Ten days after the third booster, the mice were bled as indicated in the following section and tested for antibody response to homologous rhizobia antigens by the microplate indirect ELISA method. University of Ghana http://ugspace.ug.edu.gh 55 3.8.7 Bleeding of mice and screening for antibody response The mice were bled from the tail veins and from the heart by cardiac puncture and the serum tested at 1:50 in PBS. The tip of the tail of each mouse was cut with a pair of scissors and the tail gently squeezed from the base towards the tip. Blood from the severed tail veins was aspirated using a 50nl eppendorf pipette and then transferred into an eppendorftube containing 500 |il of PBS and mixed thoroughly. Bleeding from the heart was done by killing the mice by anaesthesia using diethyl-ether. The mice were held at their back and blood was extracted from the heart through the sternum with a 5ml syringe. The blood in PBS was microfuged at 10,000 rpm for 2 min. The supernatants were pipetted into different tubes and kept at -20°C. Each antiserum was tested for antibody activity by titration in two-fold dilution on micro-titre plates previously coated with rhizobia antigen. 3.8.8 Selection of rabbits for immunization Blood was extracted from the ear vein of 30 rabbits with a syringe and emptied into screw capped tubes. The blood was allowed to clot at room temperature and centrifuged at 14,000 rpm to separate the serum from the clot The clear serum was drawn into sample bottles and kept at - 20°C. Each serum was tested against all the antigens for background antibody using the ELISA procedure as described previously. Rabbits with no or minimal background antibodies were selected for each antigen. 3.8.9 Immunization of rabbits The rabbits were injected with between 0.5 and 0.8ml of antigen emulsified with Freund's incomplete adjuvant or antigen mixed with 0 85% NaCl. The immunization schedules were as follows: University of Ghana http://ugspace.ug.edu.gh 56 Day Procedure 1 intraperitonea 7 intravenous 14 back 28 bleeding/back booster 35 final bleeding 3.8.10 Detection of antibody Microtitre plates were coated with 100 |il of each antigen in quadruplicate and held overnight at 37°C. The plates were washed twice in PBS-Tween, flipped empty and banged to dry. Rabbit serum in PBS-Tween at a predetermined optimal titre of 1:156 was added in 100 1^ aliqouts to each well and held at room temperature for Ihr. The plates were washed three times Goat anti­ rabbit globulin conjugated to peroxidase (Sigma) was diluted at a pre-deteimined concentration of 1:1600 in PBS-Tween and 100 jil added to each well. The plates were incubated at room temperature for 30 min. The plates were washed again 5 times and 100 jil of enzyme substrate solution (standard ABTS in citrate buffer with 35% H2O2) added to each well. The plates were gently shaken and held at room temperature for 30 min. Absorbances were read at 415 nm with MTP-32 ELISA reader University of Ghana http://ugspace.ug.edu.gh 57 3.9. Molecular characterisation 3.9.1 Sample preparation for DNA amplification The isolates were grown on YEM agar plates at 28°C for 24 and 48 hr respectively for the fast and slow growers. Pelleted cells of each isolate were suspended in 100 jil Tris EDTA (TE) (Ausubel et al., 1994) and the optical densities at 600 nm of all the samples were adjusted to 2.6 with sterile distilled water. The samples were then frozen for 4 min at -70°C. Afterwards, the cells were set on ice for lmin, boiled for 2 min, again set on ice for lmin and then boiled once more for 2 min. Finally, the cells were centrifuged for 2 min at 15,000 rpm and the supernatant used for the PCR assay. The above procedure did not produce optimal DNA amplification for some of the isolates In these cases, single colonies of the isolates were grown in YEM medium. The cells were pelleted by centrifugation at 15000 rpm Genomic DNA was then isolated from the pellets using DNeasy Plant Mini Kit (Qiagen) according to the supplier’s instruction. DNA concentration was adjusted spectrophotometrically to lOOng/ml 3.9.2 PCR amplification of the 16S rRNA gene Primers fDl (5 -AGAGTTTGATCCTGGCTCAG-3 ) and rDl (5' AAGGAGGTGATCCAGCC- 3') described by Weisburg et al. (1991) were used for PCR amplification. They are derived from conserved regions of the 16S rRNA genes and amplify nearly full-length 16S rRNA genes (Weisburg et al, 1991). PCR amplification was carried out in total reaction volume of lOOjil. DNA was amplified by mixing 100 ng template DNA (pure DNA) or alternatively (cell extract) 5-8 ul, with 1 x PCR buffer (50 mM KC1; 20 mM Tris.HCl, pH 8.0), 200 |iM each of dATP; University of Ghana http://ugspace.ug.edu.gh 58 dCTP; dGTP and dTTP (Boehringer Mannheim), 3 mM MgC^, 0.2fiM of each primer, and 2 U Taq DNA polymerase (Gibco.BRL). All amplifications were carried out in a Perkin-Elmer thermocycler (GeneAmp PCR System 9600). The temperature profile was as follows: an initial denaturation step at 95°C for 5 min followed by 35 cycles of 30 sec. denaturation at 94°C, 1 min annealing at 50°C and 2 min extension at 72°C and a final extension step for 4 min at 72 C (Sessitsch, 1997). 3.9.3 Electrophoresis and imaging Aliquots (5|il) of the amplified DNA were mixed with loading buffer (2 (il) and analysed by horizontal agarose gel electrophoresis inl% agarose gels stained with 0.5|ig ml'1 ethidium bromide for 30min. The gels were photographed on a UV transilluminator after the electrophoresis. 3.9.4 Restriction fragment analysis Aliquots (10 ^1) of PCR products were digested with the tetrameric restriction endonucleases: Dde 1, Hae III, Msp I, and Rsa I (Gibco BRL) in a total reaction volume of 20|il and incubation at 37°C for 2 hr. The resulting DNA fragments were analysed by horizontal agarose gel electrophoresis in 2.5 % agarose gels stained with ethidium bromide. Molecular weight markers (100 bp) were run a lane of each gel to enable calculation of the sizes of the resulting restriction fragments. Electrophoresis was carried out at 100V for 3 hr and the gels were photographed on a UV transilluminator. University of Ghana http://ugspace.ug.edu.gh 59 3.10. Effectiveness of isolates in fixing nitrogen The experiment was carried out in Leonard jars (of 250ml volume) containing 200 g acid- washed sand and N-free nutrient solution (Somasegaran and Hoben, 1985), in the bottom compartment. The isolates were grown in 100 ml YEM broth until late log phase (determined by optical density values at 600nm). Cowpea seeds were surface sterilised with 0.1% mercuric chloride (Somasegaran and Hoben, 1994) and germinated on 1% (w/v) water agar. Two of the pre-germinated seeds were planted in each jar that had been sterilised by autoclaving. The seedlings in each jar were inoculated with 1 ml (approximately 10s cell ml *) of the rhizobial culture. There were three replicate jars for each of the Rhizobium isolates. Uninoculated seedlings with or without nitrogen (70jxg m l'l KNO3) served as controls. The jars were arranged in a randomized complete block design in the green house. The inoculated plants were kept supplied with N-free nutrient solution, while the uninoculated control received nitrogen. (Somasegaran and Hoben, 1994). The plants were harvested 35 days after planting (DAP). Nodulation, shoot dry weight and shoot nitrogen content were recorded. The mean dry weight of shoot (X) was used to calculate an index of effectiveness (E) defined as: X, - XTO Ej = ---------------x 100 ( Ferreira and Marques, 1992), Xtn - Xto Where, j is the shoot dry weight of inoculated test strain, TO is that of the uninoculated control and TN that of the nitrogen control. Plant dry weight values of each isolate was compared with those of N controls and the LSD at P = 0.05 level was used to delineate isolates significantly different from the N controls (Beck et al., 1994). Classes of effectiveness were defined from comparison with the controls. Symbiotic effectiveness was high when the isolate produced plant yield equal to or greater than N- fertilised plants; moderate when slightly less than N controls University of Ghana http://ugspace.ug.edu.gh 60 and ineffective when isolates produced yields similar to uninoculated controls (Beck et al., 1994). 3.10.1. Relative effectiveness of isolates in fixing nitrogen Differences in symbiotic effectiveness of the 10 most effective isolates were compared in a separate experiment TAL 169 as a standard strain. Procedures described previously (3.10.1.) were followed. Relative effectiveness compared with strain TAL 169 was calculated by the following expression: Shoot dry wt inoculated test strain Relative Effectiveness = ----------------------------------------------- x 100 (Gibson, 1980). Shoot dry wt inoculated standard strain 3.11. Measurement of nodulation competitiveness by glucuronidase (GUS) fusion 3.11.1 Gus fusion donor strain The E. coli strain SI7-1 X-pir containing the relevant GUS transposon as donor strain (Jefferson et al, 1986) was used. The strain was obtained from the FAO/IAEA Agricultural and Biotechnology Laboratory, Seibersdorf, Austria. 3.11.2 Marking rhizobia with gusA gene Five millilitre cultures of three of the effective isolates (2 , 10 and 14) and the donor strain were prepared in a modified YEM broth (Danso and Alexander, 1974) and Luria Bertani broth (LB) containing spectinomycin (50ng/ml) (Ausubel et a l, 1995) respectively. The cultures were microfuged at 4,000 rpm for 10 min. The supernatant was carefully removed and the cells were University of Ghana http://ugspace.ug.edu.gh 61 washed in YEM medium to remove the antibiotic (spectinomycin) and microfiiged again. Plate matings were carried out as follows. The E. coli and rhizobia cells were resuspended in 1 and 0.25 ml respectively, of YEM medium. One hundred microlitre of each cell suspension was spotted together on YEM agar plates. The two drops were mixed and spread over the plate until the plates were dry The plates were incubated overnight at 28°C. After the overnight incubation, 2 ml sterile distilled water was pipetted onto the surface of the mating plate and spread around using a glass spreader. One millilitre of the suspension was then transferred from the plate into 1.5-mI sterile tubes using a micropipette. Three-fold (10x, lOOx and lOOOx) dilutions of the suspension were made in sterile distilled water. One hundred microlitre of each dilution was subsequently spotted and spread on Brown and Dilworth (B and D) medium (Brown and Dilworth, 1975) containing sucrose. The plates were incubated at 28°C and examined regularly for growth of single colonies. Transconjugants were grown on B and D medium supplemented with spectinomycin (50 ng ml‘1) to select for the insertion of the transposon. 3.113. Detection of gusA - marked Bradyrhizobium derivatives One litre YEM agar was prepared, autoclaved and allowed to cool. Fifty milligram of isopropyl- B-D- thiogalactopyranoside (IPTG) and 50 mg of 5-bromo-4-chloro-3-indolyl-B-D-glucuronide (x-gluc) were dissolved separately in 1 ml sterile distilled water and added to the YEM. One millilitre N, N-dimethyl formamide (DMF) was also added and plates poured. Single colonies of the gus A marked derivatives were then streaked on the poured plates and incubated at 28°C. Blue colonies appeared on the plates after two to three days of incubation, confirming that the University of Ghana http://ugspace.ug.edu.gh 62 Bradyrhizobium isolates were gusA marked. The gus marked isolates were thereafter represented as G2, G10, and GI 4. 3.11.4. Competition experiment Competition between the gus-marked (effective) isolates and the unmarked ineffective isolates was studied in jars containing sterile sand and N- free nutrient solution (Somasegaran and Hoben, 1994). Surface sterilised cowpea seeds were pre-germinated on 1% (w/v) water-agar plates before being transplanted into the Leonard jars Each seedling was inoculated with 1ml suspension of the marked and unmarked isolates in ratios of 1:1, 1:2 and 2 :1, to supply approximately 108 cells per seedling. The numbers were verified by the Miles and Misra drop count method (Collins and Lyne, 1985), at the time of inoculation. Jars containing the plants were arranged in a completely randomized block design in the greenhouse. There were three replicates of each treatment Controls included uninoculated plants and plants supplied regularly with 0 05% (w/v) KNO3 i.e., 70 ng ml"* nitrogen. The plants were harvested at 28 DAP. Shoot dry weight and nodule numbers were determined. 