Fact Sheet: Commercialization of Sinorhizobium (Rhizobium) Meliloti, RMBPC-2
In September 1997, the Environmental Protection Agency (EPA) approved limited commercialization of the intergeneric microorganism Sinorhizobium meliloti (S. meliloti) strain RMBPC-2. The microorganism will be used as a microbial seed inoculant to coat alfalfa seeds prior to planting. The company that will be manufacturing and distributing the new seed inoculant is Research Seeds, Inc., which is located in St. Joseph, MO. The company will be allowed to manufacture up to a maximum production volume of 500,000 pounds of the microbial seed inoculant during any consecutive 12-month period. Prior to attaining that production volume, the company is required to notify the Agency so that EPA may assess the need for additional data on the microorganism.
RHIZOBIA AND NITROGEN FIXATION
Rhizobia are a group of bacteria, encompassing the generaRhizobium, Sinorhizobium and Bradyrhizobium, normally found in soil, which establish mutually beneficial (symbiotic) relationships with legumes. Rhizobia form growths called nodules (nodulation) on the roots of the legumes, and provide usable nitrogen to the plants. In return, the plants provide a carbon and energy source for the rhizobia.
Nitrogen is an essential nutrient for healthy plant growth. Although abundant in the air and in organic matter in the soil, plants are unable to use nitrogen in these forms. Conventional methods of providing nitrogen to plants in a usable form include adding nitrogen rich fertilizers to the soil, or inoculating seed (i.e., coating the seed) with bacteria able to perform a process called nitrogen fixation. Nitrogen fixation is the process of converting atmospheric nitrogen into an inorganic form that plants can use. Nitrogen "fixing" bacteria not only provide nitrogen to the plants they nodulate, but also leave behind excess nitrogen in the soil, potentially reducing the need for nitrogen fertilizers the next growing season. Rhizobia fix nitrogen for legumes, and S. meliloti is specific to the legumes alfalfa, sweet clover and fenugreek. Prior to 1994, S. meliloti was classified as Rhizobium meliloti (R. meliloti).
Rhizobia have been used commercially as seed inoculants in the form of seed coatings for over one hundred years. Currently, about 80% of alfalfa grown in the United States is inoculated with rhizobia prior to planting.
This new microorganism was extensively reviewed by EPA pursuant to its authority under the Toxic Substances Control Act (TSCA). Section 5 of TSCA requires that information about the health and environmental effects of new chemical substances (including new microorganisms) be reviewed by EPA before the substances may be used commercially in the United States. This review is conducted by EPA's Office of Pollution Prevention and Toxics. Information on new chemical substances or new microorganisms is submitted in the form of a premanufacture notice, or PMN. Each PMN for a new chemical substance or new microorganism receives a separate numerical designation; in the case of strain RMBPC-2, the PMN received the designation P92-403. Under TSCA, new microorganisms are those formed by combining genetic material from organisms in different genera (intergeneric). A genus (pl. genera) is a level in the taxonomic classification system used to group organisms based upon their similarity to other organisms.
EPA began evaluating various intergeneric strains of Sinorhizobium (Rhizobium) meliloti beginning in 1987. Research Seeds became involved in this research beginning in 1992, and submitted several PMNs for approval to conduct several small and large scale research field trials with various strains of these microorganisms, including strain RMBPC-2. These field trials included some strains developed by another company and previously evaluated by EPA. These field trials are subject to a Consent Order issued by EPA under section 5(e) of TSCA. The Consent Order, as amended, limits the use of the intergeneric strains of Rhizobium meliloti, including strain RMBPC-2, to specific sites only for research and development (R&D) purposes. The Consent Order went into effect on April 28, 1992, (the Order) and was subsequently modified in 1993 and 1994 to permit additional field trials at different sites.
On May 26, 1994, Research Seeds submitted a request to commercialize Rhizobium meliloti strain RMBPC-2. On January 4, 1995, a subcommittee of the Biotechnology Science Advisory Committee (BSAC) met to review the Agency's draft risk assessment. The BSAC is a panel of scientists from academia and other government agencies which functions as a peer review group for risk assessments of certain biotechnology products reviewed by EPA under TSCA. The BSAC submitted its report on March 6, 1995.
In September, 1997, EPA further amended the Consent Order to allow limited commercial use of S. meliloti strain RMBPC-2. This represents the first commercial use of an intergeneric microorganism in the environment under TSCA. The basis for the Agency's decision to allow the limited commercial use of this microorganismis summarized below.
