Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations
1986 Ecology of Bradyrhizobium japonicum in Iowa soybean rhizosphere soil Patrick M. Hicks Iowa State University
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Recommended Citation Hicks, Patrick M., "Ecology of Bradyrhizobium japonicum in Iowa soybean rhizosphere soil " (1986). Retrospective Theses and Dissertations. 8252. https://lib.dr.iastate.edu/rtd/8252
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8703711
Hicks, Patrick M.
ECOLOGY OF BRADYRHIZOBIUM JAPONICUM IN IOWA SOYBEAN RHIZOSPHERE SOIL
Iowa State University PH.D. 1986
University Microfilms In t6rn &ti 0 nâl 300 N. zeeb Road, Ann Arbor, Ml 48106
Ecology of Bradyrhizobium japonicum
in Iowa soybean rhizosphere soil
by
Patrick M. Hicks
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of the
Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Department: Agronomy Major: Soil Microbiology and Biochemistry
Approved:
Signature was redacted for privacy.
In Charge of Major «Wrk
Signature was redacted for privacy.
F Signature was redacted for privacy. Iowa State University Ames, Iowa 1986 il TABLE OF CONTENTS Page GENERAL INTRODUCTION 1 LITERATURE REVIEW 3 Inoculation and Establishment of Nonindigenous Strains of Bradyrhizobium .japonicum in Soil 3 Ecology of the Rhizosphere and Bradyrhizobium .japonicum Interactions 4 Mycorrhizae-Rhizobium-legume tripartite symbiosis 4 Rhizobium survival in soil 7 Correlation of Pseudomonas spp. with B. .japonicum serogroup dominance and soil reaction 14 Fast-growing soybean nodulating Rhizobium 16 SECTION I. EFFECTS OF P FERTILIZATION AND VA MYCORRHIZAE ON MODULATION OF SOYBEAN 19 ABSTRACT 20 INTRODUCTION 22 MATERIAL AND METHODS 24 P Fertilization 24 VAM Inoculation 26 RESULTS AND DISCUSSION 28 P Fertilization 28 VAM Inoculation 32 LITERATURE CITED 35 SECTION II. SEASONAL FLUCTUATIONS OF SOYBEAN RHIZO SPHERE MICROFLORA AND INFLUENCE ON BRADYRHIZOBIUM JAPONICUM 38 ABSTRACT 39 INTRODUCTION 40 MATERIALS AND METHODS 42 Rhizosphere Populations 42 Greenhouse Screening 45 iii Page RESULTS AND DISCUSSION 49 Rhizosphere Populations 49 Greenhouse Screening 52 Sterile soil 52 Nonsterile soil 53 CONCLUSIONS 56 LITERATURE CITED 57 SECTION III. BIOLOGICAL FACTOR INFLUENCING BRADY- RHIZOBIAL NODULE OCCUPANCY IN A HIGH pH SOIL 59 ABSTRACT 60 INTRODUCTION 61 MATERIALS AND METHODS 63 RESULTS AND DISCUSSION 66 SUMMARY 69 LITERATURE CITED 70 SUMMARY AND CONCLUSIONS 71 LITERATURE CITED 74 ACKNOWLEDGMENTS 80 APPENDIX A. EXPERIMENTAL DESIGN OF FAST-GROWING RHIZOBIUM NODULATION STUDY 81 APPENDIX B. RECOVERY OF FAST-GROWING RHIZOBIUM FROM SOYBEAN NODULES 82 APPENDIX C. EXPERIMENTAL DESIGN OF FAST-GROWING RHIZOBIUM N FIXATION STUDY 87 APPENDIX D. DINITROGEN FIXATION IN FAST-GROWING RHIZOBIUM ISOLATES AND BRADYRHIZOBIUM JAPONICUM USDA 110 88 1 GENERAL INTRODUCTION Soybeans are predominantly nodulated by Bradyrhizobium japonicum serogroup 123 in Midwestern noncalcareous soils and by 135 in calcareous soils (Damirgi et al., 1967; Ham et al., 1971b; Kapusta and Rowenhorst, 1973; Weaver and Frederick, 1974a, 1974b). The factors responsible for this phenomenon have remained undiscovered to date. If soil factors influence nodule occupancy, they could be physical, chemical, or bio logical. These studies sought to investigate possible microbiological inter actions (biological factors) responsible for the dominance of serogroups 123 and 135. The first section, entitled "Effects of P fertilization and VA mycorrhizae on nodulation of soybean," examined the influence of vesicular-arbuscular mycorrhiza and P fertilization on B. .japonicum 123 nodule occupancy in both field and greenhouse soils. This section con tains an Introduction, Materials and Methods, Results and Discussion, and Literature Cited subsections. Tables also are included in the text. This manuscript will be submitted to the Soil Science Society of America Journal for publication. The second section, entitled "Seasonal fluctuations of soybean rhizosphere microflora and influence on Bradyrhizobium .japonicum," was both a field survey of soybean rhizosphere microflora and two struc tured experiments that evaluated if bacterial isolates capable of stimulating B. japonicum 123 growth on artificial media could influence nodule occupancy in both sterile and nonsterile soil. This section has the same subsection format as Section 1 and also contains a Summary and 2 tables in the text. This manuscript also will be submitted to the Soil Science Society of America Journal for publication. The third section, entitled "Biological factor influencing brady- rhizobial nodule occupancy in a high pH soil," was a preliminary study investigating fluorescent pseudomonad populations and B. .japonicum serogroup 135 dominance in calcareous soils. This section has the same format as Sections 1 and 2, and also contains a Summary subsection. Tables also are included in the text. Future submission of this manu script for publication has not been determined at time of printing. These sections are followed by an overall summary and discussion of results. Additional literature used in the General Introduction, Literature Review, and the Summary and Conclusions are cited last. Appendix A is an outline of the experimental design used to test fast-growing rhizobial strain nodulation. Appendix B lists the raw data collected in this study. Appendix C is an outline of the experi mental design used to test fast-growing rhizobial strain fixation. Appendix D lists the raw data collected in this study. These data will be published as part of a manuscript submitted by the Department of Genetics, Iowa State University. 3 LITERATURE REVIEW Inoculation and Establishment of Nonindigenous Strains of Bradyrhizobium japonicum in Soil Most soybean growers in Iowa do" not inoculate seed prior to plant ing because it is questionable whether such practices actually do produce greater yields where indigenous populations of B. .japonicum are present (Elkins et al., 1976). In other areas of the U.S. where soybeans are grown, inoculation of seed is a common practice because of the relatively low cost of commercial inoculants and the potential for yield increases in these areas after inoculation. Inoculation is sometimes referred to as "cheap insurance" (Lynch and Sears, 1952) due to potential yield in creases balanced against relatively low costs of application. Ham et al. (1971a) reported that inoculating soybean seed with B. japonicum at planting in Iowa does not significantly increase yield. Seed yield is not increased because introduced rhizobia do not compete well with naturalized B. .japonicum serogroup 123. Johnson et al. (1965) demonstrated that only 5% of nodules were colonized by inoculant rhizobia on soybeans grown in soil containing indigenous rhizobial populations. The remaining nodules were produced by rhizobia established in soil prior to planting. Recoveries of inoculant rhizobia ranging from 0 to 17% were reported by Ham et al. (1971a). Massive inoculation may result in greater success in colonization of soybeans by introduced rhizobia. As much as 800 to 1000 times the usual inoculum rate may be required to achieve 50% of nodules produced from inoculant rhizobia. Boonkerd et al. (1978) obtained increased recoveries 4 of two strains of B. .japonicum by using inoculations of 10 to 100 times normal rate. Dunigan et al. (1984) determined that 3 years of massive soil inoculation with a nonindigenous strain of B. .japonicum resulted in the permanent establishment of the strain and eventually resulted in increased competitive ability with native rhizobia. Kapusta and Rowenhorst (1973) failed to produce significant increases in seed yield with massive inoculations but did improve recoveries of two B. japonicum strains with inoculum rates of 1.2 x 10^^ cells cm ^ of row. It has been shown in both greenhouse and field studies that commer cial inoculum does not supply rhizobia in numbers sufficient to provide extensive and competitive nodulation in soils having high indigenous populations (Weaver and Frederick, 1974a, 1974b; Nelson et al., 1978; Hiltbolt et al., 1980). Ecology of the Rhizosphere and Bradyrhizobium .japonicum Interactions Mycorrhizae-Rhizobium-legume tripartite symbiosis Nodulation and N^ fixation Some soil fungi can initiate a highly specialized association with soybean roots in which infection does not adversely affect the proper functioning of roots. This root-fungus re lationship produces tissue that is comprised of both plant and fungal cells called mycorrhiza. Nonseptate, endotrophic-established mycorrhiza is termed vesicular-arbuscular mycorrhiza (VAM) and is the type most often found in association with soybeans (Barea and Azcon-Aguilar, 1983). 5 Effectivity as defined by Mosse (1974) pertains to the degree of nutritional or other advantage resulting from the symbiotic association between a particular autotroph and heterotroph. The influence of VAM on the growth response and yield potential of soybeans has been investi gated from their effect on nutrient uptake and possible influence on fixation. Effectivity in this case depends on a three-way symbiosis between soybeans, B. .japonicum, and VAM. The effect of VAM development on N concentrations in soybean plants was thought to be indicative of either 1) a positive relationship between nodular B. .japonicum and VAM, or 2) increased efficiency in N uptake of mycorrhizal roots. Schneck and Hinson (1973) found that inoculation with VAM significantly increased seed yield of nodulating but not non- nodulating isolines of soybeans. It was assumed from these findings that VAM did not increase N absorption from the soil in nonnodulating soybean plants, and it was also assumed that the same holds true for nodulating plants. Carling et al. (1978) examined the effects of VAM on the ability of soybean plants to utilize N from both biologically fixed and soil- available sources. In this experiment, they utilized nodulating and nonnodulating soybean isolines as well as sterile soil and P fertiliza tion treatments. Fertilization with P was done to counter any effect on P nutrition in non-VAM plants. It was noted that the Ng-fixing capacity of the nodulating plants increased in response to either P fertilization or VAM infection. Both nodule dry weight and acetylene reduction activ ity increased in these treatments. They concluded that VAM has no direct 6 effect on B. .japonicum but does Increase the efficiency of fixation by increasing host P nutrition and subsequently that of nodular B. .japonicum. No significant increase in N nutrition from soil as measured by leaf nitrate reductase activity Was reported. Vesicular-arbuscular mycorrhizae and B. japonicum interactions in field conditions were studied by Bagyaraj et al. (1979). Nodule number, nodule dry weight, and nodule N content were significantly greater in soybeans treated with VAM + B. .japonicum inoculant than with B. .japonicum alone. The N content of plants treated with VAM and B. .japonicum was over twice that of those receiving a monoxenic inoculum. The improvement in nodulation and plant nutrition with VAM inocula tion was related to increased P supply to the host. This experiment was conducted on soil low in available P. In many soils currently under soybean cultivation, the fertilization plan established for the annual or biannual rotation of soybeans with others crops results in an increased availability of P. This could result in less dramatic increases in soy bean growth with VAM-Rhizobium inoculants. The effect of soil fertility and VAM on soybeans was studied by Carling and Brown (1980), who evaluated 19 VAM isolates in greenhouse culture of soybeans. The relative effectivity of most isolates was not greatly influenced by P availability. Inoculation with VAM resulted in significant increases in plant growth and yield in both low and high fertility soils. Competition for nodule occupancy VAM stimulates nodulation and Ng fixation (Crush, 1974; Ganry et al., 1982), mainly through increased 7 P nutrition, but other nonnutritional interactions have also been sug gested (Ross and Harper, 1970). Extracellular polysaccharides produced by Rhizobium can increase root cell permeability, enhancing VAM formation. This takes place in the Medicago sativa-Rhizobium meliloti-VAM symbiosis (Barea and Âzc6n-Aguilar, 1983). Plant-growth-regulating substances produced by Rhizobium also increase VAM infection. The hormonal inter actions in the legume rhizospheres are complicated because VAM produce plant-like hormones, and these hormones may play a role in the infec tion mechanism of soybean roots by B. japonicum. No experiments have been reported to date investigating the effects of VAM on competition for nodule occupancy by strains of B. japonicum. Rhizobium survival in soil Persistence and stimulation in the rhizosphere B. .japonicum survive as free-living bacteria in soil prior to invasion of plant roots and therefore must be able to compete with a