3.11.5. Staining of nodules The roots of the cowpea plants were washed thoroughly with water and immersed in 40 ml GUS extraction buffer containing 50 mM sodium phosphate buffer pH 7 0, 0.1% (w/v) Triton X-100, 0.1% (w/v) Sarkosyl, 0.05% (w/v) SDS, 1 mM EDTA amended with 40 ng IPTG ml' 1 andlOO Hg X-GlcA ml"l (Jefferson et al., 1987). A vacuum was applied for 15 min from a water jet pump to facilitate penetration of the substrate into the nodules Thereafter, the roots were incubated for 24 hr at 3 7°C in the substrate-containing buffer. The roots were then transferred to fresh buffer with IPTG and X-GlcA and vacuum was again applied for 15 min followed by University of Ghana http://ugspace.ug.edu.gh 63 incubation at 37°C overnight. All nodules containing the marked isolates turned blue showing that the nodules were formed by the gusA marked effective isolates. The proportion of nodules occupied by the gusA marked isolates were counted for each inoculation ratio and competitive indices calculated by linear regression using the equation of Beattie et al. (1989). In this model, competition between two different strains is described by the relationship: Log [(Px + Pboth) / (Py + Pboth)] = Cxy + K (Ix / Iy) Where: X and Y are the two competing strains; Px and Py are the proportions of nodules occupied by X and Y in the inoculum. Pboth is the proportion of nodules by both strains; and Ix and Iy represent the concentration of X and Y in the inoculum. The intercept of this equation, Cxy, is defined as the competitiveness index and the slope is K: a statistically significant positive value indicates that strain X is more competitive than strain Y, while a negative value shows that it is less competitive 3.11.6. Effect of placement and time of placement of inoculum on competitive ability of isolates Competition for nodule occupancy among the isolates was evaluated further, by changing the relative times of exposure of the competing isolates on the seed using gusA marked isolates as described above to differentiate between the nodule forming isolates. University of Ghana http://ugspace.ug.edu.gh 64 3.11.7. Speed of infection of host Cultures of the competing isolates (designated 2, 10, 14, 29, 30, 72 and 94), were grown separately in YEM broth and used to individually inoculate pre-germinated surface sterilised seeds in plastic growth pouches containing nitrogen-free nutrient solution. There were six replicates for each isolate. The plants were maintained in the greenhouse and the seedlings were monitored for the first appearance of nodules every 12 hours. 3.12. Inoculation of cowpea with isolated indigenous cowpea bradyrhizobia isolates 3.12.1. Soils used The experiment was conducted in four of the 20 soils, Ankasa, Akuse, Bekwai and Tafali. The soils contained variable numbers of indigenous cowpea bradyrhizobia isolates (Table 4.1) and also come from different ecological zones (Fig 3.1). Plastic pots (18cm high, 15cm wide at the top and 12cm at the base) were filled with 1.5 kg of each of the soils. All the soils were fertilized with essential macro and micro-nutrients except nitrogen as described by Owiredu and Danso (1988). Three of the effective isolates 2, lOand 14, and a mixture of the three were used for inoculation. The isolates were cultured aseptically at room temperature (28V 3°C) on a wrist-action shaker for 5 - 7 days in culture bottles containing YEM broth until cell densities were about 108 cells ml'1 as determined by Misra and Miles Drop Count method (Collins and Lyne, 1985). University of Ghana http://ugspace.ug.edu.gh 65 3.122. Inoculation procedures 3.12.2.1 Seed inoculation Seeds of cowpea cultivar (Asontem) were surface-sterilised and pre-germinated on 1% (w/v) water agar. Four of the germinated seeds were planted in each pot. One millilitre aliquot of each bradyrhizobia! culture (108 cell ml"1) was then inoculated onto and around the seed before covering the seed with soil. In the case of the isolate mixture, 0.33 ml of each of the isolates was combined to achieve this 1ml aliquot. 3.12.2.2 Soil inoculation One millilitre suspension of each of the isolates was diluted to 50 ml in sterile distilled water and mixed thoroughly with the soil. Each pot was then planted with four of the pre-germinated seeds. 3.12.23. Plant growth The plants in the two different inoculation methods were thinned to two plants 4 days after germination. The treatments were each replicated 4 times and the pots were arranged in a randomised complete block design on raised benches in the open air. The plants were watered daily and harvested at 35 DAP. The roots were carefully washed and the number of nodules (plant1) counted The shoots were oven-dried at 60-65 °C for 48 hours and weighed Kjeldahl N analysis was done on ground samples (0.2 mm) of the shoot. University of Ghana http://ugspace.ug.edu.gh 6 6 CHAPTER FOUR 4.0 Results 4.1 Examination of components of nitrogen fixation in cowpea 4.1.1 Nodulation potential of cowpea in Ghanaian soils Nodulation of 45 cowpea cultivars grown by farmers in various parts of the country were evaluated in 20 different soils to assess their nodulation potential with native bradyrhizobia. The cowpea cultivars tested exhibited variable nodulation potentials in the various soils; in general nodulation in soils of the various ecozones was more than 50% (Fig. 4.1). None of the soils could support nodulation of all the cultivars tested. Generally, nodulation of the cowpea cultivars was relatively low in soils from the high rainforest zone, for the majority of which no nodules were formed. The highest nodulation was observed in soils of the Forest savanna transition zone. Cowpea cultivar, Asontem, recorded the highest nodulation frequency, nodulating in 18 out of the 20 soils. 4.1.2 Estimation of indigenous bradyrhizobia numbers in the soils Soil had a pronounce effect on the abundance of cowpea bradyrhizobia. While no cowpea bradyrhizobia were detected in soils (Ankasa and Tikobo which were sampled from the high rain forest zone with a pH of less than 4.4), at least 60% of soils contained more than 103 bradyrhizobia cells (g soil1 ) with the highest, 3.1x104 being encountered in the Akuse soil (Table 4.1). There was also a clear relationship between the abundance of cowpea bradyrhizobia and the ecological zone under which the soil developed. The lowest number of cowpea bradyrhizobia, between zero and less than 10 cells (g soil1 ) in all four cases examined, University of Ghana http://ugspace.ug.edu.gh 6 6 CHAPTER FOUR 4.0 Results 4.1 Examination of components of nitrogen fixation in cowpea 4.1.1 Nodulation potential of cowpea in Ghanaian soils Nodulation of 45 cowpea cultivars grown by formers in various parts of the country were evaluated in 20 different soils to assess their nodulation potential with native bradyrhizobia. The cowpea cultivars tested exhibited variable nodulation potentials in the various soils; in general nodulation in soils of the various ecozones was more than 50% (Fig. 4.1). None of the soils could support nodulation of all the cultivars tested. Generally, nodulation of the cowpea cultivars was relatively low in soils from the high rainforest zone, for the majority of which no nodules were formed. The highest nodulation was observed in soils of the Forest savanna transition zone Cowpea cultivar, Asontem, recorded the highest nodulation frequency, nodulating in 18 out of the 20 soils. 4.1.2 Estimation of indigenous bradyrhizobia numbers in the soils Soil had a pronounce effect on the abundance of cowpea bradyriiizobia. While no cowpea bradyrhizobia were detected in soils (Ankasa and Tikobo which were sampled from the high rain forest zone with a pH of less than 4 4), at least 60% of soils contained more than 10J bradyrhizobia cells (g soir1 ) with the highest, 3.1xl04 being encountered in the Akuse soil (Table 4.1). There was also a clear relationship between the abundance of cowpea bradyrhizobia and the ecological zone under which the soil developed. The lowest number of cowpea bradyrhizobia, between zero and less than 10 cells (g soir1) in all four cases examined, University of Ghana http://ugspace.ug.edu.gh 67 ID01Q.*ou 100 90 80 70 60 50 40 30 20 10 0 H igh Ra in fores t Sem i- deciduous Forest Guinea Savanna Ecological zone Coastal Savanna Forest-Savanna Transition Fig. 4.1 Proportion of cowpea plants which formed nodules in soils from the different ecological zones. University of Ghana http://ugspace.ug.edu.gh 6* Table. 4.1 Most probable number estimates of cowpea bradyrhizobia isolates in the various soils Soil Series Ecazone Rhizobia/g soil Adenta Coastal Savanna 100 Agawtaw 17000 Akuse 31000 Aveime 10000 Abenia High Rainforest 0 Ankasa 6 Boi 6 Tikobo 0 Akumadan Semi-deciduous forest 1000 Bekwai 58 Nzema 17 Wacri 1000 Am an tin Forest-Savanna Transition 3100 Bediesi 17000 Denteso 10000 Ejura 10000 Lima Guinea Savanna 5800 Nyankpala 1000 Siare 3100 Tafale 10 University of Ghana http://ugspace.ug.edu.gh La g rh izo bi a nu m be rs /g so il 69 4 5 4 0 3.5 3 0 2 5 2.0 15 1 0 0 5 0 0 High Rainforest Semi Guinea Savanna Forest Savanna Deciduous T rans ition Forest Ecological zone Coastal Savanna Fig 4 .2 The papulations of cowpea bradyrhizobia in soils from different ecological zones. University of Ghana http://ugspace.ug.edu.gh occurred in the High rainforest ecozone. The trend was High rainforest < Semi decidous forest < Guinea savanna < Forest savanna transition < Coastal savanna (Fig. 4.2). The soils from which the counts were made varied considerably in their properties measured (Table 3.2). 4.1.3 Response of cowpea to nitrogen fertilization The response of cowpea to nitrogen fertilization was tested at rates ranging from zero to 240 Kg N/ha. Except for Adenta soil for which no significant yield increases occurred at any of the nitrogen fertilizer rates, variable significant yield increases occurred in the other soils for the cowpea varieties used (Fig. 4.3). The highest yield response of Asontem and Sanji cultivars were recorded at 120 Kg N/ha, whilst that of Soronko and Benga cultivars were recorded at 160 Kg N/ha, when the mean response of the cowpea cultivars were evaluated on all the soils (Fig. 4.4). For Asontem, significant yield increases occurred at 80 Kg N/ha in Akuse and Wacri soils and at 120 Kg N/ha in Tafali soil (Figs 4.5; 4.6). In the case of Soronko significant yield increases occurred at 120 Kg N/ha in Akuse and Wacri soils and at 160 Kg N/ha in Tafali soil. Benga also recorded significant yield increases at 160 Kg N/ha in Akuse soil and at 80 Kg N/ha in the other soils (Figs 4.5; 4.6; 4.7). Sanji on the other hand showed significant yield increase at 120 Kg N/ha in Akuse and Tafali soils and at 40 Kg N/ha in Wacri soil (Figs 4.5; 4.6; 4.7). In all the soils, nodulation of the cowpea cultivars was generally enhanced from between 40 to 80 Kg N/ha except for Adenta soil (Fig. 4.8). Beyond 80 Kg N/ha nodulation was on the average inhibited but not completely (Fig. 4.3). 