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Role of the Nodulation Restrictive Allele Rj4, in Soybean Evolution
f.PlantPhysiol. Vol. 132.pp. 453-455(1988)
Role of the Nodulation Restrictive Allele Rj4 in Soybean Evolution)1T. E.
DEVINE
N. F. & S. G. Lab., USDA/ARS, BARC-W, Beltsville, MD 20705 USA Received September 10, 1987 . Accepted October 1, 1987
Summary The symbiosis of legumes with rhizobia has been subject to the selection pressures of 200 million years of evolution. Evidence from soybean of ecogeographic distribution of nodulation compatibilities with specific Rhizobium strains indicates that the coevolution of host and microsymbiont has played a role in the evolution of compatibilities. Some strains of bradyrhizobia that nodulate soybean also produce a substance called rhizobitoxine that interferes with chlorophyll synthesis. The Rj4 allele in soybean conditions an ineffective nodulation response with specific strains of bradyrhizobia while the rj4 allele permits normal nodulation with these strains. Two near isogenic soybean lines (99.95 % genetically identical) differing specifically in the Rj4 vs. rj4 alleles have been synthesized and used to test an array of chlorosis-inducing and non-chlorosis-inducing bradyrhizobia for response with the Rj4 allele. The Rj4 allele was found to interdict nodulation with 33.3 % of the chlorosis inducing rhizobia and only 2.7 % of the nonchlorosis inducing rhizobia. Thus, the Rj4 allele may have a positive value to the host plant in areas with significant populations of chlorosis-inducing rhizobia by protecting the plant from nodulation with some of these rhizobia. The frequency of ineffective nodulation with the chlorosis-inducing strain USDA61 shows a progressive loss with domestication from the wild species Glycine soja (63 %) to the Asiatic plant introductions of G. max (26%), and with selection for agronomic types in North America as demonstrated by the preliminary test lines (13.7 %) and the more highly selected uniform test lines (8.5 %). This may be an example of the loss of rhizobial specificity in a plant species occurring in historic time.
Key words: Bradyrhizobium japonicum, Glycine max (L.) Merr., Glycine soja Sieb & Zucc., coevolution, symbiosis.
The symbiosis between the legume host plant and the rhizobial microsymbiont is thought to have evolved approximately 200 million years ago (Pohill et al., 1981). Clearly these phylogenetically divergent organisms have a complex and highly integrated interrelationship. In such a complex relationship there are numerous points at which the symbiotic process can fail. Genotypes exhibiting these failures provide important tools for genetic analysis.
* Paper presented at a workshop in AmaIfi (April 1987), supported by the «OECD Co-operative Research Project on Food Production and Preservation». © 1988 by Gustav Fischer Verlag, Stuttgart
In soybeans, several genes controlling nodulation response and segregating according to Mendelian inheritance have been identified. The rjl allele is a recessive gene that in homozygous mode produces the nodulation-restrictive phenotype referred to as «non-nodulating» (Williams and Lynch, 1954). The rjl allele produces a powerful and pervasive restriction of nodulation with a broad spectrum of Bradyrhizobium japonicum. Only a single nodule per 1,000 or 1,500 plants was recovered from rjl plants in field soil containing a heterogeneous population of B. japonicum (Devine, 1984 a). Several other genes have been reported in which the dominant allele produces an ineffective nodulation response. Plants with an ineffective nodulation response have rudimentary nodules or cortical proliferation rather than normal
454
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nodules. The Rh allele, discovered in the cultivar «Hardee», produces an ineffective nodulation response with Bradyrhizobium strains USDA7, USDA 14, and USDA122 (Caldwell, 1966). The cultivar «Hardee» nodulates normally with other strains of B. japonicum such as USDA110, USDA142, etc. B. japonicum strains USDA7, USDA14 and USDA122 induce normal nodule development on soybean cultivars not carrying the Rh allele. In tests with 22 strains of the c1 (or 324-44) serogroup and 5 strains of the 122 serogroup, «Hardee» also displayed an ineffective nodulation response (Caldwell et aI., 1966). By extrapolation from the results with strains USDA7, and