variety of microorganisms throughout much of the year. Rhizobia can survive quite well in the absence of soybeans for extended periods of time. Norman (1942) observed soybean nodulation in fields that had not supported soybean crops for over 24 years. Weaver et al. (1972) determined most probable number counts of B, .japonicum in 52 Iowa fields. At sampling, 31 fields were planted in corn, one field was in alfalfa, one field was in oats, and 19 fields were in soybeans. Variation in B. .japonicum populations was less within a field than between different fields. Variation among soil series within single fields was not significant. Only one field 8 contained a very large population of B. .japonicum, and this field had been planted to inoculated soybeans 2 years prior to sampling. The fields contained from fewer than 10 to more than 10^ B. .japonicum g ^ soil. The numbers of rhizobia were significantly correlated to the pres ence of soybeans within a cropping history of 12 years before sampling, but not correlated to the presence of soybeans at sampling. Elkins et al. (1976) conducted greenhouse studies to test the effect of soybean crop ping histories between 0 to 11 years previous to sampling on soybean nodulation and B. .japonicum populations. No aspect of soybean cropping history was significantly correlated with the Rhizobium populations. Although rhizobia survive quite well in the absence of soybeans, a marked rhizosphere effect is often noted (Caldwell and Vest, 1968). The stimulation of Rhizobium in the legume host, nonhost legume, and nonlegume rhizosphere has been discussed by Lowendorf (1980). Peters and Alexander (1966) showed that the population stimulation varies with plant variety, soil type, and even B. japonicum strain. Growing soybeans in the field establishes a substantial population of soybean rhizobia (Tuzimura and Watanabe, 1961), and the release of rhizobia from plant nodules after the growing season increases numbers of soil rhizobia (Tuzimura and Watanabe, 1961). These populations over winter and are restimulated in successive crop rhizospheres. Competition in the rhizosphere Interactions between organisms in the rhizosphere may influence the establishment of a dominant rhizo- bial strain. It is difficult to establish and maintain an introduced strain in soil containing an indigenous rhizobial population. Various 9 strains differ in their ability to establish themselves in competition with rhizobia already present in the soil (Jenkins et al., 1954; Baird, 1955; Abel and Erdman, 1964). Strains also differ in competitiveness for nodule establishment on legume foots (Means et al., 1961; Johnson et al., 1965; Caldwell, 1969; Skrdleta and Karimoua, 1969), and competi tion for available infection sites on roots is independent of the rela tive numbers of each strain to which the root is exposed (Bhaduri, 1951; Purchase and Nutman, 1957). The more competitive strains have a definite advantage in this respect. Increasing the size of inoculant of less competitive strains will not completely overcome the dominance of the more competitive strains. Natural selection in the rhizosphere will have a profound effect on establishment and competition of strains. The combination of toler ances to adverse conditions and favorable physiological characteristics will allow a population to dominate. Other strains without these toler ances and favorable characteristics will not compete with the established strain (Vincent, 1974). Natural selection is a major factor affecting the competition that exists among B. japonicum strains in soil. Saprophytic competition in the rhizosphere occurs among rhizobia and other genera. The level of repression exerted on Rhizobium species by other organisms varies. Hatting and Louw (1969) found approximately 25% of the bacteria and actinomycetes isolated from the root region inhibited clover rhizobia. Dàmirgi and Johnson (1966) reported most isolates of actinomycetes from a soybean field did not inhibit any of eight B. .japonicum strains. Inhibition of rhizobia by other genera 10 appears to vary among locations and among species of inhibitors as well as Rhizobium species. Rhizosphere conditions produced by soybean plants influence the types and numbers of bacteria present and also promote the ability of the rhizobia to compete as free-living bacteria within the soil popula tion. Smith and Miller (1974) found eight of nine rhizosphere bacteria inhibited B. japonicum on agar plates. As a group, the rhizosphere organisms inhibited the B. japonicum strains to a greater extent than the nonrhizosphere organisms. A greater percentage of rhizosphere organ isms showed some inhibitory ability, and the average degree of rhizobial inhibition was more severe with rhizosphere isolates. However, when the inhibitory bacteria were introduced into vermiculite or sterile soil with B. japonicum. nodulation was not affected. One isolate that did not inhibit rhizobia on agar plates damaged the taproots of soybean plants but did not reduce nodulation. It appeared that changes in the rhizo sphere induced by the plant influenced the composition and size of bac terial populations and the ability of rhizobia to compete and persist as free-living bacteria. The ability of bacteria to inhibit rhizobia on culture media and not under "natural" conditions could also be due to the artificial growing conditions used in the laboratory. Root excretions exert a greater influence on rhizobia than on other rhizosphere organisms (Elkan, 1962). The suppression of nodulation with a nonnodulating soybean was due to the effect of root exudates in the rhizosphere. Other organisms in the soil appeared to have less affect 11 than root exudates on rhizobia in the rhizosphere. Root excretions may be the primary component of the rhizosphere effect presented by Smith and Miller (1974). It appears that potential effects of rhizobial inhibition by bacteria may not be realized in the legume-rhizobia symbiosis due to this plant-microbe interaction. These complex interactions between micro bial populations competing for dominance has led to controversy over the relative effect of each upon the legume-rhizobia symbiosis. Competition between strains of rhizobia can take place among 1) naturalized strains, 2) naturalized strains and introduced strains, and 3) introduced strains in inoculant (Ham, 1978). These three levels of contention, as well as the influence of the plant itself, have an influence on the legume-rhizobia symbiosis. Caldwell and Vest (1968) believed that legume-rhizobia interactions influenced the ability of a strain to nodulate and fix N^. Different cultivars of soybeans, in combination with various strains of natural ized B. japonicum, nodulated to varying degrees. Closely related soybean genotypes had similar distributions of rhizobia inhabiting their nodules. Differences existed among plant cultivars and their acceptance of B. .japonicum strains from a series of naturalized strains. Specific groups of related soybean cultivars nodulated to a greater extent with specific groups of B. japonicum strains. It is interesting to speculate on past plant breeding techniques and B. .japonicum serogroup 123 dominance in Iowa soils. Soybean breeders' field tests may have unwittingly selected for cultivars that preferen tially nodulated with B. .japonicum 123. It is thought that B. .japonicum 12 123 was in the inoculant when soybeans were first grown in Iowa (Dr. J. Burton, personal communication, The Nitragin Co., Milwaukee, WI). Over years of planting with soybean cultivars that "preferred" the naturalized Rhizobium, the soil microbial population may have become dominated by this serogroup, the effects of which we see today. Seasonal variation in rhizosphere Rhizobium populations as influenced by environmental conditions Physical and chemical properties of soil influence the magnitude and composition of microbial populations. Bradyrhizobium japonicum survive as free-living bacteria in soil prior to invasion of the plant root system, and therefore the soil environmental conditions will have a profound affect on them during this phase of their existence. Caldwell and Weber (1970) found the influence of environmet on the ecology of rhizobia was significant. Different soil environments contained different strains of B. japonicum. Other researchers have con firmed this and have attributed the variations to soil type and environ mental factors. Caldwell and Hartwig (1970) found the distribution of B. japonicum strains varied from location to location. They assumed variation in soil properties between locations was responsible for the different popula tions. Johnson and Means (1963) isolated different strains of B. japonicum from nodules of plants grown in different soils. They noted that the strain found in any particular soil remained dominant whether cropped under field or greenhouse conditions. They concluded that soil type influenced strain distribution to a greater extent than did environ mental factors. Damirgi et al. (1967) also believed that soil type had a 13 greater influence on B. japonicum strain distribution than did the envi ronment. They reported that soil type influenced the distribution, competitiveness, and nodulation by various strains of B. japonicum. Baird (1955) found that nodulation by different rhizobial strains would increase in certain Australian soils. Different soil types diminished the competitive nodulation characteristics of these strains. Weber and Miller (1972) reported soil temperature had a significant effect on the distribution of B. .japonicum. Some strains predominated in soybean root nodules at elevated temperatures, while lower root zone temperatures favored dominance of other strains. Roughly et al. (1980) found that competition for nodulation of the soybean cultivar 'Malayan' by B. .japonicum strains was influenced by root temperature. This cultivar was known to be quite promiscuous in the spectrum of strains acceptable for nodulation. Increased temperature heightened the pro pensity of certain strains to nodulate. It is evident that temperature affects the distribution, competitiveness, and Ng-fixation ability of B. .japonicum. Soil moisture also affects nodule formation. Desiccation will not only hamper the metabolic function of both symbionts but may also inter fere with the initiation of nodulation by hampering microbial movement. Hamdi (1971) concluded that nodulation of legumes could be restricted in dry soils by failure of seed inoculum to migrate through the soil to root hairs. He also stated that under water stress the root hairs themselves will undergo morphological changes that inhibit infection by rhizobia. 14 Different rhizobial strains tend to nodulate at different times throughout the season. The presence of available nodule sites over the growing season will influence the establishment and persistence of dif ferent strains. Caldwell and Weber (1970) differentiated between B. japonicum strains nodulating early in the growing season as opposed to strains nodulating later. Correlation of Pseudomonas spp. with B. japonicum serogroup dominance and soil reaction Ham et al. (1971b) discovered that B. japonicum serogroup 123 was dominant in soybean nodules of plants grown in Iowa soils below pH 7.5. Serogroup 135 dominated in soils with pH greater than 7.8. Serogroup 123 was predominant in most samples, usually accounting for more than 50% of the nodules. Other serogroups, except serogroup 135, were not present in more than 50% of the nodules of any sample. Previous studies by Damirgi et al. (1967) on soil pH and B. japonicum serogroup competition indicated that the pH effect was not strictly related to acidity and alkalinity. Truog (1946) had shown that the avail ability of Fe in soil decreases with increasing soil pH; Fe is of lower availability at the higher pH ranges that favor dominance of serogroup 135. Ham et al. (1967) reported differential behavior of B. japonicum strains 123 and 135 on soybeans in sand culture with Fe nutrition rang ing from 0 to 16 ppm. At lower Fe concentrations, serogroup 135 occupied 88% of nodules and serogroup 123 occupied very few. This trend reversed at the 3 and 4 ppm concentrations of Fe. At the 16 ppm concentration. 