70 University of Ghana http://ugspace.ug.edu.gh 71 90 80 E 70 ■o 60 © “ 50 o t 40 | 30 Z 20 10 0 o ._ . , „ „ , , r o •4 & *4 4b p # 4 a o p # 4 « n v q 4 # • r« 6 p # Nitrogen application rate (kg/ha) □ Tafali HAkuse □ Adenta □ Wacn J I L I L 17 Nitrogen application ra te (kg/ha) □ Tafali BAkuse □A denta □ Wacri Fig. 4.3 Mean responses of cowpea varieties to nitrogen application on various soil types. Error bars represent standard errors o f the means University of Ghana http://ugspace.ug.edu.gh 72 □ Asontem D Soronko □ Benga □ Sanji Fig. 4.4 Mean performance of cowpea varieties to nitrogen application. Error bars represent standard errors o f the means University of Ghana http://ugspace.ug.edu.gh 73 □ Asontem H Soronko □ Benga □ Sanji N application rate (kg/ha) 16 N application rate (kgftia) □ Asontem ■ Soronko □ Benga □ Sanji Fig. 4.5 Response of cowpea varieties to nitrogen fertilization on Aklise soil series. Error bars represent standard errors o f the means University of Ghana http://ugspace.ug.edu.gh Sh oo t dry w ei gh t (g ) Nu m be r of no du le s 74 100 30 10 0 n n t I I ] N apphcatoon rate (kgfha} □ Asontem ■Soronko DBenga □ Sami 18 17 16 >5 14 i3 12 11 10 9 8 l i O O Q CD " T OJ o o CO « - CM o o o CD ID *7 oCX) oCO N application rate (kg/ha) □ Asontem D Soronko DBenga GSanji Fig. 4.6 Response of cowpea varieties to nitrogen fertilization on Wacn soil series. Error bars represent standard errors o f the means University of Ghana http://ugspace.ug.edu.gh 75 Fig. 4 .7 Response of cowpea varieties to nitrogen fertilization on 1 atall soil senes. Error bars represent standard errors o f the means University of Ghana http://ugspace.ug.edu.gh 76 S S 8 g | ° S 2 R 8 g V ° S § R S | | N application rate (kg/ha) □ Asontem ■ Soronko □ Benga □ Sanji Fig. 4.8 Response of cowpea varieties to nitrogen fertilization on Adenta soil series. Error bars represent standard errors o f the means University of Ghana http://ugspace.ug.edu.gh 77 4.2 Diversity of indigenous cowpea bradyrhizobia isolates 4.2.1 Physiological and metabolic analysis The 100 indigenous cowpea bradyrhizobia isolates examined comprised 18% fast growing and 82% slow growing types (Table 4.2). Differences in pH tolerance are shown in Table 4.3. None of the isolates could grow at pH 3 .0. Twenty nine percent of the isolates could grow at pH 3 .5, out of which only 5% were fast growing (Table 4.2). Fifty percent of the fast growing isolates grew at pH 4.5, whilst 53% of the slow growing isolates showed growth at pH 4.5. All the isolates were capable of growth from pH 5.5 to 7.0. The maximum NaCl concentration at which the isolates could grow ranged from 1% to 5% (Table 4.2). All the fast growing isolates and 72% of the slow growing ones tolerated NaCl concentration of 3%. Only 33% of the total isolates could not grow in 5% NaCl. Almost all the isolates tested metabolised all the eight carbon compounds as sole carbon sources (Table 4.2). 4.2.2 Host range analysis The degree of compatibility of indigenous cowpea bradyrhizobia isolates when in symbiosis with some of the host legumes commonly found growing in Ghana was examined to determine their level of promiscuity. There were differences in the type of host nodulated by the different isolates (Table 4.3), with none of the isolates capable of nodulating all the nine host legumes tested. All the isolates when re-inoculated, nodulated the homologous host, indicating they were not contaminants (Fig. 4.9). Fourteen percent of the isolates were specific on cowpea and did not nodulate any other host legume species (Table 4.3). The broadest host range of seven host species was shown by six of the isolates (14, 43, 49, 51, 29 and 87). Mimosa spp was the University of Ghana http://ugspace.ug.edu.gh Table 4.2 Physiological and metabolic characteristics of cowpea bradyrhizobia isolates 78 Isolates Growth pH NaCl (% ) Carbon Sources Fast Slow 3.5 4.5 5.5 8.5 9.5 10.5 1 2 3 5 Ara Fru Gal Glu Lac M ai Man Sue 1 + - - + + + + + + + + + + + + + + + + 2 - + - - + + + + + + + + + + + + + + + + 3 - + - - + + + + + + + + + + + + + + + + 4 - + - + + + + + + + - + + + + + + + 5 - + - + + + + + - - + + + + + + + 6 - + - - + + + + + + + + + + + + + + + + 7 - + - + + + + + + + + + + + + + + + + + 8 - + - + + + + + + + + + + + + + + + + + 9 - + - + + + + + + + + + + + + + + + + 10 + - - + + + + + + + + + + + + + + + + 11 - + - - + + + + + + + + + + + + + + + + 12 - + - - + + + + + + + + + + + + + + + + 13 - + - + + + + + + + + + + + + + + + + 14 - + - - + + + + + + + - + + + + + + + + IS - + - - + + + + + + + - + + + + + + + + 16 - + - + + + + + + + + + + + + + + + + + 17 - + + + + + + + + + + - + + + + + + + + 18 - + - + + + + + + + + + + + + + + + + + 19 + - + + + + + + + + + - + + + + + + + + 20 + - - + + + + + + + + + + + + + + + + 21 - + + + + + + + + + - + + + + + + + 22 - + + + + + + + + + + + + + + + + + + + 23 + - - + + + + + + + + + + + + + + + + 24 - + - + + + + + + + + + + + + + + + + + 25 - + + + + + + + + + + - + + + + + + + + 26 - + + + + + + 4- + + - + + + + + + + + 27 + - - - + + + + + + + + + + + + + + + + 28 - + - + + + + + + + + - + + + + + + + 29 + - + + + + + + + + + + + + + + + + + + 30 - + - + + + + + + + + + + + + + + + + + 31 - + + + + + + + + + + + + + + + + + + + 32 - + + + + + + + + + + - + + + + + + + + 33 - + - - + + + + + *4- + + + + + + + + + + 34 - + - - + + + + + + + + + + + + + + + + 35 - + - + + + + + + + + + + + + + + + + 36 - + - + + + + + + + + + + + + + + + + + 37 - + - - + + + + + + + + + + + + + + + + 38 + - - + + + + + + + - + + + + + + + + 39 - + - - + + + + + + + - + + + + + + + + 40 - + - + + + + + + - - - - + - + + + + + 41 - + - - + + + + + + + + + + + + + + + + 42 - + - - + + + + + - - - - - + + - - + + 43 + - - + + + + + + + + + + + + + + + + 44 + - - + + + + + + + + + + + + + + + + 45 - + - - + + + + + + + - + + + + + + + + 46 " + - + + + + + + + + + + + + + + + + + 47 ” + - - + + + + + + + - + + + + + + + + 48 " + ■ - + + + + + + + + + + + + + + + + 49 “ + 1 - + + + + + + + + + + + + + + + +50 + + + + + + + + + + + + + + + + + University of Ghana http://ugspace.ug.edu.gh 79 Table 4 Isolates 2 com Growth1 mupjj pH NaCl (%) Ca rfaon Sounxs‘ Fast Slow 3.5 4.5 5.5 8.5 9.5 10.5 1 2 3 5 Ara Fru Gal Glu Lac Mai Man Sue 51 + - - + + + + + + + + - + + + + + + + 52 _ + + + + + + + + + + - + + + + + + -f 53 _ + _ - + + + + + - - - + + + + + + + 54 _ + _ + + + + + + + -f + + + + + + + + + 55 _ + - + + + + + + + + + + + + + + + + + 56 - + - + + + + + + + + - + + + + + + + + 57 - + + + + + + + + + + + + + + + + + + + 58 - + + + + + + + + + + + + + + + + + + + 59 + - + + + + + + + + + + + + + + + + + 60 + - + + + + + + + + + + + + + + + + + + 61 - + - - + + + + + + + + + + + + + + + + 62 - + - - + + + + + + + + + + + + + + + 63 - + - + + + + + + + + + + + + + + + + 64 - + + + + + + + + + + + + + + + + + -+■ + 65 - + - + + + + + + + + + + + + + + + + 66 - + + + + + + + + + + + + + + + + ■+- + 67 + + + + + + + + + + + + + + + + + + + 68 - + - + + + + + + + + + + + + + + + + 69 + + + + + + + + + + + + + + + + + + + 70 + + + + + + + + + + + + + + + + + + 71 - + + + + + + + + + + + + + + + + + + + 72 - + - + + + + + + + + + + + + + + + + 73 - + + + + + + + + + + + + + + + + + + 74 - + + + + + + + + + + + + + + + + + + + 75 - + + + + + + + + + + + + + + + + + + + 76 - + - + + + + + - - - + + + + + + + + 77 + + + + + + + + + + + + + + + + + + + 78 - + - + + + + + - - - - + + + + 79 + * - + + + + + + + + + + + + + + + + + 80 + + - + + + + + + + + + + + + + + + + 81 - + + + + + + + + + + + + + + + + + + 82 - + - - + + + + + + + + + - + + + + 83 + - + + + + + + + - - + + + + + + + + 84 - + - + + + + + + + + + + + + + + + + + 85 - + - + + + + + + - - + + + + + + + + 86 + - + + + + + + + + + + + + + + + + + 87 • + - + + + + + + + + + + + + + + + + 88 + - + + + + + + + + - + + + + + + + + 89 - + + + + + + + + + + + + + + + + + + + 90 + + + + + + + + + + + + + + + + + + + 91 + - - - + + + + + + + - + + + + + + + + 92 + - - + + + + + + + + + + + + + + + + 93 + + + + + + + + + + + + + + + + + + + 94 - + - - + + + + + + + - + + + + + + + 95 - + + + + + + + + + + + + + + + + + + 4* 96 • + + + + + + + + + + - + + + + + + + + 97 - + + + + + + + + + + - + + + + + + + + 98 + - - + + + + + + + + + + + + + + + + + 99 * + - + + + + + + + + - + + + + + + + + 100 + + + + + + + + + + - + + + + + + + + Ara - Arabinosc; Fru Fructose; Gal - Galactose; Lac = Lactose; Mai = Maltose; Man = Mannito ; Sue =Sucrose University of Ghana http://ugspace.ug.edu.gh 80 Table 4.3 Diversity of cowpea bradyrhizobia isolates by cross inoculation GROUP1 SUBGROUP2 ISOLATES LEGUMES3 1 24,50,56,60, 62, 67,76, 80,93,95,96,98,99,100 CP 2 2a 57,72,79,97 CP, BG 2b 81 CP, SB 2c 23 CP, CLG 2d 6,22, 25,28, 68, 69, 73, 78, 83 CP, GN 2e 92 CP, PRR 3 3a 1,3, 5, 16, 64, 65, 70, 71,77, 87,94 CP, SB, GN 3b 10 CP, GN, CLG 3c 11,90 CP, GN, PRR 3d 26,55,59, 63 CP, GN, BG 3e 58 CP, BG, PRR 4 4a 54 CP, SB, GN, CTL 4b 32,39,53,75 CP, SB, GN, CLG 4c 15, 17,21,27, 37, 48,66,74 CP, SB, GN, BG 4d 86 CP, BG, PRR, CLG 4e 88 CP, GN, CLG, CTL 4f 91 CP, GN, BG, PRR 5 5a 4,30,34,35,36,38,44, 46, 84 CP, SB, GN, BG, PRR 5b 85 CP, SB, GN, CLG, CTL 5c 7 CP, SB, GN, CLG, PRR 5d 18,19 CP, SB, GN, BG, CLG 5e 33 CP, SB, GN, LC, CLG 6 6a 9, 12, 20,31,40,41, 42, 47, 52, 82 CP, SB, GN, BG, PRR, CLG 6b 89 CP, SB, GN, BG, PRR, CTL 6c 2, 13,45 CP, SB, GN, BG, CLG, CTL 6d 8 CP, SB, GN, BG, CLG, MM 7 7a 14,43,49,51 CP, SB, GN, BG, PRR, CLG, CTL 7b 29 CP, SB, GN, BG, LC, PRR, CLG 7c 61 CP, GN, BG, LC, PRR, CLG, CTL Group number indicates number o f legumes nodulated Subgroup indicates different legume combinations Abbreviations for the legumes LEGEND Bambara Groundnut Calapogonium Cowpea Crotalaria Groundnut Leucaena Mimosa Pueraria Soyabean BG CLG CP CTL GN LC MM PRR SB University of Ghana http://ugspace.ug.edu.gh least nodulated host by the isolates, being nodulated by only one of the isolates (Isolate 8), (Fig. 4.9) and (Table 4.3). Nodulation of the legumes in terms of number of isolates capable of inducing nodule formation on a legume species was in the order cowpea > groundnut > soybean > bambara groundnut > calopogonium > pueraria > leucaena > mimosa. Based on the number of legume species nodulated by an isolate, seven major groupings with 28 sub groups were obtained (Table 4.3). Group one contained the greatest number of isolates (14%). These were all the isolates that were specific on cowpea only. The four food legumes, groundnut, soybean and bambara groundnut, were nodulated by over 50% of the isolates whilst the non-food legumes, mimosa, leucaena, crotalaria, pueraria and calopogonium were nodulated by less than 35% of the isolates (Fig. 4.9). 4.2.3. Serology 4.2.3.1 Antibody responses in immunized mice Response of the B ALB/c mice to rhizobial antigen immunization is illustrated in Fig. 4.10. High serum antibody responses, with titres far beyond 1:6000 were obtained against homologous antigens by indirect ELISA Mean antibody titre against both antigens was quite high, maintaining optical density greater or equal to 0 1 for serum dilutions of up to 1:3000. 81 University of Ghana http://ugspace.ug.edu.gh 82 eo 3-aoe cfu fti Legum es Fig. 4.9 Proportion of cowpea bradyrhizobia isolates that nodulated several legume species. University of Ghana http://ugspace.ug.edu.gh 83 cto oo vj to O' c o o o o o o mm K+t —. mm o o —.O' ON oo o o o o o o o .—. -A ro o L*J —vO “ o to o .u o o o o o o ,_, w O’ ON -4 1^ oo X* o o NO o o o o o o o — <2 oo 'O osi vO Os o *-A o O o o o o —. sO oo o sO 00OO oo o 'O o UJ NO _. o o o o o to nO o VO NO NO00 ro o V*J 'O •.A o m^ , - - - o — o o o o oIO o o to 'VI to '-A o o o o o o —- o ■—• — o _o o oo -4 — o o o o o o o —• •OI u so '-Ao 00 *—* KJ% -o «*A 29 20 5 o to - Isolate o o o o o o to -U VO sO NO oo NO o O' NO o o o o o o _ o Xk to -U w o u sO oo 00 VA o o ro o ] - , - o o o o o o VA —o o o *o 00 w _ o o , - o o -o NO o o o NOU> Uj o o oo oo O' o o , - o o o o o o o UJ 'mm 1— o to-J -o o o o u O' e o . o o o o o o -U M M o c ro00 o -o o O oo vC , . o o o o o o o Q •-* to — X*o X* u> , - o ■ o o -— o o UJ to oo 00 o o 4*'-J o V i nO -o O' NC T - o o o o o o u to ON oo a. NO oo “ ON NO >o so ON «-A c ,_. o o T - o U> o o o o O'to K/\ ~o o to tA o o o 1 - , ■ o -t- ON o NO o o -Jr-A u> to UJ s o t* o , ■ — o oo UJ o o o o oi- oo ro to & '-A o o o o o o o to — UJ — — o-0 o to w s o *-A -J o o o o o o o to itk K/% •~/l 1^ 00to — o UJ to •— 'VI o H sin' University of Ghana http://ugspace.ug.edu.gh N um be r of iso lat es ( % ) 8 6 6 0 ----- 50 F H W 30 ijij i i i i i i i 20 i ii i i j i j i j: 10 o _|-------- 1:■1 ■: : : ■ :l-------------------------------------------------------: Strongly related Differentially related Not related Cross reactivity Fig. 4.11 Relationship between cowpea rhizobia isolates as determined by reactivity of homologous and heterologous antisera University of Ghana http://ugspace.ug.edu.gh 87 Table 4.5 Restriction patterns of cowpea bradyrhizobia isolates revealed by RFLP analysis of PCR-amplified 16s rRNA genes Isolates Restriction pattern1 of amplified 16s rRNA genes digested with Dd el Hae III Msp / Rsa 1 Fast growers 19 a a a a 20 b b b a 23 c c c b 27 b b b a 29 b b b a 38 d d d c 43 e c e b 44 f e f b 51 b b b a 57 c c c b 59 g f g d 60 b b b a 69 S r f g d 79 b b b a 88 a a a a 92 h g h ? 