USDA14 in serogroup c1 and with strain 122, the ineffective nodulation response of the Bradyrhizobium strains in the c1 and 122 serogroups was ascribed to the Rj2 allele (Caldwell, 1966). The possibility that «Hardee» may have carried other genes that conditioned the ineffective nodulation response with some of the B. japonicum strains in these serogroups was not tested. Later, the cultivar «Hardee» was reported to carry the Rh allele that conditioned an ineffective nodulation response with B. japonicum strain USDA33 (Vest, 1970). The cultivar «Hill» was reported to carry the Rj4 allele that conditioned an ineffective nodulation response with B. japonicum strain USDA61 (Vest and Caldwell, 1972). A dominant allele conditioning ineffective nodulation with the fast-growing strain USDA205 of Rhizobium fredii, was found segregating in the F2 and F3 progeny of the cross «Peking» x «Kent» (Devine, 1984 b). «Kent» carried the dominant allele conditioning the ineffective response and «Peking» carried the recessive allele permitting nodulation. Since a great number of distinctive plant genotype/Rhizobium genotype interactions are possible, it would be possible to collect a whole menagerie of genes involved in host/Rhizobium interactions. However, it has seemed more important to understand the role of the nodulation response genes now available, than to accumulate a collection of genes conditioning aberrations. The question may be posed: Why do we encounter genes conditioning the failure of normal nodulation? The first explanation that comes to mind is that these genes are «inborn metabolic errors» resulting from mutation. However, most mutations are recessive. Although the rjl allele is a recessive allele and fits the pattern expected for spontaneous mutation (absence of the trait in the parental lines and a single isolated occurrence), the Rh, Rh, Rj4 alleles and the new as yet undesignated allele conditioning ineffective nodulation with the fast-growing strain USDA205 are all dominant alleles. There is, therefore, an incongruity between the dominant nature of these alleles and a simple mutation hypotheses. An alternative hypothesis may be constructed from a consideration of the evolution and domestication of soybean in Asia and its subsequent introduction as a crop in North America. It is reasonable to assume that if in Asia a soybean genotype was planted in a soil environment that lacked a population of rhizobia which was genetically compatible with the host genotype, that nodulation failure would occur and the plant would experience nitrogen deficiency symptoms. In an environment of limited soil nitrogen, such a plant genotype would not be suited to survival under natural selection or quasi-natural selection during domestication. There would
be a selection pressure for the evolution of mutually compatible populations of host plant and rhizobia. However, when these are introduced to North America or tested in the artificial environment of a «Leonard jar», it is possible, for example, that a host genotype derived from a germplasm base from south or central China may be forced into an incompatible association with a rhizobial strain derived from North China or Japan. These entities were not subject to selection pressure for mutual compatibility and this may be expressed as a nodulation failure without any restriction on the dominant or recessive nature of the genetic control system. This would appear a reasonable hypothesis; however, experience teaches that although a rational hypothesis may be most appealing to deductive reasoning, it may be entirely erroneous. How then can such a hypothesis be tested empirically? We elected to examine a sample of soybean plant introductions from several East Asian countries to determine their nodulation response with the rhizobial strains that define for the presence of the Rh and Rj4 alleles. We reasoned that if mutation alone was responsible for the ineffective nodulation responses, then the allelic frequency would be expected to be low and would be randomly distributed, since mutation is a random process. Conversely, if coevolution were the operative process responsible for the ineffective nodulation responses, then the allelic frequency of the incompatible response need not be low, and there should be a geographic pattern to the allelic frequencies rather than a random distribution. The results