15 serogroup 123 occupied the majority of nodules. The results of this study showed that high Fe availability favors serogroup 123 dominance and low Fe availability favors serogroup 135 dominance. Schroth and Hancock (1982) described a plant-disease suppression phenomenon in many soils. Root surface colonization by bacteria in the genus Pseudomonas suppressed disease. Leisinger and Mangraff (1979) showed that fluorescent pseudomonads produce secondary metabolites with a range of antibiotic activity. These metabolites were first referred to as ferric pseudobactins and later as siderophores (high-affinity Fe(III) ion chelators). Schroth and Hancock (1982) reported that sidero phores were produced by a yellow-green fluorescent Pseudomonas isolate and used the isolate to suppress Gaeumannomyces and Fusarium infection. The suppression was also demonstrated by the addition of the siderophore alone to soil. In a related experiment, Scher and Baker (1980) suppressed the growth of Fusarium spp. with additions of either a Pseudomonas sp. or ethylenediamine di-(o-hydroxy-phenlacetate)[Fe(III)EDDHA ]. They con cluded that the iron in Fe(III)EDDHA is only available to microorganisms that produce siderophores; the siderophore-producing fluorscent pseudo monads were favored while the Fusarium spp. were suppressed. These results indicated that fluorescent pseudomonads in the rhizosphere were producing siderophores that suppressed the colonization of other micro organisms by complexing Fe. Olson and Misaghi (1982) reported that soil pH affected sidero phore production by fluorescent pseudomonads. Soil extracts with pH 16 ranges of approximately 3.0 to 8.0 were amended with filter-sterilized succinate broth inoculated with Pseudomonas fluorescens. All extracts supported bacterial growth, but sidérophore production was substantially reduced at low pH values. Siderophore production was not evident at pH ranges below 7.0 but was measured at pH 7.9. The production of sidero phore by Pseudomonas fluorescens was inhibited by increased Fe avail ability at lower pH values. Fast-growing soybean nodulating Rhizobium Members of the genus Rhizobium nodulate legumes and fix Ng in symbiosis. Speciation within the genus was initially based on cross- inoculation groups, but host specificity for all rhizobia could not be firmly established. The various species are now separated into two groups based on growth rate and pH effect when grown on yeast extract mannitol medium. Slow-growing strains, with generation times of 6 to 11 hours, do not produce acid in culture and have been transferred to the genus Bradyrhizobium. Fast-growing strains, with generation times of 2 to 4 hours, produce acid in culture and remain in the genus Rhizobium. Soybeans are usually nodulated only by slow-growing strains (Bradyrhizobium .japonicum), but recently fast-growing strains of rhizobia have been isolated from soil and root nodules of soybeans collected in the People's Republic of China. Keyser et al. (1982) first reported the isolation and identifica tion of fast-growing strains of rhizobia from soybean root nodules in east-central China. These strains were obtained by other researchers 17 and were shown to have physiological characteristics very different from B. .japoniciun (Sadowsky et al., 1983; Heron and Pueppke, 1984; Scholia et al., 1984; Stowers and Eagleshara, 1984; Appelbaum et al., 1985; Buendia- Claveria and Ruiz-Sainz, 1985). These strains did not, however, form nodules on several North American commerical soybean cultivars (Jansen van Rensburg et al., 1983; Hattori and Johnson, 1984). Dowdle and Bohlool (1985) isolated fast-growing rhizobia from two soils in the People's Republic of China. The two soils were from central China and had different cropping histories. One soil had been under con stant soybean cultivation without inoculation for several decades. The second had been under continuous rice cultivation without record of prior soybean growth. Rhizobium isolates were obtained from suspensions of soil adhering to soybean seedlings grown in each soil. The rhizosphere soil suspensions were used to inoculate soybeans planted in sterile vermiculite. Soybeans were also planted directly in the soils, and rhizobial isolates were obtained from soybeans grown by both methods. Five soybean cultivars were used to test nodulation and Ng-fixation characteristics of the fast growers. Two cultivars were improved, yellow-seeded Chinese cultivars; a third was Peking (an unimproved, black-seeded cultivar); and two were common commercial cultivars grown in North America (Davis and Williams). The fast-growing isolates were highly effective on the two culti vars from China and Peking but were less effective on Williams and Davis. The important result of the various isolate-cultivar interactions was that one fast-growing isolate was as effective as strain USDA 110 on 18 each soybean cultivar. Some fast-growing isolates that Dowdle and Bohlool (1985) tested were dominant nodulating strains in the soils tested from China, although the soils contained substantial populations of slow-growing rhizobia. 19 SECTION I. EFFECTS OF P FERTILIZATION AND VA MYCORRHIZAE ON NODULATION OF SOYBEAN 20 ABSTRACT A general synergistic relationship exists between the presence of raycorrhizae and fixation, especially on soils low in P. However, no one has reported whether P nutrition and mycorrhizal infection of field-grown soybeans (Glycine max L.) affects rhizobial competition and nodule occupancy. The objective of these studies was to determine if the level of soil P and its influence on mycorrhizal development affected the competitive nature of the bacterial symbionts active in forming nodules. The effects of P fertilization on mycorrhizal development and rhizobial nodule occupancy were evaluated in a 2-year field study. A follow-up greenhouse experiment evaluated rhizobial strain competition with and without mycorrhizal inoculations. Four soils, ranging in Bray -1 PI levels from 12 to 91 mg kg and belonging to Aquic Hapludolls, Typic Argiudolls, and Typic Haplaquolls, were fertilized with triple super- phosphate at 0 or 112 kg P ha • Mycorrhizal infection, nodulation, and nodule-occupant serology were assessed at early flowering. Phosphorus fertilization significantly reduced mycorrhizal infection, but had no effect on the numbers of nodules per plant or on seed yields. Unexpectedly, there was a significant decrease in nodule occupancy of Bradyrhizobium japonicum serogroup 123, the dominant serogroup found in Midwest soils. Overall, the occupancy of 123 in nonfertilized plots averaged 72% (similar to the values reported by others), whereas the average percentage for the plots receiving the high rate of P was 37%. The significant reduction in nodule occupany by serogroup 123 was 21 accompanied by a significant increase of unknown rhizobia that did not agglutinate with commonly reacting antisera of the Midwest (USDA 142, 135, 123, 122, 110, 76, 31, and 6). Greenhouse inoculation of soybean by Glomus mosseae in sterile growth medium did not result in a competitive reduction of USDA 123 when challenged with USDA 110. 22 INTRODUCTION Many agronomie plants form endotrophic mycorrhizae (Mosse, 1974; Cox and Tinker, 1976; Schoneqt and Hattingh, 1976; Yost and Fox, 1982), in which a loose network of hyphae is established in soil adjacent to the root. In addition to this exterior network, hyphae permeate the root cortex. The interior hyphae can produce extensive coils that develop into haustoria-like structures called arbuscules, which function in nutrient exchange between plant and fungus. Vesicles that act as storage organelles or spores may form among and within cortical cells. These plant-fungal associations presumably occur in the root-rhizosphere zone at the same time that rhizobia establish themselves in the rhizo- sphere and plants. Vesicular-arbuscular mycorrhizae (VAM) stimulate both nodulation and Ng fixation of soybeans (Glycine max L.) when nodulated by Bradyrhizobium japonicum (Sutton, 1973; Crush, 1974; Daft and El-Giahmi, 1976; Carling et al., 1978; Bagyaraj et al., 1979; Carling and Brown, 1980; Gandry et al., 1982). This stimulation was attributed mainly to soybean P nutrition, but non-nutritional interactions also have been implicated (Ross and Harper, 1970). Daft and Nicolson (1966) reported that significant decreases in VAM fungal infection occur with high P fertilization. Almendras and Bottomley (1985) recently showed that the application of small amounts of P influenced nodule occupancy of indige nous serogroups of Rhibozium trifolii, but no work has been reported in soybeans on the effect of VAM or P nutrition on nodule occupancy. Since there is a close physical association of fungi and rhizobia within the 23 plant, it is reasonable to assume that the presence of one may affect growth and development of the other. In this study, the influence of P fertilization on nodule occupancy and VAM fungal infection was evaluated at two Iowa sites for 2 years. Also, a greenhouse study was conducted to evaluate nodule occupancy with and without VAM inoculation. 24 MATERIALS AND METHODS P Fertilization The field studies were conducted at four locations, two each year over 2 consecutive years (Table 1). Separate sites were used each year at the Agronomy and Agricultural Engineering Research Farm (Nicollet series) east of Boone, lA; one site was located at the Curtiss Research Farm (Webster series) south of Ames, lA; and one site was located at a private farm (Dinsdale series) south of Sheffield, lA. These locations were selected to provide a range of background P fertilities. Treatments consisted of no P and high P fertilization. The fertil ized plots received 112 kg P ha ^ as triple superphosphate (0-46-0) surface broadcast and tilled 15-cm deep. Control plots were tilled 15-cm deep as well. Corsoy 79 soybeans were planted on 17 and 31 May in 1984 for Dinsdale and Nicollet soils and on 8 and 9 May in 1985 for Nicollet and Webster soils, respectively. The seeding rate was 14 seeds per 60 cm of row. Treatment plots were 3 by 9 m with four, 76-m rows in 1984; and 3.8 by 7.6 m with five, 76-cm rows in 1985. All treatments were replicated five times except for those on the Webster soil in 1985, which were replicated four times. Treatments were arranged in randomized complete block designs. No B. japonicum inoculant was applied; therefore, nodulation was dependent on native bradyrhizobia. Plants were sampled from each plot at growth stages Rl.O and R4.5 in 1984 and 1985, respectively, and 50 nodules were selected at random to be serotyped per treatment. Antisera prepared against B. japonicum 25 Table 1. Soil characteristics used in field evaluation of P fertiliza tion and nodule occupancy Soil Taxonomical series Year family Texture pH OM K g kg — 1 --mg kg ""1-• Nicollet 1984 fine-loamy, clay 6.4 26 99 20 mixed, mesic, loam Aquic Hapludoll Dinsdale 1984 fine-silty, silty 6.4 31 115 12 mixed, mesic, clay Typic Argiudoll loam Nicollet 1985 fine-loamy, clay 6.5 38 131 42 mixed, mesic, loam Aquic Hapludoll Webster 1985 fine-loamy, clay 6.2 45 197 91 mixed, mesic, loam Typic Haplaquoll pH was determined 1:1 soil to H_0, OM by Mebius method, K by flame photometry, and P by Bray PI. USDA 142, 135, 123, 122, 110, 76, 31, and 6 (Absco Custom Antisera, USAFA, CO) were used to calculate percentage recoveries of B. .japonicum serogroups. For mycorrhizal determination, 10 root samples were randomly se lected from each plot and examined by using the clearing and staining procedure of Kormanik et al. (1980). Stained VAM vesicles were observed in 4-cm sections from each root. Seed yields were determined by harvesting two middle rows from each plot. Ends of plots were trimmed, and 6 m of each row were cut 2 by hand, giving a sample area of 9.1 m . Seed was separated by 26 a stationary thresher, weighed, and moisture content calculated. Seed weights (Mg ha were adjusted to 13% moisture for each plot. VAM Inoculation The greenhouse study evaluated nodule occupancy as related to VAM and B. japonicum inoculation with and without added nonsterile soil. In the sterile treatments, Nicollet soil was sterilized at 121°C for 1 hr in 15-cm diameter clay pots sealed with A1 foil. Bragg soybean seed was surface sterilized in 95% ethanol for 1 min followed by five, 1-min washes with sterile HgO; two seeds were planted in each pot. (Bragg soybeans were used instead of Corsoy 79 to reduce early flowering and senescence in the greenhouse.) At the unifoliate stage, seedlings were thinned to one plant per pot, and treatments applied (Table 2). Five replications of each treatment were arranged in a randomized com plete block design on the greenhouse bench. Mycorrhizal inoculant was prepared by using sorghum roots and rhizosphere soil as a carrier. Eight, 30-cm diameter plastic pots were cleaned and filled with steamed greenhouse potting mix (composed of soil, peat, and perlite in a ratio of 20:40:40 by volume). Ten sorghum seeds were planted in each pot and inoculated with 10 g of soil containing Glomus mosseae spores. (The initial inoculant was provided by Dr. Richard Schultz, Dept. of Forestry, Iowa State University.) After 8 weeks, four pots were randomly selected and the root system and adhering soil were mixed. To verify the presence of VAM, three root samples were stained and observed for vesicles. Spores were counted in three, 5-g soil samples by subjecting each sample to the nematode illutriation processes 27 Table 2. Treatments* used in sterile Nicollet soil to evaluate nodule occupancy after VAM inoculation Treatment USDA 110 USDA 123 VAM Soil number lo hi lo hi inoculation inoculation 1 X X X 2 X X X 3 X X 4 X X 5 X X X 6 X X X *low (lo) 110 and 123 were 5.9 and.5.2, high (hi) 110 and 123 were 8.9 and 8.3 log bradyrhizobia seedling , respectively. VAM inoculant was 5 g of sorghum inoculant. Soil inoculan^ was 10 g of nonsterile Nicollet containing 5.1 log bradyrhizobia g outlined by Sasser and Jenkins (1960). An average of 4.4 spores g ^ soil was counted. -1 Bradyrhizobium .japonicum USDA 110 resistant to 500 mg L of kana- -1 mycin and B. japonicum USDA 123 resistant to 500 mg L of streptomycin were grown in yeast extract mannitol (YEM) broth in shake culture for 14 days, serially diluted, and plated on YEM agar for enumeration. One milliliter of broth was applied directly to pregerminated seedlings (3-cm radicle stage) during inoculation. Plants were harvested 60 days after treatment application, and 10 nodules per pot were randomly chosen and surface-sterilized 1 min each in successive baths of 5% sodium hypochlorite, 95% ethanol, and four sterile H^O washes. The nodules were crushed and contents plated on YEM -1 -1 • agar containing 500 mg L of streptomycin or 500 mg L of kanamycin. Root samples were stained as before and vesicle counts recorded. 