98 i ? i e The different patterns detected with each enzyme among the 100 isolates analysed are designated by the lower case letters. University of Ghana http://ugspace.ug.edu.gh 87a Table 4.5 continued Isolates Restrirtinn naftern of amDlified 16s rRNA 2cnes digested with Dde/ Hae III Msp / Rsa / Slow growers 1 a b a a 2 b a b b 3 a a c b 4 b c d a 5 a a c b 6 a d a c 7 a f c d 8 a a e b 9 a f c d 10 a b a a 11 a e f a 12 a b a a 13 c a e b 14 d b d a 15 a a c b 16 a a c b 17 a a a b 18 a a a b 21 e e a b 22 a b a b 24 a b a a 25 f f J b 26 K g c b 28 h k k b 30 a b a b 31 a a a b 32 i 1 1 d 33 a a a b 34 d b d b 35 a b a b 36 a b a a 37 a b a a 39 1 J j i 40 k i g f 41 k J g f 42 a a a b 45 a a a b 46 g __ k g c 47 a a a b University of Ghana http://ugspace.ug.edu.gh alal 48 49 50 52 53 54 _55_ 56 58 61_ 62 63 64 65 66 67 68 70 21 72 73 74 75 76 77 78 80 I I 82 83 84 85 86 _87 89 90 21 93 94 _95 96 97 99 101 87b continued Restriction pattern of amplified 16s rRNA genes digested with Dd el Hae III a b Msp / Rsa I _b_ b a b_ a e a f University of Ghana http://ugspace.ug.edu.gh Restriction patterns of PCR-amplified fragment of 16S rRNA genes digested w ith Haelll (A) or Msp/ (B). The lane assignments (numbers) represent Bradyrhizobium strains. Lane M = molecular marker. University of Ghana http://ugspace.ug.edu.gh 88 Table 4 .6 Distribution of cowpea bradyrhizobia isolates among 20 genotypes identified by RFLP analysis of PCR-amplified 16s rRNA genes 16s rRNA genotype1 Isolates Fast growers A 19, 88 B 20, 27,29,51,60, 79 C 23,57 D 38 E 43 F 44 G 59, 69 H 92 I 98? Slow growers A 1, 3,5, 6, 7, 8,9, 10, 11, 12, 15, 16, 17, 18,22, 24,30, 31,35,37, 42, 45, 46, 49, 50, 53, 61, 62, 63, 64, 65, 66, 67, 74, 75, 85, 86, 94, 95 B 2,4 C 13,77, 82, 87,99 D 14, 34, 68 E 21,73,96 F 25,70,71,97, 100 G 26, 46, 52, 55, 56 H 28, 80, 81, 84, 89 I 32, 58, 83, 90 J 39, 76,91,93 K 40,41 The 16s rRNA genotypes lettered A to K represent the species group of bradyrhizobia isolates obtained with the four endonucleases used. University of Ghana http://ugspace.ug.edu.gh one to five isolates (Table 4.6) The ninth genomic species of the fast growing isolates designated I, was not included in the relative similarity analysis due to inconsistent bands it produced. The slow growing isolates produced 11 different composite genotypes (Table 4.6). The distribution of the isolates among the slow growing genotypes was highly unbalanced. The number of species in of the genotypes designated A, were more than half of the total number of the isolates, whilst each of the remaining 10 composite genotypes contained between 1.05% to 3.05% isolates (Table 4.6). Diversity among the genomic species identified in both the fest and slow growing isolates was very high, reaching 80% divergence (Figs 4.12 & 4.13). 4J Effectiveness of isolates in fixing nitrogen Symbiotic effectiveness was determined for each isolate from the mean of six plants inoculated with an isolate. Number of nodules formed varied among isolates, as well as symbiotic effectiveness. Estimated values for effectiveness (relative to uninoculated control) ranged from 23.5% to 118% per plant for the 100 isolates (Table 4.7). Effectiveness in fixing nitrogen obtained by five of the isolates was similar to, or higher than that of plants fertilized with 70 kg N/ha (Table 4.7). Based on the index of effectiveness the isolates varied from ineffective to highly effective, but with a predominance (68%) of isolates being ranked as moderately effective (Fig. 4.14). The effectiveness of the isolates in fixing nitrogen followed the trends observed with shoot dry weights (Table 4.7). Shoot dry weight and shoot nitrogen produced by the most effective isolate (isolate 44) were 50% higher than the values for the least effective isolate (isolate 28) (Table 4.7 and Plate 4.2). Correlation between effectiveness and shoot dry weight of plants inoculated with the different isolates was the highest (0.91), and more than correlations between the other parameters (Table 4.8). The effectiveness of the isolates grouped within each of the various ecological 89 University of Ghana http://ugspace.ug.edu.gh 90 0 8 0 7 0 6 0.5 0 4 0.3 0.2 0 1 I 1----1----1---- f----1----1---- 1---- 1---- 1 B ------------------------------------------------------ G A D ----------------------- C _____________________ E F H Fig 4 12 Dendrogram (UPGMA) showing relationship among genomic species of fast-growing cowpea bradyrhizobia isolates as determined by RFLP analysis of the 16s rDNA The matrix o f pairwise genetic distances was used to construct the dendrogram. University of Ghana http://ugspace.ug.edu.gh 91 0.9 0.8 0.7 0.6 0.5 0.4 03 0.2 0 1 I t 1 t 1 t t t t h A B H C D G E K J Fig. 4.13 Dendrogram (UPGMA) showing relationship among genomic species of slow-growing cowpea bradyrhizobia isolates as determined by RFLP analysis of the 16s rDNA The matrix o f pairwise genetic distances was used to construct the dendrogram. University of Ghana http://ugspace.ug.edu.gh 92 Table 4.7 Symbiotic effectiveness of cowpea bradyrhizobia isolates Isolate No. Shoot Dry No. of Nodule Weight (g) Nodule Dry Weight (g) Shoot N(%) Effectiveness (%) 1 8.61 2 2.17 1.26 54.78 2 10.44 121 2.46 1.40 95.45 3 8.57 25 2.30 0.95 53.89 4 9.42 69 2.33 2.07 72.82 5 8.76 38 2.28 0.90 58.12 6 9.06 49 2.15 0.87 64.81 7 8.88 87 2.41 0.97 60.80 8 9.11 31 2.24 1.71 65.92 9 9.41 96 2.27 1.54 72.60 10 10.48 126 2.50 1.68 96.43 11 9.07 21 2.29 1.20 65.03 12 9.82 36 2.41 1.46 81.73 13 9.62 55 2.35 1.65 77.28 14 10.86 175 2.60 1.82 104.89 15 10.58 150 2.53 1.76 98.66 16 10.14 107 2.36 1.96 88.86 17 9.28 31 2.42 2.41 69.71 18 10.14 78 2.50 1.88 88.86 19 9.95 135 2.48 1.20 84 63 20 9.24 108 2.29 1.74 68.81 21 9.07 165 2.42 1.18 65.03 22 9.63 102 2.32 2.69 77.50 23 8.99 88 2.43 1.08 63.25 24 8.63 2 2.14 0.92 55.20 25 9.26 49 2.39 1.09 69.26 26 9.30 41 2.31 1.18 62.36 27 7.58 11 2.14 0.84 31.84 28 6.51 4 2.18 0.78 23.57 29 9.04 111 2.48 1.57 46.36 30 7.89 93 2.36 0.83 38.25 31 8.01 48 2.25 0.92 41.42 32 9.15 87 2.37 1.46 66.81 33 9.19 79 2.46 1.06 67.70 34 8.91 59 2.20 0.70 61.46 35 9.31 73 2.36 1.34 70.37 36 9.14 86 2.53 1.71 66.59 37 9.51 101 2.31 1.48 66.81 38 10.44 149 2.54 1.54 95.54 39 7.79 159 2.35 1.37 36.52 40 9.19 77 2.39 1.57 67.70 41 10.26 212 2.52 2.13 99.55 University of Ghana http://ugspace.ug.edu.gh 93 Table 4.7 cont'd Isolate No Shoot Dry No. of Nodule Weight (g) Nodule Dry Weight (g) Shoot N(%) Effectiveness (%) 42 9.10 168 2.54 1.74 65.70 43 9.11 71 2.32 0.64 65.92 44 11.45 136 2.43 2.88 118 04 45 8.82 81 2.33 0.50 59.46 46 9.26 97 2.38 2.02 69.26 47 9.45 166 2.35 2.24 73.49 48 9 08 24 2.17 0.98 65.25 49 8.55 32 2.16 0.70 53.45 50 9.37 138 2.50 2.04 71.71 51 9.79 142 2.39 1.96 81 06 52 9.16 107 2.36 2.12 77.06 53 9.37 74 2.25 1.71 71.71 54 9 41 114 2.22 2 04 72.60 55 9.85 159 2.50 2.18 82.40 56 9.65 171 2.30 1.80 77 95 57 9.64 105 2.38 1.89 77.72 58 9 60 92 2.28 1.68 76.83 59 9.75 126 2.43 1.54 80.17 60 9.46 63 2.26 1.65 73.71 61 8 70 126 2.25 0.92 56.79 62 932 134 2.26 1.42 70.60 63 9.24 137 2.33 1.04 68.81 64 9.20 38 2.19 0.90 67.92 65 9 41 48 2.26 1.01 72.60 66 9.30 66 2.22 1.06 70.15 67 9 32 57 2.24 1.06 70.60 68 9.22 156 2.16 1.02 68.37 69 923 130 2.37 1.18 68.59 70 9.38 29 2.26 1.16 71.93 71 8.96 68 2.29 0.98 62.58 72 7.47 1 2.12 0.78 29.39 73 9.70 84 2.31 1.44 79.06 74 9 34 36 2.22 1.01 71.04 75 9.25 57 2.20 1.34 69.04 76 9.29 40 2.18 0 98 69 93 77 9.23 14 2.22 1.15 68.59 78 9.17 18 2.21 1.12 67.26 79 8.63 20 2.24 0.94 55.23 80 904 21 2.22 1 24 64 36 81 9.36 15 2.19 1 04 71.49 University of Ghana http://ugspace.ug.edu.gh 94 Table 4.7 cont'd Isolate No. Shoot Dry No. of Nodule Weight (g) Nodule Dry Weight (g) Shoot N(%) Effectiveness (%) 82 9.64 79 2.28 2.38 77.72 83 10.19 116 2.43 2.80 89.97 84 9.47 34 2.29 1.77 73.94 85 9.46 29 2.19 1.54 73.71 86 9.58 97 2.26 2.35 76.39 87 9.73 104 2.40 2.55 79.73 88 10.2 128 2.49 2.60 90.20 89 8.79 58 2.21 0.96 58.79 90 9.26 34 2.23 1.57 69.26 91 9.29 52 2.20 0.92 69.93 92 9.26 38 2.17 1.09 69.26 93 9.33 23 2.16 0 90 70.82 94 8.57 28 2.18 0 94 53 89 95 8.65 52 2.31 1.02 55.67 96 8.63 1 2.16 0 98 55.23 97 8.64 6 2.25 0.78 55.45 98 9.26 90 2.25 0.95 69.26 99 9.30 16 2.22 1.01 70.15 100 8.97 69 2.13 1.15 62.80 Control 6.82 0 0 078 23.60 +N 10.68 0 0 2.68 100.00 University of Ghana http://ugspace.ug.edu.gh 95 80 Highly Moderately Ineffective Effective Effective Indices of effectiveness Fig. 4.14 Percent number of cowpea rhizobia isolates for groups of effectiveness University of Ghana http://ugspace.ug.edu.gh 96 Table 4 .8 Correlation between effectiveness and some parameters of nitrogen fixation Shoot dry weight Nodule Numbers Nodule dry weight Shoot Effectiveness nitrogen Shoot dry weight 1 Nodule Numbers 0.509 1 Nodule dry weight 0.553 0.691 1 Shoot nitrogen 0.617 0.513 0.527 1 Effectiveness 0.908 0494 0.555 0.615 1 University of Ghana http://ugspace.ug.edu.gh 97 Plate 4 .2 Differences in plant size, leaf colour and plant vigour of cowpea inoculated with the most effective and least effective bradyrhizobia isolates. University of Ghana http://ugspace.ug.edu.gh zones is shown in Table 4.9. Generally, distribution of the isolates in the ecozones followed a normal distribution trend with the majority being moderately effective. A similar trend was observed when the isolates were grouped according to slow and fast growing types (Table 4.10). 4.3.1 Relative effectiveness of isolates in fixing nitrogen The results of the relative effectiveness of the 10 most effective isolates against the standard strain TAL 169 is presented in Fig. 4.15. The results indicated that six of the isolates possessed symbiotic effectiveness superior to the reference strain. Four of these differed significantly from the standard strain (Fig. 4.15). 4.4 Competition for nodule occupancy Few data exist on the impact of foreign genes on the fitness of an organism (Doyle et al., 1995). It is required that before using any marker system for ecological studies, its effects on the most important attributes of the organism have to be studied (Sessitsch et al., 1995). The potential effects of the Gus transposon on the fitness of the marked effective bradyrhizobia isolates was therefore evaluated before the actual competition studies Each gus-marked isolate and its parent isolate were co-inoculated at equal population densities onto the same cowpea plant grown in a growth pouch. The plants were harvested after 35 days and nodules that were formed typed using the gus staining assay method described previously. The results showed that mutants and the parent isolates formed nearly equal proportions of nodules (Table 4 .11), indicating that the mutants were equally competitive as the parent isolates. 98 University of Ghana http://ugspace.ug.edu.gh 99 Table 4.9 Effectiveness of cowpea bradyrhizobial isolates from soils of the different ecological zones. Number of isolates Ecological Zone Indices of effectiveness (%) Highly effective Moderately effective Ineffective Coastal Savanna 20.8 75.0 4.1 High Rainforest 75.0 25.0 0 Semi-deciduous Forest 33.3 58.3 8.3 Forest Savanna Transition 25.0 66.7 8.2 Guinea Savanna 16.7 79.2 4.1 University of Ghana http://ugspace.ug.edu.gh 100 Table 4.10 Symbiotic effectiveness profiles of cowpea bradyrhizobia isolates Isolates Number of isolates Effectiveness (%) tested Highly effective Moderately effective Ineffective Fast-growing 18 33.3 61.1 5.6 Slow-growing 82 24.4 70.7 4.9 University of Ghana http://ugspace.ug.edu.gh 101 Fig. 4.15 Symbiotic effectiveness of cowpea bradyrhizobia isolates relative to TAL 169 * Bars with same letters are not significantly different at 1% level o f significance University of Ghana http://ugspace.ug.edu.gh 1 0 2 Table 4.11 Percentage of nodule occupancy of GUS marked cowpea Bradyrhizobia isolates co-inoculated with the corresponding parent isolates onto the same plants. Gus marked isolates Blue stained nodules* (%) G2 51 ±5 G10 53 ±3 G14 49 ±2 * Means ± standard deviation of 3 replicates University of Ghana http://ugspace.ug.edu.gh 103 Results of the competition experiment between the gus-marked effective isolates and ineffective isolates are show in Table 4.12. The number of nodules formed by the different isolates and different isolate ratio combinations varied between 46 to 179 (Table 4.12). Although at the 1:1 ratio, each of the effective isolates occupied a higher proportion of nodules than by three of the five ineffective competitors, (29, 30, and 94), the reverse was true for the two remaining ineffective isolates (28 and 72), which outcompeted the three effective isolates tested. Even when the effective isolates outnumbered the more competitive ineffective isolates (28 and 72) by the ratio 2:1, the effective isolates still made no significant gains in nodule occupancy (Table 4 12). Calculated competitive indices (Beattie et al., 1989) are shown in Fig. 4.16. In each case die GUS-marked effective isolate is strain X and the unmarked ineffective isolate is designated as Y. The intercept values confirmed that the ineffective isolates 28 and 72 were more competitive than any of the effective isolates, whilst the remaining three ineffective isolates (29, 30 and 94) were less competitive. A depression of plant yield (plant height and plant biomass) was also observed when the highly competitive ineffective isolates 28 and 72 competed with the effective isolates. Interestingly, this depression in plant yield occurred even when the effective isolates occupied 50% or slightly more of the nodules. In contrast, a higher plant yield was observed with the combination of effective isolates plus ineffective isolate, 94, irrespective of the ratio of combination and proportion of nodules occupied by the competing isolates (Plate 4.3a and b). University of Ghana http://ugspace.ug.edu.gh *Table continued on next page G2 GUS-m arked effective isolates 94 72 30 29 28 Ineffective isolate designation 0 5 1 2 0.512 0.512 0.512 0.512 i Ratio (GUS-m arked isolate: ineffective isolate! 