GENETIC CHARACTERISTICS OF STRAIN RMBPC-2
The host microorganism used to produce RMBPC-2 was S. meliloti strain PC. Genes to enhance nitrogen fixation and nutrient utilization were added to the host strain. In addition, antibiotic resistance marker genes, which are used to isolate strain RMBPC-2 from other bacteria in the laboratory and in the environment, were also added to the host strain to create strain RMBPC-2. Based on evaluation of detailed information on the genetic modifications made to strain RMBPC-2, as summarized more fully below, EPA believes that these genetic changes are not expected to affect the microorganism's behavior in the environment.
SUMMARY OF THE RISK ASSESSMENT AND BSAC REVIEW
Human, Animal and Plant Health and Exposure
S. meliloti has been used as a seed inoculant for over one hundred years. There are no reports of pathogenic (disease-causing) effects on humans, animals, or plants associated with the use of these microorganisms. The genetic modifications made to strain RMBPC-2 are not expected to alter these characteristics in the microorganism.
Limited occupational exposure of workers to strain RMBPC-2 during manufacture and processing of the seed inoculant, as well as during application, is expected. However, there is little risk associated with such exposure due to the low hazard to human health posed by the microorganism.
Antibiotic resistance genes like those introduced into strain RMBPC-2 occur commonly in a wide array of naturally occurring microorganisms and are generally much more mobile than those introduced into strain RMBPC-2, due to the stability of the genes' location in this strain. Because of the stability of these genes in strain RMBPC-2, EPA does not believe that strain RMBPC-2 will contribute significantly to the naturally occurring antibiotic resistance gene pool.
As it will be used in commerce, strain RMBPC-2 will not be found in the same locations in the environment as other microorganisms that are human or animal pathogens. This further reduces the likelihood that such microorganisms might acquire antibiotic resistance characteristics from strain RMBPC-2.
The antibiotics to which strain RMBPC-2 is resistant have few uses in treatment of human or animal disease, and for the majority of these uses are not the drugs of first choice.
Effects on Alfalfa Yield
There were no adverse effects on alfalfa yield from use of strain RMBPC-2.
Field tests, lasting up to four years at some sites, have demonstrated that RMBPC-2 is able to significantly increase alfalfa yield under certain conditions (low nitrogen content in the soil and low indigenous rhizobial populations).
Overall, RMBPC-2 has been shown to perform within the normal range expected of naturally occurring commercial inoculants.
Nodulation of Plants Other Than Alfalfa
Rhizobial nodulation of legumes is species- and strain-specific. Each rhizobial strain is likely to nodulate only specific legumes (cross-inoculation group). S. meliloti is among the most restrictive in nodulation preference, normally nodulating only the legumes alfalfa, sweet clover and fenugreek. There are isolated reports in the literature, however, that S. meliloti may also be able to nodulate a few other leguminous plants such as mesquite.
Due to the potential for S. meliloti to inoculate legumes other than alfalfa, the BSAC Subcommittee considered whether to recommend additional testing of strain RMBPC-2 to determine its potential to inoculate legumes outside its cross-inoculation group. EPA believes that there is no hazard associated with the potential for inoculation of leguminous plants outside of the cross-inoculation group for S. meliloti, and consequently that such testing is not necessary prior to limited commercial use of strain RMBPC-2.
Survival, Dissemination and Nodulation in the Environment
The scientific advisory committee also considered the need for additional testing of the persistence, dissemination, competitiveness, and genetic stability of strain RMBPC-2. EPA believes that such additional testing is not necessary prior to limited commercial use of the product. The Agency's determination is based on data collected from field trials of S. meliloti strain RMBPC-2, as well as trials of similarly modified S. meliloti strains and non-modifiedrhizobia strains. These data support the conclusion that the behavior of strain RMBPC-2 is expected to be consistent with current commercially-available rhizobia strains.
Based on its assessment of available information, EPA believes that the initial commercialization of strain RMBPC-2 presents a low level of risk to health and the environment. In addition, strain RMBPC-2 has demonstrated a significant advantage over other commercial alfalfa seed inoculants in improving alfalfa yields under certain soil conditions. EPA acknowledges, however, that there are some uncertainties associated with the behavior of this microorganism in the environment, as noted above. Based on its intended use as a seed inoculant, the microorganism is expected to be produced insubstantial quantities and to be used in the environment in substantial quantities. EPA therefore believes that it is prudent at this time to limit commercial production of strain RMBPC-2, and to establish a subsequent opportunity for EPA to reexamine the product at a future date and to consider, in light of the information and understanding available at that time, whether additional action is needed to address questions about the behavior of strain RMBPC-2 in the environment.
For more information on this case, please contact the TSCA hotline at (202) 554-1404, TDD at (202) 554-0551. For more information on the TSCA Biotechnology Program, please visit the Program's homepage.