28 RESULTS AND DISCUSSION P Fertilization Vesicle counts showed that the P applications significantly reduced VAM infection by an average of 80% (Table 3). The average number of VAM in the control (nonfertilized) plots was 25.5 vesicles per 4-cm length of root, whereas the average in the fertilized plots was 5.0. The reduc tion was statistically significant at each location. Jaspaer et al. (1979) obtained similar results on subterranean clover. Soil test levels did not correlate with VAM infection. This suggests that the added fertilizer P had a greater inhibitory effect than residual soil P on VAM development. Modulation and yield data showed no significant differences between treatments (Table 3); although plants at the Dinsdale location produced less seed yield than plants at the other locations. The lower yields associated with the Dinsdale soil might be attributed to dry-weather stress that occurred at this site during pod fill. The other sites received adequate rainfall throughout the growing season and averaged 3.10 Mg ha ^ (46.2 bu ac seed yields. Counting of nodules proved tedious in 1984 and was replaced in 1985 by the ranking system. Fertilization with P resulted in decreased VAM infection but had no effect on total nodulation. This suggests that*VAM infection and rhizobial infection may function independently of one another, although they both involve cortical cells of the host. Even though total nodulation was unaffected by P additions, a change occurred in the strain of rhizobia that occupied the nodules. Table 3. Modulation, mean vesicle counts, and seed yield of field-grown soybeans Location Treatment Nodulation Vesicles Seed yield No. plant ^ No. (4-cm Mg ha~! or ranking root length)"! Nicollet 1984 Nonfertilized 37.9(1.7)^ 31.2(8.7)** 3.14(0.17) P fertilized 36.3(2.1) 9.2(2.0) 3.15(0.13) Dinsdale 1984 Nonfertilized 47.1(2.9) 27.0(5.4)** 2.30(0.12) P fertilized 50.4(3.0) 6.0(2.1) 2.19(0.09) Nicollet 1985 Nonfertilized 3.2(0.1) 15.4(5.9)* 2.97(0.09) P fertilized 3.2(0.1) 3.4(1.4) 3.01(0.10) Webster 1985 Nonfertilized 3.3(0.2) 28.5(12.1)** 3.30(0.12) P fertilized 3.2(0.1) 1.5(0.6) 3.02(0.14) _c All locations Nonfertilized 25.4(3.9)** 2.90(0.11) P fertilized 5.2(1.0) 2.83(0.11) Modules were counted on 10 plants per plot in 1984. In 1985, nodule number on five plants per plot was ranked on a scale of 1 to 5 as follows: 1 = 0 to 10 nodules; 2 = 11 to 20 nodules; 3 = 21 to 50 nodules; 4 = 51 to 75 nodules; and 5 = greater than 75 nodules per plant. ^Values in parenthesis are SE of the treatment means. Nodulation means were based on 10 obser vations in 1984 and five in 1985; vesicle counts were based.on 10 observations; and seed yields were based on five observations except Webster, 1985, which were based on four observations. ^No overall assessment possible because of different methods of measurement each year. * Nonfertilized different from P fertilized at 0.05 and 0.01 significance levels, respectively, based on single degree of freedom contrasts. 30 Nodule occupancy of B. japonicum 123 was significantly reduced by fertil ization (Table 4). Average occupancy by serogroup 123 was 72% in the nonfertilized plots compared with 37% in the fertilized plots. Only at the Nicollet site in 1984 was occupancy by serogroup 123 not statistic ally reduced with added P, although the trend was similar to those observed at the other sites. Members of serogroup 123 are the dominant rhizobia occupying nodules in the Midwest (Damirgi et al., 1967; Ham et al., 1971; Kapusta and Rowenhorst, 1973; Miller, 1979; Reyes and Schmidt, 1979; Kvien et al., 1981; Berg and Loynachan, 1985; Kamicker and Brill, 1986), and the data in Table 4 indicate that these soils have typical occupancy. Nodules with 123 occupants in the nonfertilized plots ranged from 66 to 76%. Only in calcareous soils is 123 not expected to be the dominant strain; strain 135 dominates in these soils (Damirgi et al., 1967). Nodule occupancy by 135 was not significantly influenced, however, by P fertil ization. This is the first report of field studies in which edaphic changes of the environment, other than pH, resulted in displacement of B. .japonicum 123. The significant reduction in nodule occupancy by B. .japonicum 123 in the F fertilized plots was accompanied by a significant increase in occupancy by unknown rhizobia. The eight antisera used to verify nodule occupancy were selected to include most strains of B. .japonicum reported to occur in Iowa soils (Damirgi et al., 1967; Ham et al., 1967; Weaver et al., 1972; Berg and Loynachan, 1985). What caused the reduction of 123 and the increase of the unknown rhizobia? Since Corsoy 79 was the only cultivar tested, this may be a Table 4. Mean recovery of .j aponicum serogroups in the P fertilization experiment Percentage recovery of serogroups^ Location Treatment 142 135 123 122 110 76 31 6 Unknown Nicollet Nonfertilized 32(11) 4(4) 72(9) 36(13) 4(2) 0(0) 2(2) 0(0) 10(3)* 1984 P fertilized 10(5) 0(0) 38(11) 40(5) 0(0) 0(0) 0(0) 0(0) 46(5) Dinsdale Nonfertilized 4(3) 0(0) 76(2)* 12(5) 2(2) 0(0) 0(0) 4(4) 10(5) 1984 P fertilized 12(6) 0(0) 54(5) 10(4) 0(0) 0(0) 0(0) 0(0) 42(6) Nicollet Nonfertilized 4(4) 2(2) 66(5)* 6(4) 6(2) 0(0) 0(0) 0(0) 24(5)* 1985 P fertilized 28(1) 2(2) 36(5) 34(10) 2(2) 0(0) 0(0) 2(2) 42(4) Webster Nonfertilized 10(7) 3(3) 75(10)* 10(4) 5(3) 8(5) 3(3) 0(0) 10(7) 1985 P fertilized 3(3) 0(0) 15(5) 5(3) 18(8) 3(3) 3(3) 3(3) 55(12) All Nonfertilized 13(4) 2(1) 72(3)*** 16(4) 4(1) 2(1) 1(1) 1(1) 12(3)** locations P fertilized 14(4) 0(0) 37(5) 23(5) 4(2) 0(0) 0(0) 1(1) 46(3) ^eans of five replications except Webster 1985, which had four replications. Percentages will not total 100 due to double occupancy of some nodules. Value in parenthesis are SE of treatment means. *'**'***Means significantly different at 0.05, 0.01, and 0.001 probability level, respectively, based on single degree of freedom contrasts. 32 specific response mediated by this one cultivar. Otherwise, added P may have caused chemical, physical, or biological changes in the rhizosphere, changes in competitive infection or abortion of infection threads, or changes in nodule formation. The level of P used in this study was only about three times the normal amount used in commercial production of Iowa soybeans. Also, fertilization with P may have altered soybean metabolism or physiology. Hallmark and Barber (1984) observed that soybean root surface area was increased significantly with increased soil P. Singleton et al. (1985) reported that fertilization with P enhanced nodulation of B. .japonicum USDA strains 31, 110, and 123. The rhizobial inoculants used by Singleton et al. (1985) were applied in monoculture, and so no inference on competitive ability can be drawn. The only direct evidence obtained in the present study was that added P statistically reduced VAM infection. VAM Inoculation A second study was conducted in a greenhouse with the working hypothesis that VAM infection resulted in serogroup 123 being more competitive. To avoid the confounding factor of P, sterile conditions were maintained so infection by VAM could be controlled by inoculation. Since the unknown rhizobia found in the field were not characterized (another study), a standard strain (USDA 110) was used as the challenging organisms. Field data suggested that with the few serogroup 110 found, occupancy was unrelated to P fertilization. Data from the greenhouse study revealed that nodule occupancy by strain 123 was not statistically related to VAM infection (Table 5). Table 5. Means and contrasts of greenhouse study to evaluate the effect of VAM inoculation on nodule occupancy by strains 110 and 123 Treatment No. Description 123 Recovery Vesicles No. (4-cm root length) ^ 1 hi 110 lo 123 +VAM 13.6(5.0) 41.4(4.3)* 2 lo 110 hi 123 +VAM 54.0(17.5) 31.4(4.2) 3 hi 110 lo 123 -VAM 12.5(3.7) 0.0(0) 4 lo 110 hi 123 -VAM 87.2(8.7) 0.0(0) 5 hi 110 lo 123 +Soil 60.0(24.5) 47.4(12.7) 6 lo 110 hi 123 +Soil 80.0(20.0) 40.4(9.4) Contrast % 123 recovery VAM infection 1 vs 3 NS^ *** 2 vs 4 S *** 1+2 vs 3+4 NS *** 5 vs 6 NS NS Values in parentheses are the SE of the means. ^Significant difference of contrast. Denotes not significantly different (NS), significantly different at the 0.07 level (S), and significantly different at the 0.001 level (***), respectively. 34 At the high rate of inoculation with strain 123 (treatments 2 vs 4), nodule occupancy was reduced 33% in the presence of VAM infection, but this was only significant at the 0.07 level. Glomus mossae was effec tively established, and the number of observed vesicles in the green house was slightly less than that observed in the field. When non- sterile soil was used as a VAM inoculant, the initial presence of different levels of strains 110 and 123 did not result in different levels of VAM fungal infection. Nodule occupancy by 123 was related to the population of 123 in the inoculant or inoculant and soil. High levels of P in the field decreased nodule occupancy by 123 and increased occupancy by serologically unknown rhizobia. Greenhouse evidence suggested that the relative competitive abilities of strains 123 and 110 were unrelated to VAM fungal infection. Clearly, fertiliza tion with P had a major impact on the abilities of different rhizobia to form nodules in the field, but the controlling factor was not deter mined. This research suggests that the environment can be modified to favor certain organisms or, if controlling factors in competition are better understood, organisms may be selected for enhanced competitive ness based on compatibility with the edaphic environment. 35 LITERATURE CITED Almendras, A. S., and P. J. Bottomley. 1985. Effects of phosphorus and lime upon nodulation by indigenous serogroups of Rhizobium trifolii. p. 395. In H. J. Evans, P. J. Bottomley, and W. E. Newton (eds.) Nitrogen fixation research progress. Martinus Nijhoff Publishers, Boston, MA. Bagyaraj, P. J., A. Manjunath, and R. B. Patil. 1979. Interaction between a vesicular-arbuscular mycorrhizas and Rhizobium and their effects on soybeans in the field. New Phytol. 82:141-145. Berg, R. K., and T. E. Loynachan. 1985. Serology and physiology of native Bradyrhizobium .japonicum populations in Iowa soils, p. 396. In H. J. Evans, P. J. Bottomley, and W. E. Newton (eds.) Nitrogen fixation research progress. Martinus Nijhoff Publishers, Boston, MA. darling, D. E., and M. F. Brown. 1980. Relative effect of vesicular- arbuscular mycorrhizal fungi on the growth and yield of soybean. Soil Sci. Soc. Am. J. 44:528-532. Carling, D. E., W. G. Riehle, M. F. Brown, and D. R. Johnson. 1978. Effects of a vesicular-arbuscular mycorrhizal fungi on nitrate reductase and nitrogenase activities in nodulating and non- nodulating soybeans. Phytopathology 68:159-1596. Cox, G., and P. B. Tinker. 1976. Translocation and transfer of nutri ents in vesicular-arbuscular mycorrhizas. I. The arbuscule and phosphorus transfer: A quantitative ultrastructural study. New Phytol. 77:371-378. Crush, J. R. 1974. Plant growth responses to vesicular-arbuscular mycorrhiza. VII. Growth and nodulation of some herbage legumes. New Phytol. 73:743-749. Daft, J. M., and A. A. El-Giahmi. 1976. Studies on nodulation and mycorrhiza on plant growth. Ann. Appl. Biol. 32:273. Daft, J. M., and T. H. Nicolson. 1966. Effects of Endogone mycorrhiza on plant growth. New Phytol. 65:343-350. Damirgi, S. M., L. R. Frederick, and I. C. Anderson. 1967. Serogroups of Rhizobium .japonicum in soybean nodules as affected by soil types. Agron. J. 59:10-12. Gandry, F., H. G. Diem, and Y. R. Dommergues. 1982. Effect of inocu lation with Glomus mosseae on nitrogen fixation by field grown soybeans. Plant Soil 68:321-329. 36 Hallmark, W. B., and S. A. Barber. 1984. Root growth and morphology, nutrient uptake, and nutrient status of early growth of soybeans as affected by soil P and K. Agron. J. 76:209-212. Ham, 6. E., L. R. Frederick, and I. C. Anderson. 