32.8 33.8 36.0 22.8 24.9 24.4 28.8 28.9 31.1 34.4 30.9 30.9 24.6 22.8 23.8 28.0 Plant height (cm ) 10.4 9.8 10.0 8.48.08.9 9.39.3 9.7 9.79696 9.29.29.3 9.6 Shoot dry weight (g) 127 134 161 110 6799 75142 147 969580 465054 52 Number of nodules 53.5 60.6 86 3 12.7 34.0 67.6 62.6 62.6 62.5 55.2 61.0 86.2 4.3 28.0 15.4 100 % GUS-m arked nodules 46.4 39.3 13.6 2 66 0 32.3 37.3 37.3 37.4 44.7 39.0 13.7 95.6 72.0 84.5 i % ineffective nodules H & 5T to crc £5 ' C/3 rod o5- rc 104 University of Ghana http://ugspace.ug.edu.gh *Table continued on next page Ol O GUS-m arked effective isolates 94 72 30 29 28 Ineffective isolate designation 0.512 0.512 0 5 1 2 0 5 1 2 0.512 i Ratio (GUS-m arked isolate: ineffective isolate) 35.5 37.1 35.0 Z'P Z L6 Z 68 Z 34.4 34.6 32.9 28.8 30.1 35.4 21.7 26.0 27.8 30.2 Plant height (cm ) 10 8 98 10.1 9.19.2 8.6 8.4?29.6 8 99 1 9.4 8.5848.9 9.2 Shoot dry weight (g) 118 148 146 122 124 114 141 123 142 1076780 11498 102 106 Number of nodules 55.9 54.0 70.5 39.3 45.1 60.5 53.1 78.8 83.0 35.5 59.7 762 33.3 45.9 66.6 100 % GUS-m arked nodules 44.1 46.0 29.4 60.6 54.8 39.4 46.8 21.1 17.0 64.4 40.2 23.7 66.6 54.0 33.3 l % ineffective nodules 105 University of Ghana http://ugspace.ug.edu.gh G14 GUS-m arked effective isolates 94 72 30 29 28 Ineffective isolate designation Z I £ 0 w - P 0.512 0.512 0.512 j i Ratio (GUS-m arked isolate: ineffective isolate) 38.4 32.3 40.2 Y9 Z ZO Z ZZ Z 29.9 26.8 27.5 33.5 32.0 33.6 21.0 23.6 24.6 28.8 Plant height (cm ) I II 86 roi *sD 00 ooi—> Lft 9.79.19.3 10.0 9.7 10 0 L 8 6 8 6 8 9.4 Shoot dry weight (g) 115 136 179 5 I g 4896109 ^ s§ —•V* ^ v£) 887386 96 Number of nodules 66.9 63.7 75.4 12.2 32.1 57.1 72.9 69.4 78.8 63.0 60.6 84.8 33 0 16 4 62.7 100 % GUS-m arked nodules 33.1 36.2 24.5 87.7 67.8 42.8 27.0 30.5 21.1 36.9 39.3 15.2 67.0 83.5 37.2 i % ineffective nodules H cr rT to 8 106 University of Ghana http://ugspace.ug.edu.gh 107 I s o la te Cl2 ♦ It No 28 u No 29 —A - u No 30 ■ If NO 72 — n No 94 Isolate G10 ! LogCMy) Isolate GI A l j> t fliTy) NO 2« H NO » —A— IS No 30 —X— K No T i —* — II No 9* Fig. 4.16 Linear regression of Log [(Px+Pboth)/(Py+Pboth)] against Log (Ix/Iy) for GUS-marked Isolates 2,10,14 when competed against ineffective Isolates 28, 29, 30, 72 and 94. University of Ghana http://ugspace.ug.edu.gh Plate 4.3 a Depression of plant height and biomass associated with ineffective isolate 28 in competition with gus-marked effective isolate 2 (G2). University of Ghana http://ugspace.ug.edu.gh Plate 4.3b Higher plant yield associated with ineffective isolate 94 in competition with gus-marked effective isolate 2 (G2). University of Ghana http://ugspace.ug.edu.gh 108 4.4.1. Effect of placement and time of placement The yield enhancement or suppression effect observed with the inoculation of isolates 94 and 28, respectively, was further examined by evaluating the effect of time of placement of the competing isolates relative to the other on the seed. It was observed that the suppressive effect of isolate 28 was removed as its placement was delayed, whilst the enhancement effect of 94 was also reduced as its relative time of placement was delayed (Figs. 4.17 to 4.22). The suppressive effect of 28 was more pronounced when it was placed first followed by the effective isolate. This was, however, not the case with the beneficial effect exerted by isolate 94. 4.4.2. Time of formation of nodules by competing isolates There were differences in the time of appearance of nodules formed by the different strains The earliest nodule was observed 9 days after inoculation, by the ineffective isolate 29 (Fig. 4.23). The longest period for the first nodule formed to be observed was, 14 days after inoculation and this was by isolate 72. The total number of nodules formed during the 14 days post inoculation period by all the isolates (Fig. 4.23) showed no significant inter-strain differences (mean nodule number plant"1 = 8). University of Ghana http://ugspace.ug.edu.gh 109 105 _ 10 m S 95 I Z 9 Time (days) □ 94 ■ G2 □ G2 94 105 _ 102» 3 9 5 tn 2 4 Time (days) 0 □ 94 ■ G2 0 9 4 G2 Fig. 4.17 Effect o f placement and time o f placement o f effective isolate (G2) and ineffective isolates (28 and 94) on shoot biomass. University of Ghana http://ugspace.ug.edu.gh 110 Fig 4 .18 Effect of placement and time o f placement o f effective isolate (GI 0) and ineffective isolates (28 and 94) on shoot biomass. University of Ghana http://ugspace.ug.edu.gh I l l 10 9 5 t 8 5 1•C(O □ 94 ■ G14 □ G14 94 Time (days) 10 9 5 8 9 - 8 5 8£(/) „ 7 5 Time (days) Fig. 4.19 Effect of placement and time of placement o f effective isolate (G14) and ineffective isolates (28 and 94) on shoot biomass University of Ghana http://ugspace.ug.edu.gh Ill □ 94 ■ G14 □ G14 94 10 9 5 0 2 4 6 Time (days) □ 94 ■ G14 □ 94 G14 Fig. 4.19 Effect of placement and time o f placement of effective isolate (GI 4) and ineffective isolates (28 and 94) on shoot biomass University of Ghana http://ugspace.ug.edu.gh 1 1 2 25 2 4 6 Time (days) □ 94 ■ G2 □ G9 94 2 5 _ 2 £ c* 1 5 *_ Z 1 0 2 4 6 Time (days) 0 94 ■ G2 □ 94: G2 Fig. 4.20 Effect o f placement and time of placement o f effective isolate (G2) and ineffective isolates (28 and 94) on shoot nitrogen University of Ghana http://ugspace.ug.edu.gh 113 2 5 2 0 2 4 6 Time (days) □ 94 ■ G10 □ G10:94 □ 94 ■ G10 □ 94G10 Fig. 4.21 Effect of placement and time of placement of effective isolate (G10) and ineffective isolates (28 and 94) on shoot nitrogen University of Ghana http://ugspace.ug.edu.gh 114 1 8 1 6 — 1 4 JS 0 2 4 6 Time (days) 2 1 8 1 6 0 2 4 6 Time (days) 1 4 0 2 4 6 Time (days) 2 1 8 _ 1 6 £ 14 0 2 4 6 Time (days) Fig. 4.22 Effect of placement and time of placement of effective isolate (G14) and ineffective isolates (28 and 94) on shoot nitrogen University of Ghana http://ugspace.ug.edu.gh Da ys af ter in oc ul at io n 115 14 G2 G10 G14 28 29 30 72 94 Isolates Fig. 4.23 Time when nodules were first visible on cowpea roots inoculated with the competing isolates in single cultures University of Ghana http://ugspace.ug.edu.gh 116 4.5 Inoculation of cowpea with indigenous Bradyrhizobium isolates Inoculation generally increased the number of nodules produced by cowpea plants in all the soils (averaged) as shown in Fig. 4.24. However, the soil-inoculated treatments recorded higher nodule numbers than the seed-treated ones. In certain cases, for instance in the Lima soil series, the mean number of nodules produced by plants in the soil-inoculated treatment was double that of the seed-inoculated ones. In both seed and soil inoculation, the mixed strain inoculation consisting of the three isolates produced higher number of nodules recorded. Response to inoculation in terms of nodulation was best realised in Lima and Ankasa soils and poorest in Akuse soil (Fig. 4.24). Shoot dry weight of inoculated cowpea plants showed a greater yield over those of the uninoculated control (Fig. 4.25). The trend of shoot dry matter yield of the inoculated plants was similar to that obtained for nodulation (Fig. 4.24). Table 4.13 summarises the data for percent and total nitrogen of cowpea plants and shows that percent nitrogen values associated with the various inoculation treatments were significantly different from each other in the different soils. Plants inoculated with the mixed inoculants were superior in percent nitrogen content (Table 4.15). Again significant differences between the inoculated and the uninoculated control were best seen in Lima soil (Table 4.14). University of Ghana http://ugspace.ug.edu.gh N um be r of no du le s/ pl iin t 117 160 140 120 100 80 60 40 20 0 Akuse Lima Ankasa ■ Soil □ Seed Bekwai Lima Inoculated isolates Fig 4.24 Nodulation of cowpea (Asontem) plants inoculated with isolated native cowpea bradyriiizobia isolates. Io = Uninoculated; In 2, In 10 and In 14 = isolates 2, 10 and 14, respectively University of Ghana http://ugspace.ug.edu.gh Sh oo t dry we ig ht ( g) 118 12 10 Akuse Bekwai Lima Ankasa J i i Akuse Bekwai Lima Inoculated isolates Ankasa 8 2 B Soil □ Seed Fig.4.25 Shoot yield of cowpea (Asontem) plants inoculated with isolated native cowpea bradyrhizobia isolates. Io = Uninoculated; In 2, In 10 and In 14 — isolates 2, 10 and 14, respectively University of Ghana http://ugspace.ug.edu.gh 119 Table 4.13 Total N and % N in shoots of inoculated and uninoculated cowpea plants Soil Series Soil inoculation Seed inoculation inocula * %N Total N (mg/kg) %N Total N (mg/kg) Io 4.48 a 286.72 a 4 48 a 286.72 a In 2 4.78 a 344.16 a 4 61 a 285.82 a Akuse In 10 4.90 a 372.40 ab 5.12 b 337.92 b In 14 5.58 b 390 60 b 4 85 b 339.50 b In (2+10+14) 5.61 b 403.92 c 5.24 b 377.28 c Io 2.68 a 155.44 a 2 68 a 155.44 a In 2 2 90 ab 179.80 ab 2.72 a 163.20 ab Bekwai In 10 3.20 be 204.80 b 3 01 b 186.62 ab In 14 2.97 ab 207.90 b 2.99 b 185.38 ab In (2+10+14) 3.42 c 246.24 c 3.01 b 204.68 b Io 4.42 a 300 56 a 4.42 a 300.56 a In 2 4.50 a 378.00 b 5 48 b 394.56 b Lima In 10 5.86 b 539.12 c 5.29 b 402.04 b In 14 5.77 b 507 76 c 5 86 c 468.80 c In (2+10+14) 6.14b 626 28 d 6.02 c 505.68 d Io 2.99 a 155.48 a 2 99 a 155.48 a In 2 4.00 b 216.00 b 4.20 b 235.20 b Ankasa In 10 4.42 b 251.94 c 4.40 b 255.20 b In 14 4.74 b 265 44 c 4.48 b 268 80 b In (2+10+14) 5.80 c 336.40 d 5.68 c 352.16c Means followed by same letters in a column are not significantly different at 0.01 level for same soil series. * Io “ uninoculated; In 2, In 10 and In 14 = Inocula 2, 10 and 14, respectively. University of Ghana http://ugspace.ug.edu.gh 120 Table 4.14 Comparison of Means of Total N and %N by Soil Series Series Total N (mg/kg) %N Soil inoculation Seed inoculation Soil inoculation Seed inoculation Lima 469.88 a 414.37 a 5.33a 5.38a Akuse 359.56 b 325.45 b 5.10b 4.89b Ankasa 245.05 c 253.37 c 4.39c 4 35c Bekwai 198.84 d 179.06 d 3 03d 2 .88d Means followed by same letters in a column are not significantly different at 0.01 level for same soil series. University of Ghana http://ugspace.ug.edu.gh »?1 Table 4.15 Comparison of Means of Total N and %N by Inocula TOTAL N (mg/kg) %N Inocula* Soil inoculation Seed inoculation Soil inoculation Seed inoculation Io 224.55 a 224.55 a 3.68a 3.68a In 2 279.49 b 269.69 b 4.05b 4.21b In 10 342.06 c 295.44 c 4.59c 4.45c In 14 342.35 c 315.62 d 4.77c 4.55c In (2+10+14) 403.21 d 360.00 e 5.24d 4.99d Means followed by same letters in a colamn are not significantly different at 0.01 level for same soil series. * Io = uninoculated; In 2, In 10 and In 14 = Inocula 2 ,1 0 and 14, respectively. University of Ghana http://ugspace.ug.edu.gh 1 2 2 CHAPTER FIVE 5.0 Discussion 5.1 Potential to improve nitrogen fixation of cowpea The potential to improve nitrogen fixation of cowpea in Ghanaian soils was evaluated in this study by conducting experiments to assess the natural nodulation potential of cowpea, estimate bradyrhizobia numbers capable of nodulating cowpea in the soils and to determine the response of cowpea to inoculation and nitrogen fertilization. The formation of adequate number of nodules on legumes in any soil is dependent on the presence of high numbers of the homologous rhizobia in the soil. Estimating rhizobial population density is an indirect means of predicting whether or not a legume will respond to inoculation (Thies et al., 1991). Although there is no direct way of measuring the density of indigenous rhizobial populations in soils, the most probable number (MPN) plant infection assay provides an indirect estimate (Vincent, 1970). The most probable number infection test is based on the assumption that organisms axe randomly distributed and that the presence of one Rhizobium cell is capable of