of screening over 847 plant introductions showed that the Rj4 ineffective nodulation response occurred in 29 % of the plant introductions and was most frequent in the population from southeast Asia, i.e., Burma, Malaya, Indonesia, Thailand and Vietnam (all over 60 %) and lower in frequency in northern Asia, i.e., 0 % in U.S.S.R. (Devine and Breithaupt, 1981). The Rh ineffective response occurred in only 2 % of the 847 plant introductions tested, but showed a center of allelic frequency in the vicinity of the Chinese cities of Nanking and Hangchow (31 % of the lines). In the case of the ineffective nodulation response with the fast-growing strain of Rhizobium fredii, USDA205, higher frequencies (over 80%) of nodulation-compatible lines were found in southeast Asia, i.e., Thailand, Malaysia, and Indonesia, than in northern Asia, i.e., China 38 %, U.S.S.R. 50 % (Devine, 1985 b). The evidence, then, for ecogeographic variation for specificity of nodulation response in the case of the Rj2 and Rj4 responses and the strain USDA205 response is compatible with the concept that coevolution has played a role in the development of nodulation compatibility. Some strains of slow-growing rhizobia that nodulate soybean also produce a chemical termed rhizobitoxine Gohnson et al., 1959 and Owens et al., 1972). Rhizobitoxine interferes with chlorophyll synthesis in young soybean leaves producing a distinctive foliar chlorosis. This foliar chlorosis was reported as having occurred in producers' fields in the states of Alabama, Arkansas, California, Florida, Georgia, Mississippi, North and South Carolina, and Tennessee in the United States (Erdman et aI., 1957). Chlorosis-inducing strains of rhizobia nodulate cowpea and fix nitrogen in symbiosis with cowpea in such a manner as to produce very vigorous growth but do not produce chlorosis
Nodulation-Restrictive Soybean Evolution symptoms on cowpea. These bradyrhizobia would appear to have a physiology that is more compatible with cowpea than with soybean. Hollis et ai. (1981) have used DNA homology to characterize a number of the slow-growing rhizobia that nodulate soybean. They found two major subgroups and suggested that these two groups should receive separate taxonomic classification. Our research (Devine, 1985 a) has shown that none of the strains in group I produce chlorosis symptoms, but most of the strains in group II produce chlorosis. Apparently, then, the rhizobia producing chlorosis symptoms on soybean belong to a distinct DNA homology group. A ten-year program at Beltsville, Maryland has produced, by back-crossing and self-pollination, a set of lines isogenic except for the Rj4 vs rj4 alleles (Devine and O'Neill, 1986 b). These lines are calculated to be 99.95 % identical in their nuclear DNA and completely identical in their cytoplasmic DNA. With these lines as biological tools, it is possible to address the question whether other strains of bradyrhizobia in addition to USDA61 specifically elicit the Rj4 ineffective nodulation response. Since these isolines essentially differ only in the allele present at the Rj4 locus, any difference between the lines in nodulation response with individual rhizobial strains can be attributed specifically to the Rj4 allele. To date, five strains in addition to strain USDA61 have been identified that elicite the Rj4 interdiction of the nodulation response. These six strains are not restricted to a single serogroup (Devine et aI., 1986); rather strains USDA61 and USDA83 are in serogroup 31, strain USDA62 is in serogroup 62, and strain USDA94 is in serogroup 94. The serogroups for strains USDA238 and USDA259 are not known. Other strains in serogroups 31, 62 and 94 do not elicit the Rj4 incompatibility response. Therefore, the Rj4 allele is not serogroup-specific. Interestingly, of the six strains of bradyrhizobia that have been identified to date as eliciting the Rj4 incompatibility response, five strains produce rhizobitoxine-induced chlorosis (Devine and O'Neill, 1986 a). Of the fifteen chlorosis-inducing strains tested, five (33 %) elicit the Rj4 interdiction. Of the thirty-five strains that do not induce chlorosis, only one (2.7 %) elicits the Rj4 interdiction. This suggests that the Rj4 allele, rather than being merely an abberation from normal nodulation, may in fact have a positive selective value to the host plant in acting as a shield to protect the