1967. Serogroups of Rhizobium .japoniciun in relationship to soil properties and soybean varieties. Bact. Proc. 31:4. Ham, G. E., L. R. Frederick, and I. C. Anderson. 1971. Serogroups of Rhizobium .japonicum in soybean nodules sampled in Iowa. Agron. J. 63:69-72. Jaspaer, D. A., A. D. Robson, and L. K. Abbot. 1979. Phosphorus and the formation of vesicular-arbuscular mycorrhizas. Soil Biol. Biochem. 11:501-505. Kamicker, B. J., and W. J. Brill. 1986. Identification of Bradyrhizobium .japonicum nodule isolates from Wisconsin soybean farms. Appl. Environ. Microbiol. 51:487-492. Kapusta, G., and D. L. Rowenhorst. 1973. Influence of inoculum size on Rhizobium japonicum serogroup distribution frequency in soybean nodules. Agron. J. 65:916-919. Kormanik, P. P., W. C. Bryan, and R. C. Schultz. 1980. Procedure and equipment for staining large numbers of plant root samples for endomycorrhizal assay. Can. J. Microbiol. ,26:536-538. Kvien, C. S., G. E. Ham, and J. W. Zambert. 1981. Recovery of intro duced Rhizobium japonicum strains by soybean genotypes. Agron. J. 73:900-905. Miller, R. H. 1979. Field survey of the distribution of Rhizobium japonicum serogroups in Ohio. p. 2. In R. W. Weaver and D. A. Zuberer (eds.) Proceedings of the 7th North American Rhizobium conference, College Station, TX. Mosse, B. 1974. Specificity in mycorrhizas. pp. 469-484. In F. E. Sanders, B. Mosse, and P. B. Tinker (eds.). Endomycorrhizas. Academic Press, Inc., London. Reyes, V. G., and E. L, Schmidt. 1979. Populations of Rhizobium japonicum associated with the surfaces of soil-grown roots. Plant Soil 61:71-80. Ross, J. P., and J. A. Harper. 1970. Effect of Endogone mycorrhizae on soybean yield. Phytopathology 60:1552-1556. Sasser, J. N., and W. R. Jenkins. 1960. Estimating nematode popula tions by some selected methods. In M. Gostenbrink (ed.) Nematology. Univ. of North Carolina Press, Chapel Hill. 37 Schonect, J. D., and M. J. Hattingh. 1976. X-ray micro-analysis of elements in cells of VA mycorrhizal and nonmycorrhizal onions. Mycologia 68:296-303. Singleton, P. W., H. M. Abdel-Magid, and J. W. Tavares. 1985. Effect of phosphorus on the effectiveness of strains of Rhizobium .japonicum. Soil Sci. Soc. Am. J. 49:613-616. Sutton, J. C. 1973. Development of vesicular-arbuscular mycorrhizae in crop plants. Can. J. Bot. 51:2487-2493. Weaver, R. W., L. R. Frederick, and L. C. Dumenil. 1972. Effect of soybean cropping and soil properties on number of Rhizobium .japonicum in Iowa soils. Soil Sci. 114:137-141. Yost, R. S., and R. L. Fox. 1982. Influence of mycorrhizae on the mineral contents of cowpea and soybean grown in an Oxisol. Agron. J. 74:475-481. 38 SECTION II. SEASONAL FLUCTUATIONS OF SOYBEAN RHIZOSPHERE MICROFLORA AND INFLUENCE ON BRADYRHIZOBIUM JAPONICUM 39 ABSTRACT Microbiological interactions in Iowa soybean (Glycine max L.) rhizospheres were investigated in field and greenhouse studies. Other researchers have reported that bacterial isolates are capable of inhibit ing Bradyrhizobium .japonicum on artificial media. In the present study, isolates that stimulated B. .japonicum serogroup 123 were used to deter mine if they affected nodule occupancy by serogroup 123. Rhizosphere populations of total bacteria and actinomycetes on tryptic soy broth agar, gram-negative bacteria on crystal violet agar, enterobacteria on methylene blue agar, and pseudomonads on SI agar were measured in field- grown soybeans six times over the season at approximately 3-week inter vals. Samples of rhizosphere soil were collected from Webster and Nicollet soils, Typic Haplaquolls and Aquic Hapludolls, respectively, and microorganisms were enumerated. Climatic variables significantly influenced microbial populations but did not affect antagonistic or stimulatory organisms of USDA 123 as determined in plate culture by using an agar-overlay technique. The numbers of antagonistic and stimulatory organisms declined over the season, whereas the total population of bac teria and actinomycetes increased. Early in the season, 31% of the rhizosphere bacteria were inhibitory to 123 and 8% were stimulatory. Nine of the stimulatory organisms were tested in the greenhouse in sterile soil for their ability to alter occupancy of soybean nodules by strain 123 when challenged by USDA 110. Two organisms significantly enhanced nodulation by strain 123, but further tests indicated that the results were not reproducible in nonsterile soil. 40 INTRODUCTION Factors allowing one bradyrhizobial strain to establish itself while preventing the establishment of a second strain remain, for the most part, obscure. Although rhizobia survive well in the absence of soybeans (Norman, 1942), a marked rhizosphere effect is often noted (Peters and Alexander, 1966; Caldwell and Vest, 1968). Saprophytic com petition in the rhizosphere presumably occurs between rhizobia and other genera. Hattingh and Louw (1969) found approximately 25% of the bac teria and actinomycetes in the root region inhibited clover rhizobia when grown in plate culture. Damirgi and Johnson (1966) reported most isolates of actinomycetes from a soybean field were not inhibitory when tested against eight strains of B. japonicum. Smith and Miller (1974) found eight of nine rhizosphere bacteria inhibited B. .japonicum on agar plates. Their study also revealed that, as a group, rhizosphere organ isms inhibited B. .japonicum to a greater extent than inhibition produced by nonrhizosphere organisms. Members of serogroup 123 are the common soybean rhizobia found in Iowa soils (Johnson et al., 1965; Damirgi et al., 1967; Ham et al., 1971; Weaver and Frederick, 1974a, 1974b; Boonkerd et al., 1978; Berg and Loynachan, 1985). What has allowed these organisms to dominate? Were the organisms first to be introduced to the area, or is there something about these organisms and their compatibility with soils or cultivars of the region that has allowed them to become established? If soil factors are important, they may be chemical, physical, or biological. To determine if biological factors in Iowa soils favored growth of 41 serogroup 123, various groups of organisms were isolated from soybean rhizospheres over the growing season and evaluated for stimulation or repression of B. japonicum 123. Initial tests were conducted on culture plates; those that were stimulatory in sterile greenhouse culture were tested in nonsterile culture. Standard USDA strain 110 was used for comparison. 42 MATERIALS AND METHODS Rhizosphere Populations Four sampling areas were randomly assigned at the Agronomy and Agricultural Engineering Research Farm (Nicollet soil) east of Boone, lA, and at the Curtiss Research Farm (Webster soil) south of Ames, lA (Table 1). All areas were situated within uniform soil series to minimize variability among samples. Areas were located adjacent to one another and were 3.5 by 7.6 m, consisting of five rows spaced 76-cm apart. A 2-m border of bare soil was maintained around each area. Corsoy 79 soybeans were planted on 8 and 9 May 1985 in the Nicollet and Webster soils, respectively. Plant roots were collected on 21 May, 13 June, 9 July, 30 July, 20 August, and 18 September. Single roots were removed randomly from each area in a cylinder of soil 7.6 by 15.2 cm by using a soil-core extractor centered over the taproot after the plant top was removed. The core extractor was rinsed in 95% ethanol and allowed to dry between sample collections. The soil and root samples were sealed in plastic bags and stored on ice during trans port to the laboatory. Soil samples from borders between sample areas were collected as nonrhizosphere controls. Total time for sample collection and transport to the laboratory was approximately 1 hr. At the laboratory, each soil cylinder was saturated with sterile distilled H^O to loosen the soil. A flamed scalpel was used to tease the taproot and any attached lateral roots from the soil. Each root 43 Table 1. Characteristics of soils used in field evaluation of rhizo- sphere microbial populations Soil Series Family Texture pH OM K P , -1 , -1 g kg —mg kg - Nicollet fine-loamy, loam 6.5 38 131 42 mixed, mesic, Aquic Hapludoll Webster fine-loamy, clay loam 6.2 45 196 91 mixed, mesic, Typic Haplaquoll ^pH was determined 1:1 soil to H.O, OM by Mebius method, K by flame photometry, and P by Bray PI. and adhering soil was transferred to a wide-mouth dilution bottle con taining sterile H^O and 10 g of 6-mm sterile glass beads. The samples were mechanically shaken for 15 min and serially diluted. Duplicate dilutions were plated in the media listed in Table 2. Counts of rhizo- sphere organisms were determined on six dates, but growth chamber space limited determination of rhizobia by most probable number (Weaver and Frederick, 1972) to four dates: 21 May, 9 July, 20 August, and 18 September. Colony forming units were counted 3 days after the plates were poured. Incubation temperature was 30®C. The selective triple-agar layer technique described by Pantier et al. (1979) was then used to enumerate organisms antagonistic or stimulatory to B. .japonicum USDA 110 and USDA 123. Shake cultures of B. .japonicum were grown in yeast extract mannitol (YEM) broth for 14 days before plating. One milliliter 44 Table 2. Media used to enumerate rhizosphere microorganisms Microorganisms Medium* enumerated Tryptic Soy Agar (10% TSA) Total bacteria and actinomycetes Crystal Violet Agar (10% TSA ^ Gram-negative bacteria plus 2 mg crystal violet L ) Methylene Blue Agar (10% TSA) ^ Enterobactericeae plus 3 mg methylene blue L ) SI Agar Pseudoraonads ^Gould et al., 1985. of broth was added to the surface of each plate and spread to provide a uniform inoculant. After 10 days incubation, the plates were inspected for inhibition or stimulation of B. .japonicum growth. Nine colonies showing relatively high stimulation were streaked for purity and stored on agar slants at 3°C for further testing in the greenhouse. Climatological data were collected at the Agronomy and Agricultural Engineering Research Farm Headquarters approximately 2 and 7 km from the Nicollet and Webster soils, respectively. Daily rainfall, temperature, solar intensity, wind velocity, and evaporative index measurements were taken and averaged for sampling dates. The data recorded from planting to 21 May were averaged for the first date, the data recorded from 22 May to 13 June were averaged for the second date, and so on. This was repeated throughout the growing season so that climatological factors within a period of approximately 3 weeks prior to sampling could be cor related with microbial populations. 45 Greenhouse Screening A greenhouse study was conducted to test nine 123-stimulating bac terial isolates on nodule occupancy of bradyrhizobia (Table 3). Six were rhizosphere isolates and three were nonrhizosphere isolates that stimulated 123 when grown on agar plates. Clay pots (15-cm diameter) were filled with a mixture of two parts of Nicollet soil and one part of coarse sand. The pots were sealed with A1 foil and heated in an autoclave for three, 1-hr cycles at 121°C, with a 24-hr cool-down period between each cycle. Corsoy 79 soybean seeds were surface sterilized for 1 min in 95% ethanol followed by four, 2-min washes in sterile distilled HgO. Three seeds were planted in each pot; the plants were later thinned to one plant per pot. When seedlings reached the open cotyledon stage, a 2-cm depression was made immediately adjacent to the hypocotyl and the test organism and B. .japonicum treat ments were applied. All test organisms were applied at log 7 to log 8 organisms per seedling and rhizobia were added at either log 5.2 (lo) or log 8.2 (hi) cells per seedling (Table 3). Bradyrhizobium .japonicum USDA 110 resistant to 500 mg L ^ kanamycin and B. .japonicum USDA 123 resis tant to 500 mg L ^ streptomycin were grown in YEM broth in shake cul ture for 10 days prior to inoculation. After the inoculants were applied, the depressions were covered with soil and sterile sand was placed over the soil surface to minimize contamination. All treatments were replicated four times and arranged in a completely randomized block design on the greenhouse bench. 