inducing nodulation on an appropriate host (Woomer et al., 1988). Counts of indigenous bradyrhizobial population capable of nodulating cowpea showed that most of the soils in Ghana harbour large populations that are able to nodulate cowpea. This is not unusual since Vincent (1980) reported the presence of rhizobia even in virgin soils, although in general, in such soils, they may occur in low numbers. In this study, cowpea bradyrhizobia were found in soils never planted to cowpea (Bekwai and Nzema). This indicates that either bradyrhizobia are naturally part of the indigenous soil microflora or that other native legumes University of Ghana http://ugspace.ug.edu.gh 123 (including grain and cover legumes, shrubs and trees) are serving as hosts and thus inoculant sources of bradyrhizobia for cowpea. The higher numbers that have previously supported the growth of a host legume has been attributed to the high rate of rhizobial multiplication in the host rhizosphere compared to non-host rhizosphere (Cregan and Keyser, 1989). However, the use of legume inoculants is not common in Ghana, and non-existent at the locations where the soils used in this study were sampled. It is therefore reasonable to assume that the high bradyrhizobia cells encountered in many of these soils truly represent long established indigenous populations However, as much as forty percent of the soils examined contained 100 or less rhizobia per gram of soil, which gives an indication that a yield response to inoculation is most likely to be obtained in these soils. This does not preclude the possibility that response to inoculation in the other soils is possible. Response to inoculation can be obtained if competitive and highly effective strains are introduced in high quality inoculants. The studies of Woomer et al. (1988) have confirmed, with five different legume species (Trifolium repens, Medicago sativa, Vida sativa, Leucaena leucocephala, and Macroptilum atropurpureum), the importance of the appropriate host legume on the occurrence and preponderance of a particular Rhizobium species It is therefore not surprising that the soils that contained the highest numbers of bradyrhizobia happened to be in those from areas where cowpea is commonly cultivated. Other factors such as soil pH, soil organic matter, soil nitrogen, soil phosphorus and soil texture may also influence the population of indigenous rhizobia (Danso and Alexander, 1974) Depending upon their severity, these factors may bring about wide differences in the rhizobial University of Ghana http://ugspace.ug.edu.gh 124 numbers in different soils. Low soil pH for instance results in poor persistence of rhizobia and can affect in addition to population, the spatial distribution of indigenous rhizobia (Munns and Keyser, 1981). This was evident in this study as very low (e.g. 6 rhizobia/g of soil) and in some instances complete absence of bradyrhizobia was recorded in the soils with pHs of less than 4.5. There was a direct relationship between nodulation and bradyrhizobial numbers; nodulation was observed to be low in soils, such as Ankasa, Boi and Tafali, with low incidence of native cowpea bradyrhizobia. This is in contrast to the abundant nodulation observed in soils like Akuse, Agawtaw, and Bediesi where high bradyrhizobia counts were obtained. These result indicate that nodulation and nitrogen fixation of cowpea can be improved significantly through studies to assess the population densities of bradyrhizobia in different soils as an indication of the potential need for inoculation to achieve higher nodulation. In similar studies by Thies et al. (1991), the results suggested that cowpea inoculation response is likely to be obtained in soils containing less than 100 cells of bradyrhizobia per gram of soil, with reduced chances of improving nodulation by inoculation above this threshold value. Further improvements in the benefits of inoculation can be expected through the exploitation of the enormous genetic variability for nodulation and nitrogen fixation that would be found to exist between the indigenous bradyrhizobia and also in host genotypes. The response of cowpea to nitrogen fertilizer as measured was the net effect of nitrogen uptake and nitrogen fixation over the growing period. Of all the mineral nutrients, nitrogen has the most pronounced influence on nitrogen fixation in legumes (Horst, 1986). Although in general mineral nitrogen depresses nodulation and nitrogen fixation (Eardly et a l , 1984; Streeter, 1985; University of Ghana http://ugspace.ug.edu.gh 125 Carrol and Mathews, 1990), low levels have been found in some cases to exert beneficial effects on the symbiotic process and on yield (Hardarson et al., 1984). When the nitrogen levels of the soils were increased in this study from 0 to 40 kg N/ha, shoot dry weight of the cowpea cultivars increased except Benga, which did not increase in Akuse and in Tafali soils. The observed increment was not realised in Adenta soil. The shoot dry weight significantly increased in some of the cultivars and in some of the soils as the nitrogen levels were increased from 40 kg N/ha till a peak was reached. However, the N level at which a peak was attained differed in the different soils, reflecting the influence of the different levels of soil nitrogen already present in the different soils. Similarly, the cultivars showed different responses to different levels of nitrogen application. The interesting observation may be an indication that some of the cultivars were capable of high N2 fixation at both low and high soil inorganic levels, as observed in soybean by Hardarson et al. (1984). The response of the cultivars to increasing levels of nitrogen is an indication that nitrogen fixation was not supplying the plants with all the nitrogen they required for maximum yield. The diminishing of the positive response beyond a certain nitrogen level is not an unexpected result As the levels of nitrogen increase, nitrogenase activity, as an expression on nitrogen fixation rate has been found to decline drastically (Horst, 1986) High external concentrations of nitrogen inhibit root infection by Rhizobium (Carrol and Gresshoff, 1983). It is therefore not surprising that nodulation of cowpea in the present study declined as the nitrogen levels were increased (Figs. 4.3 and 4 .8). Some species and varieties of legumes support greater nitrogen fixation than others when soil nitrogen is high (Hardarson et University of Ghana http://ugspace.ug.edu.gh 126 a l, 1984; Senaratne et a l, 1987). Proper use when made of known genetic variability among existing species and cultivars can result in high N2 fixation and high crop yields even when soil nitrogen is high or when fertilizer nitrogen needs to be applied to associated non-fixing crop In this study, cowpea displayed a remarkable ability to support nodulation at as high as 160 kg N/ha. Lower levels up to 100 kg N/ha have been reported to severely decrease nodulation in other legumes such as soybean (Hardarson et a l, 1989) and common bean (Ruschel et a l, 1979). The results obtained in this study and that of Awoniake et al. (1990) may indicate that cowpea is a suitable candidate for intercropping. 5.2 Diversity of indigenous cowpea bradyrhizobia isolates The results of the physiological and metabolic analysis obtained have demonstrated and confirmed the results of other workers such as Mpepereki et al. (1997), that cowpea is nodulated by both fast and slow growing rhizobia. As much as 18% of the isolates were the fast growing type. A significant proportion (73%) of the isolates (both fast and slow growing) tolerated acidic conditions which seems to confirm the reports of Keyser et a l (1979); Lowendor£ (1980) and Zablotowicz and Focht, (1981), that cowpea rhizobia are acid-tolerant This acid tolerance may be an indication of adaptation to acid environments, although in the present study, only two of the isolates were isolated from soils with pH of less than 4.5. The mechanism for rhizobial tolerance to low pH is not well understood, even though plasmids (Chen et a l, 1993) and extracellular polysaccharide slime (Cunningham and Munns, 1984) have been implicated. Graham et al., (1994) reported that acid tolerance was not an adaptive response nor was it plasmid mediated nor was it correlated with extracellular polysaccharide slime production, or related to synthesis of polyamines. Graham et al, (1994) suggested that pH tolerance may be University of Ghana http://ugspace.ug.edu.gh 127 associated with outer membrane composition and structure after observing that strain UMR 1899 cells accumulated glutamate under acid stress and became more hydrophobic. This adaptive response according to Tiwari et al. fl 996), is induced by an acid protection system controlled by different pH regulated genes, which cause increase resistance to acid stress. The growth exhibited by some of the isolates in laboratory media at low and high pH suggest the versatility of these indigenous isolates to survive under different soil conditions. High salt tolerance was also found among the isolates. This contrasts reports of low and narrow salt tolerance ranges observed among rhizobial populations from other regions such as Nigeria, where cowpea isolates failed to grow at 2% NaCl (Eaglesham et al., 1987). The results, however, are in agreement with that of Mpepereki etal. (1997), who found that the majority of both fast and slow growing indigenous cowpea rhizobia from Zimbabwe tolerated 5.5% NaCl. None of the soils used in this study was sampled from a saline environment. The observed NaCl tolerance of the isolates could therefore be an indication of intrinsic resistance to high osmotic stress. Fast growing rhizobia are reported to be able to utilize as sole carbon sources certain carbon compounds (Elkan, 1992). However, no nutritional diversity differences were found between the fast and slow growing isolates in this study. The legume Rhizobium symbiosis exhibits widely differing degrees of specificity. In some instances, the symbiosis is highly specific in that a particular Rhizobium species can form a symbiotic association with only one particular legume species. This was evident in the results University of Ghana http://ugspace.ug.edu.gh 128 obtained. Fourteen percent of the isolates failed to nodulate any of the other eight host legumes except their homologous host, cowpea. Isolate 8 was also the only isolate among the 100 isolates that nodulated Mimosa spp. The feet that Mimosa was nodulated by only one out of the 100 isolates is an indication that Mimosa spp probably associates with a highly specific sub­ group of the indigenous rhizobial population. There are also intermediate cases where varying degrees of cross inoculation capabilities are exhibited as demonstrated in the results obtained. At the opposite extreme are those with broad host range, in which a diversity of legumes may be infected by one or more of several rhizobia. In this study however, none of the isolates could nodulate all the tested legumes. The high cross inoculation affinity of soybean with cowpea isolates, suggested by Norris (1965) and Christian et al. (1997), was confirmed with the present results This is in contrast with the results obtained by Bromfield and Roughley (1980), and Eaglesham (1985). Also 75% of the cowpea isolates tested nodulated groundnut. This observation is at variance with the results of Habish and Khari (1968), and Doku (1969), who reported that groundnut was not nodulated by isolates from cowpea nodules. Five out of the eight legume hosts Mimosa, Leucaena, Crotalaria, Pueraria, Calopogonium tested were on the other hand nodulated by less than 40% of the cowpea bradyrhizobia isolates tested. This results disagrees with the assumption made by Dobereiner (1978) and Halliday (1985), that tropical legumes are non-selective in the rhizobial types they require for effective symbiosis and emphasised that more work is needed on diversity of rhizobia isolates from tropical soils. University of Ghana http://ugspace.ug.edu.gh 129 The indirect ELISA method was used to determine the serogrouping of the isolates. The initial step in the determination involved testing for ability of the isolates to induce antibody production in mice. Results obtained showed that immunization of mice with rhizobial antigens induced species specific antibodies. The results of the serological work indicated that only a small fraction (8.5%) of isolates