plant from nodulation by some of the chlorosis-inducing rhizobia. Such a hypothesis, however, presents an enigma. The frequency of incompatibility with Bradyrhizobium strain USDA61 shows a progressive decrease with domestication (Devine, 1987) from the wild progenitor of soybeans Glycine soja (63 %) to the comparable Asiatic plant introductions of soybean (26 %) to the North American lines bred for industrial agriculture e.g. the preliminary test lines (13.7 %) and the even more highly selected uniform test lines (8.5 %). This would suggest that there has not been a positive selection pressure to retain the Rj4 allele during domestication and subsequent agronomic selection in North America. The reduction that has occurred in the frequency of Rj4 could be attributed to random drift, to genetic linkage with traits selected against during domestication and agronomic improvement (such as seed shattering, viney growth habit, small seed,
455
etc.) or to a negative selection value for the Rj4 allele per se that is not currently recognized. However this may be, it should be recognized that the dominant allele Rj4 for incompatible nodulation with several Bradyrhizobium strains is the most common allelic form in the wild species, G. soja. The recessive allele rj4, permitting nodulation with these Bradyrhizobium strains, may be a mutant form derived from the ancestral allele Rj4. With the development of the domesticated species G. max and subsequent selection for agronomic types in North America, we may be witnessing an example of the loss of rhizobial specificity in a plant species. References CALDWELL, B. E.: Inheritance of a strain-specific ineffective nodulation in soybeans. Crop Sci. 6, 427 -428 (1966). CALDWELL, B. E., KUELL HINSON, and H. W. JOHNSON: A strain-specific ineffective nodulation reaction in the soybean Glycine max L. Merr., Crop Sci. 6, 495-497 (1966). DEVINE, T. E.: Genetics and breeding of nitrogen fixation. In: ALEXANDER, M. (ed.), Biological Nitrogen Fixation, Plenum Publishing Co., New York, N.Y. (1984 a). - Host range and compatibility of soybean with rhizobial microsymbionts. p. 484-492. In: Proc. World Soybean Research Conference III, Ames, IA. (1985 a). - Inheritance of soybean nodulation response with a fast-growing strain of Rhizobium. J. Hered. 75, 359-361 (1984 b). - Nodulation of soybean (Glycine max L. Merr.) plant introduction lines with the fast-growing rhizobial strain USDA205. Crop Sci. 25, 354 - 356 ( 1985 b). - A comparison of rhizobiaI strain compatibilities of Glycinemaxand its progenitor species Glycine soja. Crop Sci. 27, 635-639 (1987). DEVINE, T. E. and B. H. BREITHAUPT: Frequencies of nodulation response alleles, Rjz and Rj4, in soybean plant introduction and breeding lines. USDA Tech. Bull., No. 1628 (1981). DEVINE, T. E. and J. J. O'NEILL: Host genetic systems for control of rhizobial specificity. Agron. Abst. 61-62 (1986 a). - - Registration of BARC-2 (Rj4) and BARC-3 (rj4) soybean germplasm. Crop Sci. 26, 1263-1264 (1986 b). DEVINE, T. E., J. J. O'NEILL, and L. D. KUYKENDALL: Characteristics of soybean nodulation incompatibility gene Rj4. Abstracts NEBASA p. 14 (1986). ERDMAN, L. W., H. W. JOHNSON, and F. CLARK: Varietal responses of soybeans to a bacterial-induced chlorosis. Agron. J. 49, 267 - 271 (1957). HOLLIS, A. B., W. E. KLOOS, and G. H. ELKAN: DNA: DNA hybridization studies of Rhizobium japonicum and related Rhizobiaceae. J. Gen. Microbiol. 123, 215-222 (1981). JOHNSON, H. W., U. M. MEANS, and F. E. CLARK: Responses of seedlings to extracts of soybean nodules bearing selected strains of Rhizobium japonicum. Nature 183, 308 -309 (1959). OWENS, L. D., J. F. THOMPSON, R. G. PITCHER, and T. WILLIAMS: Structure of rhizobitoxine, an antimetabolic enol-ether amino acid from Rhizobium japonicum. J. Chern. Soc. Chern. Commun. p. 714 (1972). POHILL, R. M., P. H. RAVEN, and C. H. STIRTON: Evolution and systematics of the Leguminosae. In: Advances in Legume Systematics, Part 1. POHILL, R. M. and P. H. RAAVEN (eds.). Royal Botanic Gardens, Kew, London, pp. 1-26 (1981). VEST, G.: Rh-a gene conditioning ineffective nodulation in soybean Crop Sci. 10,34-35 (1970). VEST, G. and B. E. CALDWELL: Rj4-a gene conditioning ineffective nodulation in soybean. Crop. Sci. 12, 692-694 (1972). WILLIAMS, L. F. and D. L. LYNCH: Inheritance of a non-nodulating character in the soybean. Agron. J. 46, 28-29 (1954).