46 Table 3. Experimental design* and characteristics of isolates applied in the sterile-soil greenhouse study Isolate B. japonicum Treatment ID Source Inoculant 110 123 Log no. applied- 1 Rir MBA(N)^ 7.1 8.2 (hi) 5.2 (lo) 2 RIl MBA(N) 7.1 5.2 (lo) 8.2 (hi) 3 RI2 MBA(N) 8.1 8.2 (hi) 5.2 (lo) 4 RI2 MBA(N) 8.1 5.2 (lo) 8.2 (hi) 5 Rill MBA(N) 8.8 8.2 (hi) 5.2 (lo) 6 Rill MBA(N) 8.8 5.2 (lo) 8.2 (hi) 7 RI13 S1(W) 7.7 8.2 (hi) 5.2 (lo) 8 RI13 S1(W) 7.7 5.2 (lo) 8.2 (hi) 9 RI17 TSA(W) 8.0 8.2 (hi) 5.2 (lo) 10 RI17 TSA(W) 8.0 5.2 (lo) 8.2 (hi) 11 RI19 MBA(N) 8.1 8.2 (hi) 5.2 (lo) 12 RI19 MBA(N) 8.1 5.2 (lo) 8.2 (hi) 13 Nil TSA(N) 8.2 8.2 (hi) 5.2 (lo) 14 Nil TSA(N) 8.2 5.2 (lo) 8.2 (hi) 15 NI4 TSA(N) 8.2 8.2 (hi) 5.2 (lo) 16 NI4 TSA(N) 8,2 5.2 (lo) 8.2 (hi) 17 NI5 TSA(N) 8.2 8.2 (hi) 5.2 (lo) 18 NI5 TSA(N) 8.2 5.2 (lo) 8.2 (hi) 19 None — — — — — — 8.2 (hi) 5.2 (lo) 20 None 5.2 (lo) 8.2 (hi) *Each treatment was replicated four times and established in a com pletely randomized fashion on the greenhouse bench. ^Organisms identified as RI were isolates from rhizosphere soil and those identified NI were isolates from nonrhizosphere soil. ^Indicates medium on which isolated and soil of origin: MBA is methylene blue agar, SI is pseudomonad medium, TSA is tryptic soy agar, and (N) is Nicollet and (W) is Webster soil. 47 The plants were maintained in the greenhouse under natural light ing for 8 weeks; H^O was applied as needed. Plants were harvested and placed in plastic bags, and approximately 25 g of soil adhering to each root system was collected in a sterile whirl-pak bag. Both plants and soil were maintained at S^C until analyzed. Twelve nodules from each root system were surface sterilized for 1 min each in successive baths of 5% sodium hypochlorite, 95% ethanol, and four washes of sterile H^O. The nodule contents were then streaked on plates containing either 500 mg L ^ of kanamycin or 500 mg L ^ of streptomycin. Growth on specific antibiotic agar was interpreted to be positive recovery for that inoculated strain. The percentage recoveries of B. japonicum 110 and 123 were calculated, and contrast coefficients were used to compare treatment means with control means. Nodule number, nodule weight, root weight, top weight, pod number, and pod weight were also measured and analyzed similarly to nodule occupancy. Rhizosphere competency of each isolate was tested by serially diluting rhizosphere soil from each pot and plating it on the medium originally used to isolate the organism. Colonies morphologically similar to the original isolate were counted as positive. Control soils did not contain similar organisms. Results of the sterile-soil experiment prompted a second greenhouse study in which two isolates were used in nonsterile soil. These two isolates had resulted in increased nodule occupancy of 123. Clay pots (15-cm diameter) were filled with a Nicollet soil-sand mixture (60:40 by volume) containing 6.3 and 5.3 log bacteria and actinomycetes (TSA medium) 48 Table 4. Experimental design and inoculants applied in the nonsterile greenhouse experiment , Log B. japonicum Isolate inoculant Treatment ID Source Inoculant 110^ 123^ Log no. applied- 1 RI2 MBA(N) 8.5 6.0 (lo) 8.6 (hi) 2 RI2 MBA(N) 8.5 7.9 (hi) 6.6 (lo) 3 RIll MBA(N) 8.5 6.0 (lo) 8.6 (hi) 4 Rill MBA(N) 8.5 7.9 (hi) 6.6 (lo) 5 None 6.0 (lo) 8.6 (hi) 6 None 7.9 (hi) 6.6 (lo) *Each treatment was replicated five times and established in a pletely randomized fashion on the greenhouse bench. ''isolates identified in Table 3. c ~ 1 B. .japonicum llO^was resistant to 500 mg L kanamycin and 123 was resistant to 500 mg L streptomycin. g -1, respectively. The mixture also contained 6.2 log B. .japonicum g -1 Two surface-sterilized Corsoy 79 soybean seeds were planted and thinned to one plant as previously described. At the open cotyledon stage, inoculants were applied (Table 4) as before. The plants were harvested 8 weeks later, and 16 nodules from each plant were plated on antibiotic media to determine recovery. Nodule isolates not growing on either antibiotic medium were assumed to have originated from the nonsterile soil. Previous studies showed that the soil bradyrhizobia were not tolerant to either antibiotic at the levels used. Rhizosphere competency could not be calculated in this study because of interference from microorganisms in the nonsterile soil. 49 RESULTS AND DISCUSSION Rhizosphere Populations Only stimulatory isolates were investigated for their influence on nodule occupancy by B. .j aponicum because Smith and Miller (1974) investi gated antagonistic organisms previously with limited results. No work with stimulatory organisms has been reported. Mean counts of rhizosphere organisms differed significantly among groups studied and over time (Table 5). Because no significant differ ences existed between soils or among sampling areas, the data were pooled for analysis. Enterobacteria and total bacterial populations were a major component of the rhizosphere microflora throughout the season. Gram-negative bacteria and actinomycetes were next in abundance, followed by pseudomonads. These populations were generally consistent with the findings of Gould et al. (1985) for nonrhizosphere soils. Populations of B. japonicum decreased slightly during mid-season, but rose to a peak at plant senescence. Release of rhizobia from nodules at the end of the season presumably accounted for the rise in B. japonicum rhizosphere numbers at the final sampling. Throughout the growing season, higher percentages of the rhizosphere isolates were inhibitory than stimulatory (Table 5). Approximately 30% of the isolates on the highest dilution plate were inhibitory at the 21 May sampling, whereas only 5% were stimulatory. Percentages of both inhibitory and stimulatory organisms decreased during the season. By Table 5. Mean rhizosphere populations of six bacterial groups as determined at six sampling dates Sampling date 21 May 13 June 9 July 30 July 20 August 18 September Group (VI.1)* (V5.3) (Rl.2) (R5.1) (R6.4) (R8.0) ^Log no. g ^ soil Total bacteria on TSA 5.5(4.7r 5.1(4.6) 5.2(4.4) 6.7(6.4) 6.2(5.6) 6.4(5.9) Gram-negative bacteria on CVA 4.1(3.4) 3.6(2.8) 4.6(4.2) 4.5(3.7) 4.8(4.3) 4.9(3.8) Enterobacter bacteria on MBA 5.5(4.5) 6.3(4.5) 5.7(5.3) 5.8(5.0) 6.0(5.4) 6.4(5.9) Fseudomonad bacteria on 81 2.3(1.8) 1.6(1.1) 2.9(1.9) 3.2(2.3) 2.8(2.4) 4.0(3.9) Total actinomycetes on TSA 4.9(4.2) 4.6(4.0) 4.9(4.3) 5.4(4.8) 6.0(5.3) 5.3(4.9) japonicum 5.4(4.9-5.9) 4.6(4.1-5.1) 4.8(4.3—5.3) 6.4(5.9-6.9) ^Growth stage of soybeans is indicated in parentheses. ^Mean of eight replications ± SE. ^ean of eight replications by most probable number with five observations per dilution; values in parentheses are 95% confidence limits (Weaver and Frederick, 1972). Table 5. Continued Sampling date 21 May 13 June 9 July 30 July 20 August 18 September Group (VI.1) (V5.3) (R1.2) (R5.1) (R6.4) (R8.0) Inhibitory Organisms Against 110 29(8.3) 10(3.3) 7(2.0) 9(2.6) 5(0.9) 2(0.5) Against 123 31(8.3) 16(3.9) 6(2.2) 9(2.3) 4(1.2) 3(0.8) Stimulatory Organisms^ For 110 2.1(2.1) 4.8(2.8) 0.0(0.0) 2.8(1.1) 1.5(0.6) 0.4(0.2) For 123 8.3(6.4) 5.6(3.9) 1.6(1.1) 1.9(1.0) 1.0(0.4) 0.7(0.3) Ln ^Inhibitory or stimulatory organisms as a percentage of total bacteria plus total actinomycetes found on the highest dilution plate (20 to 200 colonies) as determined by an agar-overlayer technique. Mean of eight replications ± SE. 52 season's end, approximately 2.5% were inhibitory and only 0.5% were stimulatory. Climatological data were compared with rhizosphere populations to determine the effect of climatic variables on seasonal fluctuations in bacterial counts (Table 6). Mean daily rainfall was significantly cor related with rhizosphere gram-negative bacteria, enterobacteria, and pseudomonad populations. The mean daily evaporative index was negatively correlated with gram-negative, enterobacteria, and actinomycetal popula tions. Mean daily temperature was significantly correlated to total, gram-negative, and enterobacterial bacterial populations but not to pseudomonads or total actinomycetal populations. The stress factor was negatively correlated with gram-negative bacterial and actinomycetal populations. The gram-negative bacteria were the most sensitive to environmental conditions. The enterobacter and actinomycetes were some what affected by environmental fluctuations, and the pseudomonads and total bacterial populations were least affected. Greenhouse Screening Sterile soil Two isolates produced significant increases in nodule occupancy by B. japonicum 123 (Table 7, treatments 3 and 5 vs. 19). These two isolates averaged a 42% increase in 123 occupancy over controls and a 42% decrease in 110 occupancy over controls in sterile soil. This phenomenon was observed when the ratio of applied cells was serogroup 110 at 8.2 log cells mL ^ and 123 at 5.2 log cells mL If the ratio was reversed, no significant change was observed. 53 Table 6. Effects of selected climatic variables on rhizosphere micro- floral populations Climatic variables* Evaporative Mean Precipitation index temperature r value Total bacteria 0.13 -0.21 0.37** Gram-negative 0.48*** -0.61*** 0.29* Enterobacter 0.57*** -0.60*** 0.30* Pseudomonads 0.32* -0.23 0.19 Total actinomycetes 0.10 -0.49*** 0.04 ^Precipitation was mean daily rainfall; evaporative index was HgO loss from Class A evaporation pan; mean temperature was daily max-min temperature. "A* icff *** ' ' Significant at 0.05, 0.01, and 0.001 levels of probability, respectively. Differences in rhizosphere competency between isolates were not significant (Table 7). All test organisms established themselves in rhizosphere soil at 7.5 to 8.0 log cells g . No significant differences in plant weights or other traits measured were found in the sterile-soil greenhouse experiment. Nonsterile soil When RI2 and Rill (Table 3) were added to nonsterile soil, no in crease in 123 nodule occupancy was observed (Table 8). Nodule occupants of 110 and 123 do not total 100% in Table 8 presumably because some of the nodules were occupied by indigenous bradyrhizobia. A significant reduction of occupancy by strain 110 was observed in plants inoculated with Rill compared with the control. The influence of these two isolates in sterile soil, therefore, was not repeatable in nonsterile soil. 54 Table 7. Influence of nine rhlzosphere isolates on B. japonicum 110 and 123 nodule recovery in sterile soil Isolate Nodule occupancy* Treatment ID Recovery 110 123 Pr > F Log g soil ------% hi 110 lo 123 Vk 1 B.I1 7.8 81 19 NS^ 3 RI2 7.6 40 60 *** 5 Rill 7.6 67 33 *** 7 RI13 7.8 77 23 NS 9 RI17 7.6 94 6 NS 11 RI19 8.0 87 13 NS 13 Nil 7.8 90 10 NS 15 NI4 7.6 98 2 NS 17 NI5 7.8 94 6 NS 19 None — 96 4 --- lo 110 hi 123 2 RIl 7.5 8 92 NS 4 RI2 7.6 10 90 NS 6 RIll 7.8 6 94 NS 8 RI13 7.5 12 88 NS 10 RI17 7.5 0 100 NS 12 RI19 7.6 0 100 NS 14 Nil 7.5 8 92 NS 16 NI4 7.8 2 98 NS 18 NI5 7.5 13 87 NS 20 None 2 98 ^Rhizosphere population of test organisms in medium of isolation at 8 weeks. Percentage of 12 nodules per plant, five plants per treatment. c Based on single degree of freedom contrasts between treatment and corresponding control. ***Significant at the 0.001 level of probability. 55 Table 8. Influence of two isolates on nodule occupancy by B. .japonicum 110 and 123 in nonsterile soil Isolate Nodule occupancy* Treatment inoculant 110 123 1 RI2 28 52 2 RI2 14 58 3 Rill 9** 65 4 Rill 23 55 5 -- 43 46 6 — — 38 38 ^Percentage of 16 nodules per plant, five plants per treatment; totals do not equal 100 because of indigenous soil rhizobia. ** Significantly different from control at 0.01 level of probability based on single degree of freedom contrasts. Unfortunately, because of the technique used, the ability of the added test organisms to establish in the nonsterile soil could not be deter mined. The results obtained may simply mean that the test organisms could not compete against organisms already established. 56 CONCLUSIONS Rhizosphere populations of organisms varied significantly over the growing season and among groups tested. Precipitation, evaporation index, and mean temperature during the period 3 weeks before sampling were significantly related to populations of one or more of the groups examined. The percentage of rhizosphere isolates antagonistic to B. japonicum was greater than the percentage of stimulatory isolates. Both groups of isolates declined in number over the season whereas total rhizosphere populations increased. No correlation existed between B. j aponicum populations and total percentages of antagonistic or stimula tory organisms. The agar-overlay technique showed a maximum of 30% of rhizosphere organisms were antagonistic to B. .japonicum early in the season whereas only 5% were stimulatory; these percentages decreased during the season. Two isolates that stimulated growth of strain 123 on culture plates resulted in increased 123 nodule occupancy in sterile soil. These isolates did not increase nodule occupancy by B. .japonicum 123 in nonsterile soil. 57 LITERATURE CITED Berg, R. K., and T. E. Loynachan. 1985. Serology and physiology of native Bradyrhizobium japonicum populations in Iowa soils, p. 396. In H. J. Evans, P. J. Bottomley, and W. E. Newton (eds.) Nitrogen fixation research progress. Martinus Nijhoff Publishers, Boston, MA. Boonkerd, N., D. F. Weber, and D. F. Bezdicek. 1978. Influence of Rhizobium japonicum strains and inoculation methods on soybeans grown in rhizobia populated soil. Agron. J. 70:547-549. Caldwell, B. E., and G. Vest. 1968. Modulation interactions between soybean genotypes and serogroups of Rhizobium japonicum. Crop Sci. 8:680-682. Damirgi, S. M., and H. W. Johnson. 