tested reacted strongly with antisera of each other and therefore closely related. These isolates could be classified as belonging to the same serogroup. The remaining 91.5% showed differential relatedness or did not share detectable antigenic determinants. The feet that only 8.5% of the isolates were closely related suggests that the isolates are highly diverse. Early reports by Jordan (1982), indicated that the micro symbionts of cowpea were typically slow growing bacteria with the characteristics of Bradyrhizobium species. Later studies however, have suggested fast growers as typical symbionts Nevertheless a high predominance at Bradyrhizobium species has generally been observed. The previous descriptions were based solely on growth features and the cross inoculation concept and thus did not provide precise information about the real nature and structure on the rhizobial population. Considering the broad range of specificities either of rhizobial species towards their host or of the legume species towards their symbionts, molecular identification has become a prerequisite to any study of rhizobial population structure (Lafay and Burdon, 1998). In this study the 16S rRNA gene of the isolates were analysed in order to characterise the isolates. Four tetrameric restriction enzymes (Dde/, Hae/77, Msp/ and Rsa/) were used to determine similarities among the isolates. The choice of the enzymes was based on the results of Laguerre et al. (1994) and a recent computer based study (Moyer et al., 1996), which evaluated the efficacy of selected tetrameric University of Ghana http://ugspace.ug.edu.gh 130 restriction enzymes for rDNA-RFLP analysis of rhizobial isolates. Results obtained using dendrogrames constructed from the similarity matrix of the isolates by the method of Nei and Li (1979) indicated that diversity of 100 isolates was high, suggesting the presence of several yet unidentified rhizobial strains in our soils. The high genotypic diversity revealed by the PCR-RFLP analysis in this study was in good agreement with the great diversity based on serotyping and host range analyses. However, isolates that had identical rDNA genotypes did not display similar phenotypic characteristics. This illustrates lack of correlation between phenotypic and genotypic based methods for grouping Bradyrhizobium strains Similar observations were made by So et al. (1994) and van Rossaum et al. (1995) Several reasons may account for this observation. For instance, it was observed in this study that the patterns of serological response to isolates used as immunogens was not always reciprocal Also the indirect ELISA techniques used in this study is dependent on interaction between an antibody and an antigen, which is attached to a solid phase (microtitre plate) by passive adsorption. Results may therefore be biased depending on the type of microtitre used. During a trial in this study, it was found that ELISA plates type Dynatech and sumilon C, gave different optical density results. Furthermore, grouping of isolates is based on a range of absorbance values, which may fail to detect widespread differences among the isolates. The cross inoculation grouping method may also be influenced by the number of test legumes used and also, different rhizobial cell number may be required to initiate nodules on different legumes. Age of cell cultures and different batches of PCR reagents were also observed in this study to affect the reproducibility of PCR results. Further studies may therefore University of Ghana http://ugspace.ug.edu.gh 131 be necessary to assess the use or otherwise of these methods for grouping bradyrhizobial isolates. 5.3 Symbiotic effectiveness Symbiotic effectiveness of indigenous rhizobial population is an important parameter for the selection of strains for inoculant production. It is also a primary factor for the determination of incidence and magnitude of legume response to inoculation (Singleton and Tavares, 1986, Thies et al., 1991). Twenty six percent (26%) of the isolates tested showed high nitrogen fixing capabilities, which were comparable to plants fertilized with nitrogen. This implies the presence of potential indigenous rhizobia that can be used for inoculant production. The pattern of effectiveness of the isolates followed a normal poisson distribution, with most (68%) of the isolates being moderately effective, few of the high (26%) or low (6%) effectiveness. This is in agreement with the results of Thies et al., (1991), who showed that the effectiveness patterns of indigenous rhizobia on cowpea followed a normal distribution curve. The results indicate however, that cowpea in the various soils used in the study are nodulated primarily by rhizobia that exhibited suboptimal symbiotic effectiveness. The wide range of effectiveness obtained and the feet that some of the isolates showed superiority in symbiotic effectiveness relative to the standard strain TAL 169 give the indication that a potential exits for developing legume inoculants from indigenous cowpeaBradyrhizobium strains. Obviously much more information about the indigenous rhizobial population is needed. Nevertheless, these native isolates, highly adapted to the environmental conditions ofthese soils may be a useful source of strains to resolve practical problems in field inoculation of cowpea University of Ghana http://ugspace.ug.edu.gh 132 The results are in agreement with those obtained in other countries (Chatel and Parker, 1973, Chatel et a l, 1973). Fredericks et al (1990), working with isolates from native Ethiopia clover species found significant differences among the rhizobial strains, which as a group, showed higher rates of nitrogen fixation than commercial Nitragin inoculant. The results also showed that the soil type did not influence the distribution of isolates in terms of effectiveness in nodule formation. Rather the results pointed to the heterogeneity of the native clover riiizobia isolates in symbiotic effectiveness. The feet that the fast growing isolates were as effective in fixing nitrogen as the slow growers on cowpea indicates that the fest growing isolates are not a relic association. S.4 Competition for nodule occupancy The abilities of some rhizobial strains to occupy greater percentages of nodules on the legume than other competing strains has been well established (Dowling and Broughton, 1986; Triplett and Sadowsky, 1992). What is most uncertain is sometimes, the reliability and ease of the methods used. The results of the present competition studies suggest that the P-Glucuronidase (GUS) marking technique is a potential tool for ecological studies of rhizobia, as it was able to distinguish between differences in the competitive abilities of different cowpea bradyrhizobia isolates. Interstrain variation in nodulating ability was demonstrated by high differences in the percentage of nodule attributable to the different competing isolates that were used in this study. Although the genetic make up o f & Rhizobium strain has been found to influence its competitive success (Danso and Owiredu, 1988), nodulation success by many strains on the other hand is University of Ghana http://ugspace.ug.edu.gh 133 influenced by the relative number of the strains in the mixture of strains (Amarger and Lobreau, 1982; Owiredu and Danso, 1989; Beattie et al., 1989; George and Robert, 1992). In this case, relative nodule occupancy can change if the ratio of strain is changed (Furhmann and Wollum, 1989; George and Robert, 1992) When the ratios of the competing isolates were changed in the inoculum mixture in this study, we observed that the percentages of nodules formed by the individual isolates also changed by about 50% in most cases, especially the ineffective isolates. The depression of plant yield associated with isolate 28 is interesting but not strange. Similar results have been reported in a study in Uruguay by Labandera and Vincent (1975). Such traits are by themselves sometimes useful indicators of success of competing strains of contrasting effectiveness and could provide insight into how and when some ineffective strains might exert a negative effect on legume growth in the field, including the influence of an effective inoculant strain. Other studies on clover plants by Demezas and Broughton (1986), showed that subclover plants grew poorly when less than half of the nodules were occupied by the suboptimally effective strain, WS2-01, and with more than 50% of the nodules on the same plants occupied by strain WS1-01 which exhibited superior effectiveness. There have also been reports of strains on B. japonicum (Teaney and Furhrmann, 1992) and R. tropici (O’Connel et al., 1990) that exert negative effects on legume growth What accounted for the plant growth suppressive effect of isolate 28 and perhaps of similar strains in other studies need further studies. In the case of a strain of R leguminosarum bv trifolii, Triplett and Bartha (1987) reported that this strain inhibited the growth and nodulating ability of other strains of the same biovar it competed with. If this should similarly apply in this study, then this would possibly explain why the detrimental influence of isolate 28 diminished when its inoculation was delayed relative to the effective University of Ghana http://ugspace.ug.edu.gh 134 isolates. The stimulation of plant growth by isolate, 94 is not only a contrast to that of isolate 28, but makes studies on microbial (rhizobial) interaction more interesting and more difficult Similar symbiotic and plant growth enhancement by ineffective rhizobial strain has been reported (Wardisirisuk et al., 1989), but no detailed and convincing evidence as to the underlying reason were given. Whether these beneficial changes have genetic basis, for example, the transfer of plasmids between such rhizobia in the rhizosphere (Broughton et al., 1987, Sullivan et al., 1995) would need to be investigated. On a practical although on a somewhat cautious note, the results obtained would suggest that it should be possible to formulate more effective inoculants containing selected ineffective and effective strains. Cautious, because unless well documented in several test and the potential consequence have been well established, the presence and persistence of an otherwise symbiotically incapable bradyrhizobial strain could in the long run, prove disastrous. Nevertheless, the science behind this is worth pursuing. Many reasons could account for a strain being highly competitive. One that has been shown in this study and discussed above is, the relative abundance of the different strains in soil or at the nodulation site. Another one that has been examined is the speed of infection. However, the result of the time course studies even though revealed differences in nodulation with regards to speed of formation of the first nodule, these were not related to competitiveness in nodule fomation For instance, although isolate 72 was the slowest in forming the first nodule, it was more competitive than all the isolates except 28. University of Ghana http://ugspace.ug.edu.gh 135 S.S Inoculation response of cowpea Maximum nitrogen fixation in a legume requires that the legume be adequately nodulated. Where nodulation is poor and scanty, rhizobial inoculation is necessary to ensure optimum nitrogen fixation. Results obtained in the inoculation experiment in soils in pots showed that a good percentage of the nodules formed on cowpea plants were attributed to the inoculated isolates. None of the inoculated isolates showed a consistent trend in its influence on nodulation, shoot dry matter yield or percent nitrogen of the plants. This suggests that soil characteristics may have influenced the performance of the isolates. The dominance of the mixed inoculant in attaining the highest number of nodules, the highest shoot dry matter yield and the highest total nitrogen content of the inoculated plants, may be explained by the synergistic effect of the three isolates that make up the mixed inocula, which together were probably more competitive and effective than when they were inoculated as single isolates. The advantage of using mixed inocula over single inocula had earlier been indicated by Owiredu and Danso (1988), and its outcome may be due to the interaction among them. Values obtained for nodule numbers, shoot dry weight and percent nitrogen in Ankasa soil were consistently lower than those obtained in the other three soils. The persistence