Role of the Nodulation Restrictive Allele Rj4 in Soybean Evolution)1T. E.
DEVINE
N. F. & S. G. Lab., USDA/ARS, BARC-W, Beltsville, MD 20705 USA Received September 10, 1987 . Accepted October 1, 1987
Summary The symbiosis of legumes with rhizobia has been subject to the selection pressures of 200 million years of evolution. Evidence from soybean of ecogeographic distribution of nodulation compatibilities with specific Rhizobium strains indicates that the coevolution of host and microsymbiont has played a role in the evolution of compatibilities. Some strains of bradyrhizobia that nodulate soybean also produce a substance called rhizobitoxine that interferes with chlorophyll synthesis. The Rj4 allele in soybean conditions an ineffective nodulation response with specific strains of bradyrhizobia while the rj4 allele permits normal nodulation with these strains. Two near isogenic soybean lines (99.95 % genetically identical) differing specifically in the Rj4 vs. rj4 alleles have been synthesized and used to test an array of chlorosis-inducing and non-chlorosis-inducing bradyrhizobia for response with the Rj4 allele. The Rj4 allele was found to interdict nodulation with 33.3 % of the chlorosis inducing rhizobia and only 2.7 % of the nonchlorosis inducing rhizobia. Thus, the Rj4 allele may have a positive value to the host plant in areas with significant populations of chlorosis-inducing rhizobia by protecting the plant from nodulation with some of these rhizobia. The frequency of ineffective nodulation with the chlorosis-inducing strain USDA61 shows a progressive loss with domestication from the wild species Glycine soja (63 %) to the Asiatic plant introductions of G. max (26%), and with selection for agronomic types in North America as demonstrated by the preliminary test lines (13.7 %) and the more highly selected uniform test lines (8.5 %). This may be an example of the loss of rhizobial specificity in a plant species occurring in historic time.
Key words: Bradyrhizobium japonicum, Glycine max (L.) Merr., Glycine soja Sieb & Zucc., coevolution, symbiosis.
The symbiosis between the legume host plant and the rhizobial microsymbiont is thought to have evolved approximately 200 million years ago (Pohill et al., 1981). Clearly these phylogenetically divergent organisms have a complex and highly integrated interrelationship. In such a complex relationship there are numerous points at which the symbiotic process can fail. Genotypes exhibiting these failures provide important tools for genetic analysis.
* Paper presented at a workshop in AmaIfi (April 1987), supported by the «OECD Co-operative Research Project on Food Production and Preservation». © 1988 by Gustav Fischer Verlag, Stuttgart
In soybeans, several genes controlling nodulation response and segregating according to Mendelian inheritance have been identified. The rjl allele is a recessive gene that in homozygous mode produces the nodulation-restrictive phenotype referred to as «non-nodulating» (Williams and Lynch, 1954). The rjl allele produces a powerful and pervasive restriction of nodulation with a broad spectrum of Bradyrhizobium japonicum. Only a single nodule per 1,000 or 1,500 plants was recovered from rjl plants in field soil containing a heterogeneous population of B. japonicum (Devine, 1984 a). Several other genes have been reported in which the dominant allele produces an ineffective nodulation response. Plants with an ineffective nodulation response have rudimentary nodules or cortical proliferation rather than normal