1966. Effect of soil actinomycetes on strains of Rhizobium japonicum. Agron. J. 58:223-224. Damirgi, S. M., L. R. Frederick, and I. C. Anderson. 1967. Serogroups of Rhizobium japonicum in soybean nodules as affected by soil types. Agron. J. 59:10-12. Gould, W. D., C. Hagedorn, T. R. Bardinelli, and R. M. Zablotowicz. 1985. New selective media for enumeration and recovery of fluores cent pseudomonads from various habitats. App. Environ. Microbiol. 49:28-32. Ham, G. E., V. B. Cardwell, and H. W. Johnson. 1971. Evaluations of populations of Rhizobium japonicum inoculants in soils containing naturalized populations of rhizobia. Agron. J. 53:301-305. Hattingh, M J., and H. A. Louw. 1969. Clover rhizoplane bacteria antagonistic to Rhizobium trifolii. Can. J. Microbiol. 15:361-364. Johnson, H. W., V. M. Means, and C. R. Weber. 1965. Competition for nodule sites between strains of Rhizobium japonicum applied as inoculum and strains in the soil. Agron. J. 57:179-185. Norman, A. G. 1942. Persistence of Rhizobium japonicum in soil. J. Am. Soc. Agron. 34:449. Pantier, J. J., H. G. Diem, and Y. Dommergues. 1979. Rapid method to enumerate and isolate soil actinomycetes antagonistic towards rhizobia. Soil Biol. Biochem. 11:443-445. Peters, R. J., and M. Alexander. 1966. Effect of legume exudates on the root nodule bacteria. Soil Sci. 102:380-387. 58 Smith, R. S., and R. H. Miller. 1974. Interactions between Rhizobium japonicum and soybean rhizosphere bacteria. Agron. J. 66:564-567. Weaver, R. W., and L. R. Frederick. 1972. A new technique for most- probable-number counts of rhizobia. Plant Soil. 36:219-222. Weaver, R. W., and L. R. Frederick. 1974a. Effect of inoculum rate on competitive nodulation of Glycine max. I. Greenhouse studies. Agron. J. 66:229-232. Weaver, R. W., and L. R. Frederick. 1974b. Effect of inoculum rate on competitive nodulation of Glycine max. II. Field studies. Agron. J. 66:233-236. 59 SECTION III. BIOLOGICAL FACTOR INFLUENCING BRADYRHIZOBIAL NODULE OCCUPANCY IN A HIGH pH SOIL 60 ABSTRACT Fluorescent pseudomonad populations and Bradyrhizobium japonicum serogroup 135 dominance were investigated in a calcareous and noncal- careous soil. Other researchers have attributed the dominance of 135 in alkaline soils to the availability of reduced Fe, but the results of more recent studies indicated that fluorescent pseudomonads produce siderophores (Fe chelators) in high pH soils. The objective of this study was to assess possible biological factors affecting nodule occu pancy of serogroup 135. Two soils, Webster (pH 6.6) and Canisteo (pH 7.5), both Typic Haplaquolls, were inoculated under sterile and non- sterile conditions with B. japonicum USDA 123 and USDA 135; nodule occupancy was assessed serologically. Sterilization of the Canisteo soil significantly reduced pseudomonad populations as expected. However, soil sterilization had no affect on Fe availability, as measured by chemical extraction. Sterilization also significantly increased nodule occupancy by strain 123 80% when equal numbers of strains 123 and 135 were added. The soybeans grown in nonsterile Canisteo soil had only 22.5% of their nodules occupied by 123. Nodule occupancy by strain 135 in the Webster soil was not affected by sterilization, and approximately 75% of the nodules on soybeans grown in both sterile and nonsterile soils were occupied by 123. The ability of B. japonicum 135 to dominate nodule occupancy on plants grown in a high pH soil could be attributed to a biological factor(s). 61 INTRODUCTION Nodule occupancy in soybeans grown in noncalcareous soils (pH below 7.5) in the Midwest is dominated by B. japonicum 123, and serogroup 135 dominates in plants grown in calcareous soils (pH above 7.5) (Damirgi et al., 1967; Ham et al., 1971; Weaver et al., 1972). Damirgi et al. (1967) indicated that the pH effect on nodule occupancy was not strictly related to alkalinity, but that soil type also had a significant influence. The availability of Fe to plants is reduced in alkaline soils (Truog, 1946). Ham et al. (1967) contrasted the behavior of B. .japonicum strains 123 and 135 when they were added to soybeans in sand cultures containing different levels of Fe. High Fe availability favored serogroup 123 and low Fe availability favored serogroup 135. Pseudomonads are common soil inhabitants, and Leisinger and Mangraff (1979) showed that fluorescent pseudomonads produce secondary metabol ites with a range of antibiotic activities. These metabolites were first termed ferric pseudobactins and later siderophores (high-affinity Fe(III) ion chelators). Scher and Baker (1980) showed that either a Pseudomonad sp. or Fe(III) EDDHÂ would suppress Fusarium in soil. They concluded that the iron in Fe(III) EDDHA was available primarily to microorganisms that produced siderophores. These results indicated that fluorescent pseudomonads in the rhizosphere produced siderophores that suppressed the colonization of other microorganisms by complexing Fe. Schroth and Hancock (1982) described a plant-disease suppression phenom enon in soils correlated to root surface colonization by Pseudomonas spp. 62 Olson and Mlsaghi (1982) studied the effect of soil pH on sidero- phore production by fluorescent pseudomonads. Sterile soil extracts with pH values between 3.0 and 8.0 were inoculated with Pseudomonas fluorescens. Siderophore production was substantially reduced when the pH lowered and not detected below pH 7.0. Increased Fe availability at lower pH ranges was reported to inhibit siderophore production by Pseudomonas fluoréscens. A preliminary study was designed to determine if chemical or bio logical factors were more important in allowing serogroup 135 to be more competitive in a high pH soil. The effects of Fe availability, pH, and fluorescent pseudomonad populations on nodule occupancy by B. .japonictim 123 and 135 were also examined. 63 MATERIALS AND METHODS Two soils were collected at the Agronomy and Agricultural Engi neering Research Farm east of Boone, lA (Table 1). Each soil was placed in eight, 15-cm diameter clay pots, four of which were sealed with A1 foil and sterilized at 121°C for four, 1-hr sterilization cycles with a 24-hr cool-down period between each cycle. Corsoy 79 soybean seeds were sterilized in 95% ethanol for 1 min followed by four, l-tiiin washes with sterile HgO. Four seeds were planted in each pot; the plants were thinned at the open cotyledon stage to one plant per pot. When the cotyledons opened, a 2-cm deep hole was made in the soil adjacent to each seedling and 8.2 and 8.1 log B. japonicum 123 and 135, respectively, were applied to each root system. The hole was covered and sterile sand was applied to the soil surface to minimize cross con tamination. Four replications of each treatment were arranged on a greenhouse bench in a completely randomized block design. Sixty-six days after the inoculants were applied, the plants were harvested and approximately 20 g of soil adjacent to each root system was collected in a sterile whirl-pak bag and stored at 1° to 5°C. Twenty-four hours after the soil samples were collected, the samples were serially diluted and plated on SI agar (Gould et al., 1985) to enumerate fluorescent pseudomonads. Remaining soil samples were air- dried, the pH was determined by using a 1:1 soilzHgO suspension, and available Fe was measured by using the DTPA method described by Lindsay and Norvell (1978). Table 1. Characteristics^ of soils Soil Fluorescent Series Family Texture pH japonlcum pseudomonads -1 —Log g soil—— Webster fine-loamy, clay loam 6.6 6.2 4.3 mixed, mesic Typic Haplaquoll Canisteo fine-loamy, mixed clay loam 7.5 6.1 4.7 (calcareous), mesic Typic Haplaquoll ^pH determined by 1:1 soiliH^O; JB- japonicum determined by most probable number method (Weaver and Frederick, 1972); fluorescent pseudomonads enumerated on SI agar (Gould et al., 1985). 65 Ten nodules were randomly selected from each root system and serotyped with antisera prepared against B. .japonicum USDA 142, 135, 123, 122, 110, 76, 31, and 6 (Absco Custom Antisera, USAFA, CO). 66 RESULTS AND DISCUSSION Plants grown in nonsterile Webster and Canisteo soils produced nodules that contained approximately 25% nodule occupancy by strains other than 123 or 135, but inoculated sterile soils produced plant nodules that contained only 123 and 135 (Table 2). Sterilization of the Canisteo soil statistically increased nodule occupancy by strain 123 when equal numbers of 123 and 135 were added. Occupancies' by 123 were similar in both nonsterile and sterile Webster soils. These data suggest that the factor(s) allowing 135 to be more competitive in the higher pH Canisteo soil was biological because sterilization did not affect pH and did not reduce Fe availability in the Canisteo soil. Sterilization did reduce Fe availability of the Webster soil but this did not affect 123 occupancy. The data also suggest that strain 123 was more competitive than strain 135 in forming nodules on Corsoy 79 when the biological factor was removed. This is consistent with other data col lected in the Midwest indicating that serogroup 123 is dominant in nodules of soybeans in nonalkaline soils. Nodule occupancies by other serogroups were not significantly different between sterile and nonsterile soils (data not shown). The biological factor(s) increasing 135 competitiveness in non sterile, alkaline soil was not identified. Populations of fluorescent pseudomonads in the rhizospheres of nonsterile Webster and Canisteo soils were similar at the end of the study. The ability of these organisms to produce siderophores was not determined, so the percentages, if any, of these organisms capable of siderophore production was not known. Work by 67 Table 2. Means of parameters measured in the pseudomonad study (FE III) B. japonicum Fe Fluorescent nodule occupancy Soil pH availability pseudomonads 123 135 -1 ! 1 1 1 1 1 1 1 mg Kg"^ Log no. g f Webster 6.6a® 45.9a 4.7a 70.0a 2.5c Sterile 6.6a 30.8b 2.8b 75.0a 25.0b Webster Canisteo 7.5b 10.8c 5.0a 22.5b. 52.5a Sterile 7.5b 11.2c 2.7b 80.0a 20.0b Canisteo ^Means followed by the same letter are not different at the 0.05 significance level based on Tukey's HSD. Percentage nodule occupancy does not total 100 because serogroups other than 123 and 135 occupied nodules in nonsterile soil. Olson and Misaghi (1982) suggested that a specific Pseudomonas sp. pro duced siderophores only at pH values above 7.0. Even though sterility was verified at the beginning of the study, terminal plate counts indi cated that the sterile treatments had acquired fluorescent pseudomonads; it is difficult to grow soybeans axenically in the greenhouse for extended periods. The sterilized treatments did have fewer fluorescent pseudo monads than the nonsterile soils at the end of the study (Table 2). The biological factor influenced nodule occupancy to a greater degree than did Fe availability (Table 2). Although the Fe level was significantly lower in the sterile Canisteo soil than in sterile Webster soil, no significant reduction in 123 nodule occupancy occurred between the soils. Possibly siderophores produced by pseudomonads in the higher pH soil sequestered enough Fe to render it unavailable for B. .japonicum 68 123 in the nonsterile Canisteo soil. The high availability of Fe in the nonsterile Webster soil may have inhibited siderophore production by pseudomonads, as well as provided enough Fe for 123 to compete for nodulation. The actual effects of Fe availability and siderophore production are interlinked and difficult to separate. Siderophores are not pro duced in environments with available Fe, and when produced, tend to amplify the lack of Fe by sequestering what little Fe is present. 69 SUMMARY Evidence is presented that a biological factor(s) is important in allowing serogroup 135 to occupy nodules of soybeans when grown in high pH soils. Sterilization of Canisteo soil (pH 7.5) resulted in 80% nodule occupancy of 123 when 123 and 135 were added in equal numbers. Nonsterile Canisteo soil had only 22.5% occupancy by 123. Nodule occupancy of soy beans in Webster soil (pH 6.6) was not affected by sterilization; approxi mately three-fourths of the nodules were occupied by 123. 70 LITERATURE CITED Damirgi, S. M., L. R. Frederick, and I. C. Anderson. 1967. Serogroups of Rhizobium .japonicum in soybean nodules as affected by soil types. Agron. J. 59:10-12. Gould, W. D., C. Hagedorn, T. R. Bardinelli, and R. M. Zablotowicz. 1985. New selective media for enumeration of fluorescent pseudo- monads from various habitats, App. Environ. Microbiol. 49:28-32. Ham, G. E., L. R. Frederick, and I. C. Anderson. 1967. Serogroups of Rhizobium .japonicum in relationship to soil properties and soybean varieties. Bact. Proc. 31:4. Ham, G. E., L. R. Frederick, and I. C. Anderson. 1971. Serogroups of Rhizobium japonicum in soybean nodules sampled in Iowa. Agron. J. 63:69-72. Leisinger, T., and R. Mangraff. 1979. Secondary metabolites of the fluorescent pseudomonads. Microbiol.Rev. 43:422-442. Lindsay, W. L., and W. A. Norvell. 