of the difference suggests that some property of Ankasa soil had a depressing effect on nodulation and nitrogen fixation of the isolates The observation has been made that the number of nodules formed on plants and the effectiveness of Rhizobium strain is affected by soil pH (Keyser et a l, 1979). It has also been proved that not only infection of the host plant is adversely affected by acidity (Lie, 1971), but also the functioning of the established nodule can be inhibited (Munns, 1970). These facts may explain why lower values of nodule numbers, shoot dry weight and percent University of Ghana http://ugspace.ug.edu.gh 136 nitrogen were obtained in Ankasa soil, which has a pH of 4 0. The results outlined in Figs. 4.24 -4.26 and Tables 4.13 and 4.14 showed that there was no significant difference between the uninoculated control and the inoculate treatments in Akuse and Bekwai soils. This may suggest that the native bradyrhizobia isolates in these soils may be efficient in fixing nitrogen or that the native strains outcompeted the inoculated strains in nodule formation. This as well as any underlying factors need to be studied. The method of inoculation has been shown to influence nodulation parameters of legumes (Kamicker and Brill, 1987; Danso and Owiredu, 1988; Hardarson et al., 1989). Results obtained in this study indicate that soil was superior to seed inoculation. This might probably be due to the uniform distribution of rhizobial cells in the whole soil in the case of the soil inoculation method. Consequently, the chance of a growing root and a rhizobial cell getting into contact was increased as opposed to when the inoculation was localised on the seed. Similar observations were made on soybean by Danso and Bowen (1989), Hardason et al. (1989) and Danso et al. (1990). Practical ways of inoculating rhizobia directly into the bulk of the soil may therefore enhance nodulation response and nitrogen fixation, especially where native strains abound. Several reports have shown that because cowpea Bradyrhizobium strains abound in most soils of the major cowpea growing areas (Doku, 1969; Sellschop, 1962), cowpea rarely responds to bradyrhizobial inoculation (Doku, 1969; Rhodes and Nangju, 1979). Even though substantial quantities of nodules were observed on the uninoculated plants, which gives a good reflection of the number of indigenous cowpea bradyrhizobia present in the different soils, the results University of Ghana http://ugspace.ug.edu.gh 137 obtained in Lima and Ankasa soils contrast the postulate of no response to inoculation in tropical soils. Rather the results indicated that response to inoculation in the presence of native rhizobia can occur in some soils if not all. The type of inoculant strain used, the variety of cowpea used and the type of soil may all influence the results that may be obtained. University of Ghana http://ugspace.ug.edu.gh 138 CHAPTER SIX 6.0 CONCLUSION The primary aim of this work was to study the characteristics of the indigenous bradyrhizobia population in relation to cowpea, in order to enhance its nitrogen fixation and yield. A preliminary study was done to assess the potential to improve nitrogen fixation by cowpea. Following this, 100 indigenous bradyrhizobia isolates were obtained from nodules of cowpea grown in 20 different soils sampled from five major ecological zones in Ghana. The diversity of the isolates was examined using phenotypic, serological and molecular characters To understand the potential to improve nitrogen fixation of a legume, the ranges of effectiveness of the indigenous rhizobia is also required. Consequently the symbiotic effectiveness of the 100 isolates was determined. Low symbiotic nitrogen fixation is in many cases a result of the superior competitive abilities of ineffective over effective rhizobia for nodule occupancy (May and Bohlool, 1983). Understanding the interaction between the effective and ineffective rhizobial isolates so as to bias competition in favour of the more effective ones is therefore essential for improving symbiotic efficiency Competitive abilities of selected isolates of known effectiveness were therefore assessed. Exotic rhizobia inoculants have been considered as aliens and less successful (Brockwell, 1981). The strains most competitive in nodule formation and persistent in a particular environment are often those isolated from similar environments (Chatel and Parker, 1973). This hypothesis was also tested using selected isolates in selected soils. University of Ghana http://ugspace.ug.edu.gh 139 Results of the natural nodulation of cowpea indicated that large variations in nodulation exist in cowpea. Such variations are not uncommon in legume species, but have practical implications. In this study, for instance, cowpea cultivar Asontem was found to be the most prolific nodulator which makes it a favourable candidate for breeding purposes. The feet that none of the 20 soils used could support nodulation of all the 45 cowpea cultivars suggests the paucity of the soil resident rhizobia that were compatible with all cowpea cultivars. The nodulation in each soil therefore alludes to the nature of the rhizobial composition in affinities of the cultivars that the soil supports. The pattern of nodulation reflected the varying sizes of indigenous bradyrhizobial populations in the different soils. Inadequate infectivity or efficacy of the indigenous bradyrhizobia isolates was demonstrated by the positive response of cultivars to nitrogen fertilization which together with the variability in nodulation and population sizes of indigenous bradyrhizobia indicate a potential for improvement by use of bradyrhizobial inoculants. Although cowpea is generally thought to symbiose with slow-growing rhizobia, it was clear from the investigations of the phenotypic characteristics of the isolates that both fast and slow growing rhizobia were represented in nodules of cowpea. This provides evidence to dismiss the myth that all cowpea rhizobia are slow growing. The results also provide a contribution towards dismissing the view about the exaggerated nodulation promiscuity of tropical rhizobia. It is possible that subgroups of rhizobia exist based on symbiotic specificity. The diversity of the isolates revealed by the phenotypic characteristics was further supported by results of PCR analysis that target specific chromosomal loci of the 16S rRNA gene. Twenty distinct genotypic groups were found according to the RFLP analysis of the 16S rRNA gene. Thus one intriguing observation made in this thesis is that irrespective of the method employed to analyse for University of Ghana http://ugspace.ug.edu.gh 140 diversity the results showed great diversity among the indigenous bradyrhizobia isolates, even though there was lack of correlation among the methods. This points to the possible existence of several unique and as yet unidentified species of Bradyrhizobium strains in our soils. The presence of large diversity of native bradyrhizobial strains may suggest the need for diverse bradyrhizobia strains as inoculants for successful inoculation of cowpea. Again, the presence of large diversity of bradyrhizobial strains may contribute to the negative response of cowpea to inoculation observed in some soils in the tropics Even though the soils studied with the exception of those from the high rainforest region contain sufficient numbers of indigenous rhizobia that could nodulate and efficiently fix nitrogen in cowpea, determination of the symbiotic effectiveness of the isolates showed that the majority (68%) were moderately effective. Interestingly, the proportion contained both fast- and slow-growing isolates which were somewhat evenly distributed in all the soils. The utilization of the GUS A marker technique to assess competitiveness between effective and ineffective isolates for nodule occupancy resulted in a high differentiation of isolates as all the nodules on each plant were analysed for nodule occupancy. The results showed that the insertion of the GUS A marker gene did not affect the nodulation behaviour of the marked isolates making the technique highly suitable for the study of rhizobial competition. Competitive ability of rhizobia strains as observed in this study was not linked to effectiveness of isolates. An interesting feature about interaction between effective and ineffective isolates as revealed in this study was that such an interaction could lead to either depressed or enhanced University of Ghana http://ugspace.ug.edu.gh 141 plant yield which suggests the possibility of formulating mixed inoculants containing such selective strain combinations. Response of cowpea to inoculation has been a controversial issue for some time now. Lack of a positive response is attributed to the presence of large population of indigenous rhizobia. However, other causes such as (1) the unnecessary application of inoculants on cultivars capable of nodulating effectively with indigenous strains, (2) the use of wrong strains in the inoculants, (3) diversity of the indigenous rhizobia and (4) an unknown factor limiting full expression of symbiosis may be responsible. The present study has shown that cowpea responds positively to inoculation in some soils. Further work will allow identification of cultivars requiring inoculation or not in certain areas and to evaluate the importance of the environment on the performance of particular cultivar/rhizobia strain combinations. Meanwhile, as revealed in this study, the use of selected indigenous rhizobia isolates for the preparation of inoculants may be considered a better option. It is clear, from the foregoing discussion, that to ensure that N2 fixation by cowpea is enhanced, several factors have to be considered. These may include the selection of efficient Nr-fixing cultivars and a comprehensive knowledge of the diversity of the indigenous rhizobia particularly their effectiveness and competitiveness. Inoculant strains have often been recommended based on good symbiotic performance in a particular environment, while the soil status or the agro- ecological zone of the final application has not been considered. The soils of the tropics contain a vast genetic pool of indigenous rhizobia which have not yet been identified. Such strains and species can provide a variety of inoculant strains that may show better performance. Correlation University of Ghana http://ugspace.ug.edu.gh 142 between the size of the indigenous rhizobia population and performance of inoculant strain has been established (Thies et a l, 1991) However, the effect of the diversity of indigenous rhizobia on the performance of an inoculant strain has not been determined. 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Intraspecific variability for nitrogen fixation in southern pea. Journal of American Society of Horticultural Science. 103: 806-808. Zdor, R E., and Pueppke, S. G., 1990. Competition for nodulation of soybean by Bradyrhizobium japonicum 123 and 138 in soil containing indigenous rhizobia. Soil Biology and Biochemistry 22: 607-613. Zewdu, T , Nick, G , Suomalainen, S., Paulin, L., and Lindstrom, K , 1998 Phylogeny of Rhizobium galegae with respect to other rhizobia and agrobacteria. International Journal of Systematic Bacteriology 48: 349-356 University of Ghana http://ugspace.ug.edu.gh 193 a p p e n d i x Composition and preparation of media used 1. Yeast Extract mannitol agar (for the isolation of rhizobia) Mannitol 1 0 0 g Dipotassium phosphate 0.5 g Magnesium sulphate 0.2 g Sodium chloride 0.1 g Yeast extract 0.5 g Agar 15g Distilled water 1000 ml. 2. PBS. (Phosphate Buffered Saline) Nacl 8.0 g Na2HP0 412H20 2.7g Na.H2PC>4 0.4g Distilled water 1000ml pH 7.2-7.4 3 Running buffer (10 x TBE) Tris base I08g Boric acid 55g 0 5MEDTA 40ml (pH 8.0) 4. Loading Buffer Bromophenol blue 0.25% Sucrose (w/v) 4 0% University of Ghana http://ugspace.ug.edu.gh 194 5. LB Medium Bacto-tiyptone 1 Og Yeast extract ^g Sodium chloride 5g 6 N-free Nutrient solution Stock solution Form 1 CaCl2.2H20 294.1 g/1 2 KH2PO4 135.1 g/1 3 Fe-citrate 6.7g/l MgS047H20 123 3 g/1 K2SO4 87.0g/l MnS04H20 0.33 8g/l 4 H3BO3 0.247g/l ZnS047H20 0.288g/l CuS04.5H20 0.100g/l CoS04.7H20 0.056g/l Na2Mo02 2H20 0.048g/l For each 10 litres of full strength culture solution, take 5 .0ml each of solutions 1 to 4, then add to 5.0 litres of water, then dilute to 10 litres. Use IN NaOH to adjust the pH to 6.6-6.8. University of Ghana http://ugspace.ug.edu.gh