454
T. E.
DEVINE
nodules. The Rh allele, discovered in the cultivar «Hardee», produces an ineffective nodulation response with Bradyrhizobium strains USDA7, USDA 14, and USDA122 (Caldwell, 1966). The cultivar «Hardee» nodulates normally with other strains of B. japonicum such as USDA110, USDA142, etc. B. japonicum strains USDA7, USDA14 and USDA122 induce normal nodule development on soybean cultivars not carrying the Rh allele. In tests with 22 strains of the c1 (or 324-44) serogroup and 5 strains of the 122 serogroup, «Hardee» also displayed an ineffective nodulation response (Caldwell et aI., 1966). By extrapolation from the results with strains USDA7, and USDA14 in serogroup c1 and with strain 122, the ineffective nodulation response of the Bradyrhizobium strains in the c1 and 122 serogroups was ascribed to the Rj2 allele (Caldwell, 1966). The possibility that «Hardee» may have carried other genes that conditioned the ineffective nodulation response with some of the B. japonicum strains in these serogroups was not tested. Later, the cultivar «Hardee» was reported to carry the Rh allele that conditioned an ineffective nodulation response with B. japonicum strain USDA33 (Vest, 1970). The cultivar «Hill» was reported to carry the Rj4 allele that conditioned an ineffective nodulation response with B. japonicum strain USDA61 (Vest and Caldwell, 1972). A dominant allele conditioning ineffective nodulation with the fast-growing strain USDA205 of Rhizobium fredii, was found segregating in the F2 and F3 progeny of the cross «Peking» x «Kent» (Devine, 1984 b). «Kent» carried the dominant allele conditioning the ineffective response and «Peking» carried the recessive allele permitting nodulation. Since a great number of distinctive plant genotype/Rhizobium genotype interactions are possible, it would be possible to collect a whole menagerie of genes involved in host/Rhizobium interactions. However, it has seemed more important to understand the role of the nodulation response genes now available, than to accumulate a collection of genes conditioning aberrations. The question may be posed: Why do we encounter genes conditioning the failure of normal nodulation? The first explanation that comes to mind is that these genes are «inborn metabolic errors» resulting from mutation. However, most mutations are recessive. Although the rjl allele is a recessive allele and fits the pattern expected for spontaneous mutation (absence of the trait in the parental lines and a single isolated occurrence), the Rh, Rh, Rj4 alleles and the new as yet undesignated allele conditioning ineffective nodulation with the fast-growing strain USDA205 are all dominant alleles. There is, therefore, an incongruity between the dominant nature of these alleles and a simple mutation hypotheses. An alternative hypothesis may be constructed from a consideration of the evolution and domestication of soybean in Asia and its subsequent introduction as a crop in North America. It is reasonable to assume that if in Asia a soybean genotype was planted in a soil environment that lacked a population of rhizobia which was genetically compatible with the host genotype, that nodulation failure would occur and the plant would experience nitrogen deficiency symptoms. In an environment of limited soil nitrogen, such a plant genotype would not be suited to survival under natural selection or quasi-natural selection during domestication. There would
be a selection pressure for the evolution of mutually compatible populations of host plant and rhizobia. However, when these are introduced to North America or tested in the artificial environment of a «Leonard jar», it is possible, for example, that a host genotype derived from a germplasm base from south or central China may be forced into an incompatible association with a rhizobial strain derived from North China or Japan. These entities were not subject to selection pressure for mutual compatibility and this may be expressed as a nodulation failure without any restriction on the dominant or recessive nature of the genetic control system. This would appear a reasonable hypothesis; however, experience teaches that although a rational hypothesis may be most appealing to deductive reasoning, it may be entirely erroneous. How then can such a hypothesis be tested empirically? We elected to examine a sample of soybean plant introductions from several East Asian countries to determine their nodulation response with the rhizobial strains that define for the presence of the Rh and Rj4 alleles. We reasoned that if mutation alone was responsible for the ineffective nodulation