1978. Development of a DTPA soil test for zinc, iron, manganese, and copper. Soil Sci. Soc. Am. J. 42:421-428. Olson, M. W., and J. J. Misaghi. 1982. Siderophore production by Pseudomonas fluorescens in soil extracts. Phytopathology 72:997. Scher, F. M., and R. Baker. 1980. Mechanism of biological control in a Fusarium suppressive soil. Phytopathology 70:412-417. Schroth, M. N., and J. G. Hancock. 1982. Disease-suppressive soil and root colonizing bacteria. Science 216:1376-1381. Truog, E. 1946. Soil reaction influence on availability of plant nutrients. Soil Sci. Soc. Am. Proc. 11:305-308. Weaver, R. W., and L. R. Frederick. 1972. A new technique for most- probable-number counts of rhizobia. Plant Soil 36:219-222. Weaver, R. W., L. R. Frederick, and L. C. Dumenil. 1972. Effect of soybean cropping and soil properties on number of Rhizobium japonicum in Iowa soils. Soil Sci. 114:137-141. 71 SUMMARY AND CONCLUSIONS The effects of P fertilization on mycorrhizal development and rhizobial nodule occupancy were evaluated in a 2-year field study. A follow-up greenhouse experiment evaluated rhizobial strain competition with and without mycorrhizal inoculation. Other researchers have shown a general synergistic relationship exists between the presence of mycor- rhizae and N^ fixation, especially on low P soils. However, no one has reported whether P nutrition and mycorrhizal infection of field-grown soybeans (Glycine max L.) affects rhizobial competition and nodule occupancy. The objective of these studies was to determine if the level of soil P, and its influence on mycorrhizal development, affected the competitive nature of the bacterial symbionts active in forming nodules. Four soils, ranging in Bray PI levels from 12 to 92 mg kg ^ and belonging to Aquic Hapludolls, Typic Argiudolls, and Typic Haplaquolls, _i were fertilized with triple superphosphate at 0 or 112 kg P ha Mycorrhizal infection, nodulation, and nodule-occupant serology were assessed at early flowering. Fertilization with P significantly reduced mycorrhizal infection, but had no effect on the numbers of nodules per plant or on seed yields. Unexpectedly, there was a significant decrease in nodule occupancy by Bradyrhizobium .japonicum serogroup 123, the domi nant serogroup found in Midwest soils. Over all locations, the occupancy of 123 in nonfertilized plots averaged 72% (similar to values reported by others), whereas the average percentage for the plots receiving the high rate of P was 37%. The significant reduction in 123 nodule occupany was 72 accompanied by a significant increase of unknown rhizobia that did not agglutinate with commonly reacting antisera of the Midwest (USDA 142, 135, 123, 122, 110, 76, 31, and 6). Greenhouse inoculation of soybean by Glomus mosseae in sterile growth medium did not result in a competitive reduction of USDA 123 when challenged with USDA 110. Microbiological interactions in Iowa soybean rhizospheres were in vestigated in field and greenhouse studies. Other researchers reported that bacterial isolates are capable of inhibiting B. japonicum on artifi cial media, but the present studies involved isolates that stimulated B. .japonicum serogroup 123. The question of whether these stimulating organisms could explain why serogroup 123 dominates Midwest soybean nodule occupancy was studied. Rhizosphere populations of total bacteria and actinomycetes on tryptic soy broth agar, gram-negative bacteria on crystal violet agar, enterobacteria on methylene blue agar, and pseudomonads on SI agar were measured in field-grown soybean rhizospheres six times over the season at approximately 3-week intervals. Rhizosphere soil samples were collected from Webster and Nicollet soils, Typic Haplaquolls and Aquic Hapludolls, respectively, and the microorganisms were enumerated. Climatic variables significantly influenced microbial populations but did not affect antagonistic or stimulatory organisms of USDA 123 as deter mined in plate culture by using an agar-overlay technique. The numbers of antagonistic and stimulatory organisms declined over the season, whereas the total population of bacteria and actinomycetes increased. Early in the season, 30% of the rhizosphere bacteria were stimulatory to 123 and 5% were inhibitory. 73 Nine of the stimulatory organisms were tested in the greenhouse in sterile soil for their ability to alter 123 nodule occupancy of soy beans when challenged by USDÂ 110. Two organisms enhanced 123 nodula- tion when added to sterile soil, but further tests indicated that the results were not reproducible in nonsterile soil. The effect of fluorescent pseudomonad populations on nodulation by B. .japonicum serogroup 135 was studied in calcareous and noncalcareous soils. Other researchers have attributed the dominance of 135 in high pH soils to availability of reduced Fe, but the results of more recent studies indicated that fluorescent pseudomonads produce siderophores (Fe chelators) in high pH soils. Two soils, Webster (pH 6.6) and Canisteo (pH 7.5), both Typic Haplaquolls, were inoculated under sterile and non- sterile conditions with B. japonicum USDA 123 and USDA 135; nodule occupancy was assessed serologically. Sterilization of the Canisteo soil significantly reduced pseudomonad populations, as was expected. However, sterilization had no affect on Fe availability, as measured by chemical extraction. Sterilization also resulted in a significant in crease of 80% in nodule occupancy of strain 123 when equal numbers of 123 and 135 were added. Plants from the nonsterile Canisteo soil had only 22.5% of the nodules occupied by 123. Nodule occupancy in the Webster soil was not affected by sterilization, and approximately 75% of the nodules from plants grown in the sterile and nonsterile soils were occupied by 123. A biological factor(s) was important in allowing B. .japonicum 135 to occupy nodules and dominate in a soil of high pH. 74 LITERATURE CITED Abel, G. H., and L. H. 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Weaver, R. W., and L. R. Frederick. 1974a. Effect of inoculum rate on competitive nodulation of Glycine max L. Merrill. I. Greenhouse studies. Agron. J. 66:229-232. Weaver, R. W., and L. R. Frederick. 1974b. Effect of inoculum rate on competitive nodulation of Glycine max L. Merrill. II. Field studies. Agron. J. 66:233-236. 79 Weaver, R. W., L. R. Frederick, and L. C. Dumenll. 1972. Effect of soybean cropping and soil properties on numbers of Rhizoblum .1 aponicum in Iowa soils. Soil Scl. 114:137-141. Weber, D. F., and V. L. Miller. 1972. Effect of soil temperature on Rhizoblum japonicum serogroup distribution in soybean nodules. Agron. J. 64:796-733. 80 ACKNOWLEDGMENTS The author wishes to acknowledge the advice and suggestions of Dr. Tom Loynachan, Dr. Irv Anderson, Dr. Paul Hartman, Dr. Richard Schultz, and Dr. Lois Hattery Tiffany in the completion of this study and preparation of this manuscript. Also acknowledged is the encourage ment and support offered by the author's wife, Carol. The author further wishes to acknowledge the financial assistance of the Iowa Agricultural Experiment Station, Allied Corporation, and Iowa State University during the course of this study. 81 APPENDIX A. EXPERIMENTAL DESIGN OF FAST-GROWING RHIZOBIUM NODULATION STUDY I. Five replications II. Two soybean cultivars A. Corsoy 79 B. Williams 82 III. Three fast-growing rhizobial isolates IV. Two inoculum rates A. Eight log cells ml ^ B. Five log cells ml ^ V. Three samples at 4-week intervals A. Four weeks B. Eight weeks C. Twelve weeks 82 APPENDIX B. RECOVERY OF FAST-GROWING RHIZOBIUM FROM SOYBEAN NODULES 83 Rhizobium Nodule Fast-growing Soybean Inoculant isolate number nod recovery cultivar rate Mi No. % ; 4 4 100.0 Williams 8 1 k 1 25.0 Williams 8 2 3 1 33.3 Williams 8 3 4 3 • 75.0 Williams 8 4 5 5 100.0 Williams 8 5 4 0 0.0 Williams 5 1 3 0 0.0 Williams 5 2 3 0 0.0 Williams 5 3 6 1 16.7 Williams 5 4 1 0 0.0 Williams 5 5 2 7 2 28.6 Williams 8 1 2 2 1 50.0 Williams 8 2 2 3 1 33.3 Williams 8 3 2 6 0 0.0 Williams 8 4 2 2 0 0.0 Williams 8 5 2 6 0 0.0 Williams 5 • 1 2 3 2 66.7 Williams 5 2 2 8 4 50.0 Williams 5 3 2 5 3 60.0 Williams 5 4 2 6 0 0.0 Williams 5 5 3 2 1 50.0 Williams 8 1 3 2 1 50.0 Williams 8 2 3 5 3 60.0 Williams 8 3 3 3 1 33.3 Williams 8 4 3 8 1 12.5 Williams 8 5 3 1 0 0.0 Williams 5 1 3 4 1 25.0 Williams 5 2 3 7 0 0.0 Williams 5 3 3 6 1 16.7 Williams 5 4 7 1 14.3 Williams 5 5 4 1 25.0 Corsoy 8 1 4 4 100.0 Corsoy 8 2 4 0 0.0 Corsoy 8 3 4 0 0.0 Corsoy 8 4 4 0 0.0 Corsoy 8 5 4 0 0.0 Corsoy 5 1 4 0 0.0 Corsoy 5 2 3 0 0.0 Corsoy 5 3 0 0 0.0 Corsoy 5 4 1 1 100.0 Corsoy 5 5 2 1 0 0.0 Corsoy 8 1 2 2 1 50.0 Corsoy 8 2 2 8 0 0.0 Corsoy 8 3 2 6 3 50.0 Corsoy 8 4 2 2 0 0.0 Corsoy 8 5 2 1 3 1 33.3 Corsoy 5 1 2 1 6 0 0.0 Corsoy 5 2 2 1 2 1 50.0 Corsoy 5 3 2 1 2 0 0.0 Corsoy 5 4 2 1 4 2 50.0 Corsoy 5 5 3 1 8 0 0.0 Corsoy 8 1 3 1 1 0 0.0 Corsoy 8 2 3 1 3 0 0.0 Corsoy 8 3 3 1 1 1 100.0 Corsoy 8 4 3 1 1 0 0.0 Corsoy 8 5 3 1 4 3 75,0 Corsoy 5 1 3 1 1 0 0.0 Corsoy 5 2 3 1 2 2 100.0 Corsoy 5 3 3 1 4 3 75.0 Corsoy 5 4 3 1 1 1 100.0 Corsoy 5 5 1 2 22 9 40.9 Williams 8 1 1 2 14 6 42.9 Williams 8 2 1 2 10 5 50.0 Williams 8 3 1 2 10 5 .50.0 Williams 8 4 1 2 6 3 50.0 Williams 8 5 1 2 18 5 27.8 Williams 5 1 1 2 4 0 0.0 Williams 5 2 1 2 10 6 60.0 Williams 5 3 1 2 18 6 33.3 Williams 5 4 1 2 13 6 46.2 Williams 5 5 2 2 10 4 40.0 Williams 8 1 2 2 12 6 50,0 Williams 8 2 2 2 10 5 50.0 Williams 8 3 2 2 10 5 50.0 Williams 8 4 2 2 3 2 66.7 Williams 8 5 2 2 3 2 66o7 Williams 5 1 2 2 17 6 35.3 Williams 5 2 2 2 16 7 43.8 Williams 5 3 2 2 16 9 56.3 Williams 5 4 2 2 16 7 43.8 Williams 5 5 3 2 8 4 50.0 Williams 8 1 3 2 14 6 42.9 Williams 8 2 3 2 10 5 50.0 Williams 8 3 3 2 12 6 50.0 Williams 8 4 3 2 9 2 22.2 Williams 8 5 3 2 5 4 80.0 Williams 5 1 3 2 10 4 40.0 Williams 5 2 3 2 6 3 50.0 Williams 5 3 3 2 10 4 40.0 Williams 5 4 3 2 2 2 100.0 Williams 5 5 1 2 3 0 0.0 Corsoy 8 1 1 2 11 3 27.3 Corsoy 8 2 1 2 10 7 70.0 Corsoy 8 3 1 2 2 0 0.0 Corsoy 8 4 2 13 8 61.5 Corsoy 8 5 85 1 2 9 3 33.3 Corsoy 5 1 1 2 9 0 0.0 Corsoy 5 2 1 2 3 0 0.0 Corsoy 5 3 2 6 0 0.0 Corsoy 5 4 1 2 14 6 42.9 Corsoy 5 5 2 2 10 2 20.0 Corsoy 8 1 2 2 11 10 90.9 Corsoy 8 2 2 2 1 1 100.0 Corsoy 8 3 2 2 6 2 33.3 Corsoy 8 4 2 2 1 0 0.0 Corsoy 8 5 2 2 20 5 20.0 Corsoy 5 1 2 2 1 0 0.0 Corsoy 5 2 2 2 3 2 66.7 Corsoy 5 3 2 2 3 1 33.3 Corsoy 5 4 2 2 15 8 53.3 Corsoy 5 5 3 2 15 10 66.7 Corsoy 8 1 3 2 1 1 100.0 Corsoy 8 2 3 2 10 6 60.0 Corsoy 8 3 3 2 1 0 0.0 Corsoy 8 4 3 2 8 4 50.0 Corsoy 8 5 3 2 15 5 33.3 Corsoy 5 1 3 2 1 0 0.0 Corsoy 5 2 3 2 9 3 33.3 Corsoy 5 3 3 2 15 7 46.7 Corsoy 5 4 3 2 16 5 31.3 Corsoy 5 5 3 20 7 35.0 Williams 8 1 3 12 8 66.7 Williams 8 2 3 6 3 50.0 Williams 8 3 3 10 6 60.0 Williams 8 4 3 16 12 75.0 Williams 8 5 3 12 9 75.0 Williams 5 1 3 1 0 0.0 Williams 5 2 2 16 4 25.0 Williams 5 3 2 10 2 20.0 Williams 5 4 2 10 2 20.0 Williams 5 5 n 3 12 9 75.0 Williams 8 1 1 3 20 7 35.0 Williams 8 2 2 3 16 9 56.3 Williams 8 3 2 3 16 10 62.5 Williams 8 4 2 3 20 10 50.0 Williams 8 5 2 3 7 5 71.4 Williams 5 1 2 3 21 10 47.6 Williams 5 2 2 3 1 0 0.0 Williams 5 3 2 3 16 5 31.3 Williams 5 4 2 3 16 7 43.8 Williams 5 5 3 3 16 7 43.8 Williams 8 1 3 3 16 8 50.0 Williams 8 2 3 3 16 6 37.5 Williams 8 3 3 3 16 5 31.3 Williams 8 4 5 3 3 16 7 43.8 Williams 8 86 3 K 6 42.9 Williams 5 1 3 18 9 50.0 Williams 5 2 3 16 7 43.8 Williams 5 3 3 14 6 42.9 Williams 5 4 3 14 7 50.0 Williams 5 5 3 16 3 18.8 Corsoy 8 1 3 16 4 25.0 Corsoy 8 2 3 16 4 25.0 Corsoy 8 3 3 8 2 25.0 Corsoy 8 4 3 24 10 41.7 Corsoy 8 5 3 16 4 25.0 Corsoy 5 1 3 16 4 25.0 Corsoy 5 2 3 16 6 37.5 Corsoy 5 3 3 16 5 31.3 Corsoy 5 4 3 16 4 25.0 Corsoy 5 5 2 3 16 3 18.8 Corsoy 8 1 2 3 16 4 25.0 Corsoy 8 2 2 3 16 6 37.5 Corsoy 8 3 2 3 16 5 31.3 Corsoy 8 4 2 3 16 5 31.3 Corsoy 8 5 1 2 3 16 6 37.5 Corsoy 5 2 2 3 8 3 37.5 Corsoy 5 2 3 16 3 18.8 Corsoy 5 3 5 4 2 3 24 5 20.8 Corsoy 5 2 3 16 4 25.0 Corsoy 5 3 3 16 6 37.5 Corsoy 8 1 3 3 12 4 33.3 Corsoy 8 2 3 3 20 4 20.0 Corsoy 8 3 3 3 16 4 25.0 Corsoy 8 4 3 3 16 3 18.8 Corsoy 8 5 3 3 4 1 25.0 Corsoy 5 1 3 3 28 12 42.9 Corsoy 5 2 3 3 16 12 75.0 Corsoy 5 3 3 3 16 7 43.8 Corsoy 5 4 3 3 1 0 0.0 Corsoy 5 5 87 APPENDIX C. EXPERIMENTAL DESIGN OF FAST-GROWING RHIZOBIUM Ng FIXATION STUDY I. Five replications II. Rhizobial strains A. Three fast-growing isolates B. One Bradyrhizobium japonicum 110 isolate III. One sterile control treatment IV. Sampled 3 weeks after inoculated 88 APPENDIX D. DINITROGEN FIXATION IN FAST-GROWING RHIZOBIUM ISOLATES AND BRADYRHIZOBIUM JAPONICUM USDA 110 ( 89 Replication Nodule Nodule Ethylene Rhizobial number dry weight produced isolate -1-1 hr g nodule • dry weight • 1 13 0.0033 22.7 2 22 0.0135 15.6 3 36 0.0197 5.3 U 71 0.0375 10.3 5 3 0.0011 0.0 1 43 0.0283 7.5 2 2 7 0.0033 45.5 2 3 14 0.0047 13.9 2 4 22 0.0091 0.0 2 5 14 0.0071 0.0 2 1 96 0.0561 12,1 3 2 32 0.0091 18.7 3: 3 29 0.0157 15.6 3 4 16 0.0034 10.3 3 5 35 0.0148 13.5 3 1 37 0.0200 16.3 110 2 17 0.0072 63.2 110 3 39 0.0167 28.1 110 A 43 0.0207 74.2 110 5 11 0.0052 74.5 110 I, 2, and 3 were fast-growing isolates, and 110 was B. .japonicum 110.