responses, then the allelic frequency would be expected to be low and would be randomly distributed, since mutation is a random process. Conversely, if coevolution were the operative process responsible for the ineffective nodulation responses, then the allelic frequency of the incompatible response need not be low, and there should be a geographic pattern to the allelic frequencies rather than a random distribution. The results of screening over 847 plant introductions showed that the Rj4 ineffective nodulation response occurred in 29 % of the plant introductions and was most frequent in the population from southeast Asia, i.e., Burma, Malaya, Indonesia, Thailand and Vietnam (all over 60 %) and lower in frequency in northern Asia, i.e., 0 % in U.S.S.R. (Devine and Breithaupt, 1981). The Rh ineffective response occurred in only 2 % of the 847 plant introductions tested, but showed a center of allelic frequency in the vicinity of the Chinese cities of Nanking and Hangchow (31 % of the lines). In the case of the ineffective nodulation response with the fast-growing strain of Rhizobium fredii, USDA205, higher frequencies (over 80%) of nodulation-compatible lines were found in southeast Asia, i.e., Thailand, Malaysia, and Indonesia, than in northern Asia, i.e., China 38 %, U.S.S.R. 50 % (Devine, 1985 b). The evidence, then, for ecogeographic variation for specificity of nodulation response in the case of the Rj2 and Rj4 responses and the strain USDA205 response is compatible with the concept that coevolution has played a role in the development of nodulation compatibility. Some strains of slow-growing rhizobia that nodulate soybean also produce a chemical termed rhizobitoxine Gohnson et al., 1959 and Owens et al., 1972). Rhizobitoxine interferes with chlorophyll synthesis in young soybean leaves producing a distinctive foliar chlorosis. This foliar chlorosis was reported as having occurred in producers' fields in the states of Alabama, Arkansas, California, Florida, Georgia, Mississippi, North and South Carolina, and Tennessee in the United States (Erdman et aI., 1957). Chlorosis-inducing strains of rhizobia nodulate cowpea and fix nitrogen in symbiosis with cowpea in such a manner as to produce very vigorous growth but do not produce chlorosis
Nodulation-Restrictive Soybean Evolution symptoms on cowpea. These bradyrhizobia would appear to have a physiology that is more compatible with cowpea than with soybean. Hollis et ai. (1981) have used DNA homology to characterize a number of the slow-growing rhizobia that nodulate soybean. They found two major subgroups and suggested that these two groups should receive separate taxonomic classification. Our research (Devine, 1985 a) has shown that none of the strains in group I produce chlorosis symptoms, but most of the strains in group II produce chlorosis. Apparently, then, the rhizobia producing chlorosis symptoms on soybean belong to a distinct DNA homology group. A ten-year program at Beltsville, Maryland has produced, by back-crossing and self-pollination, a set of lines isogenic except for the Rj4 vs rj4 alleles (Devine and O'Neill, 1986 b). These lines are calculated to be 99.95 % identical in their nuclear DNA and completely identical in their cytoplasmic DNA. With these lines as biological tools, it is possible to address the question whether other strains of bradyrhizobia in addition to USDA61 specifically elicit the Rj4 ineffective nodulation response. Since these isolines essentially differ only in the allele present at the Rj4 locus, any difference between the lines in nodulation response with individual rhizobial strains can be attributed specifically to the Rj4 allele. To date, five strains in addition to strain USDA61 have been identified that elicite the Rj4 interdiction of the nodulation response. These six strains are not restricted to a single serogroup (Devine et aI., 1986); rather strains USDA61 and USDA83 are in serogroup 31, strain USDA62 is in serogroup 62, and strain USDA94 is in serogroup 94. The serogroups for strains USDA238 and USDA259 are not known. Other strains in serogroups 31, 62 and 94 do not elicit the Rj4 incompatibility response. Therefore, the Rj4 allele is not serogroup-specific. Interestingly, of the six strains of bradyrhizobia that have been identified to date as eliciting the Rj4 incompatibility response, five strains produce rhizobitoxine-induced chlorosis (Devine and O'Neill, 1986 a). Of the fifteen chlorosis-inducing strains tested, five (33 %) elicit the Rj4 interdiction. Of the thirty-five strains that do not induce chlorosis, only one (2.7 %) elicits the Rj4 interdiction. This suggests that the Rj4 allele, rather than being merely an abberation from normal nodulation, may in fact have a positive selective value to the host plant in acting as a shield to protect the plant from nodulation by some of the chlorosis-inducing rhizobia. Such a hypothesis, however, presents an enigma. The frequency of incompatibility with Bradyrhizobium strain USDA61 shows a progressive decrease with domestication (Devine, 1987) from the wild progenitor of soybeans Glycine soja (63 %) to the comparable Asiatic plant introductions of soybean (26 %) to the North American lines bred for industrial agriculture e.g. the preliminary test lines (13.7 %) and the even more highly selected uniform test lines (8.5 %). This would suggest that there has not been a positive selection pressure to retain the Rj4 allele during domestication and subsequent agronomic selection in North America. The reduction that has occurred in the frequency of Rj4 could be attributed to random drift, to genetic linkage with traits selected against during domestication and agronomic improvement (such as seed shattering, viney growth habit, small seed,
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etc.) or to a negative selection value for the Rj4 allele per se that is not currently recognized. However this may be, it should be recognized that the dominant allele Rj4 for incompatible nodulation with several Bradyrhizobium strains is the most common allelic form in the wild species, G. soja. The recessive allele rj4, permitting nodulation with these Bradyrhizobium strains, may be a mutant form derived from the ancestral allele Rj4. With the development of the domesticated species G. max and subsequent selection for agronomic types in North America, we may be witnessing an example of the loss of rhizobial specificity in a plant species. References CALDWELL, B. E.: Inheritance of a strain-specific ineffective nodulation in soybeans. Crop Sci. 6, 427 -428 (1966). CALDWELL, B. E., KUELL HINSON, and H. W. JOHNSON: A strain-specific ineffective nodulation reaction in the soybean Glycine max L. Merr., Crop Sci. 6, 495-497 (1966). DEVINE, T. E.: Genetics and breeding of nitrogen fixation. In: ALEXANDER, M. (ed.), Biological Nitrogen Fixation, Plenum Publishing Co., New York, N.Y. (1984 a). - Host range and compatibility of soybean with rhizobial microsymbionts. p. 484-492. In: Proc. World Soybean Research Conference III, Ames, IA. (1985 a). - Inheritance of soybean nodulation response with a fast-growing strain of Rhizobium. J. Hered. 75, 359-361 (1984 b). - Nodulation of soybean (Glycine max L. Merr.) plant introduction lines with the fast-growing rhizobial strain USDA205. Crop Sci. 25, 354 - 356 ( 1985 b). - A comparison of rhizobiaI strain compatibilities of Glycinemaxand its progenitor species Glycine soja. Crop Sci. 27, 635-639 (1987). DEVINE, T. E. and B. H. BREITHAUPT: Frequencies of nodulation response alleles, Rjz and Rj4, in soybean plant introduction and breeding lines. USDA Tech. Bull., No. 1628 (1981). DEVINE, T. E. and J. J. O'NEILL: Host genetic systems for control of rhizobial specificity. Agron. Abst. 61-62 (1986 a). - - Registration of BARC-2 (Rj4) and BARC-3 (rj4) soybean germplasm. Crop Sci. 26, 1263-1264 (1986 b). DEVINE, T. E., J. J. O'NEILL, and L. D. KUYKENDALL: Characteristics of soybean nodulation incompatibility gene Rj4. Abstracts NEBASA p. 14 (1986). ERDMAN, L. W., H. W. JOHNSON, and F. CLARK: Varietal responses of soybeans to a bacterial-induced chlorosis. Agron. J. 49, 267 - 271 (1957). HOLLIS, A. B., W. E. KLOOS, and G. H. ELKAN: DNA: DNA hybridization studies of Rhizobium japonicum and related Rhizobiaceae. J. Gen. Microbiol. 123, 215-222 (1981). JOHNSON, H. W., U. M. MEANS, and F. E. CLARK: Responses of seedlings to extracts of soybean nodules bearing selected strains of Rhizobium japonicum. Nature 183, 308 -309 (1959). OWENS, L. D., J. F. THOMPSON, R. G. PITCHER, and T. WILLIAMS: Structure of rhizobitoxine, an antimetabolic enol-ether amino acid from Rhizobium japonicum. J. Chern. Soc. Chern. Commun. p. 714 (1972). POHILL, R. M., P. H. RAVEN, and C. H. STIRTON: Evolution and systematics of the Leguminosae. In: Advances in Legume Systematics, Part 1. POHILL, R. M. and P. H. RAAVEN (eds.). Royal Botanic Gardens, Kew, London, pp. 1-26 (1981). VEST, G.: Rh-a gene conditioning ineffective nodulation in soybean Crop Sci. 10,34-35 (1970). VEST, G. and B. E. CALDWELL: Rj4-a gene conditioning ineffective nodulation in soybean. Crop. Sci. 12, 692-694 (1972). WILLIAMS, L. F. and D. L. LYNCH: Inheritance of a non-nodulating character in the soybean. Agron. J. 46, 28-29 (1954).