Fixation by Non-leguminousPlants

CharleneVan Raalte

ALL STUDENTS OF BIOLOGY learn about , ation in the non-legumes, I could find little information. Downloaded from http://online.ucpress.edu/abt/article-pdf/44/4/229/339852/4447478.pdf by guest on 03 October 2021 including such agriculturally essential as , My interest in these plants subsequently led me to several , and that form a with nitrogen- teams of biologists actively researching many aspects of fixing nodule . But most biology students the biology and ecology of the non-leguminous nitrogen never learn about another abundant, widespread, and fixers. These scientists have recently made some impor- perhaps equally important group of plants that have tant discoveries of theoretical as well as immediate prac- nitrogen-fixingroot nodules quite different from those of tical interest. Some of these findings will be described the legumes. This group of non-leguminous nitrogen- here. fixing plants includes trees and shrubs (Alnus sp.), bayberry and sweet gale ( sp.), and sweet-fern (Comptonia peregrina). These plants are rarely men- TABLE1. The BiologicalCharacteristics of NitrogenFixation tioned in basic biology or ecology texts, despite the fact The initialreaction N2 + 6H+-2NFL that their nitrogen-enrichingability makes them important FinalProducts components of their ecosystems and potentially very Aminoacids and proteins useful to farmers and foresters. OrganismsResponsible Onlybacteria (many types) The so-called nitrogen-fixing plants are of special in- EnzymeInvolved Nitrogenase(common to all N terest to me because I study vegetation tolerant of fixers) nutrient-poor . These plants have an advantage in Energetics Energyrequired to breakthe tripleN2 bond such soils since they associate with bacteriathat can con- vert or "fix" nitrogenous gas to ammonium (table 1). Importance Untilthe industrialfixation of N in the 1940s, mostof the N in the The harbors the bacteria in nodules that develop biospherehad been fixedby on its ; the bacteria obtain respiratory carbon sub- bacteria. strates from the plant, and the plant obtains nitrogen containing amino acids from the bacteria. Most of the nitrogen-fixingplants growing in the habitats I have been This story is about plants, bacteria, nitrogen, and even studying-sand , burned sites, and eroded soils sheep; perhaps more importantly,it is also a story about in the Northeast-are not legumes. But several years our ignorance until recent years of a basic biological ago when I attempted to learn more about nitrogen fix- phenomenon, recognition of that ignorance, and the rapid development of a new field in both basic and ap- Charlene Van Raalte (no photograph available) is an assistant professor plied research. of ecology at Hampshire College, Amherst, Massachusetts 01002. She has also taught at Dalhousie University and at the Marine Biological Non-leguminous Laboratory, Woods Hole, Massachusetts. She was awarded a B.A. Nitrogen-fixing degree with highest honors from Skidmore College and her Ph.D. Plants vs. Legumes degree from the Boston University Marine Program at the Marine Bio- logical Laboratory. She was the recipient of the Mellon Foundation Since the legumes are at least somewhat familiar to Faculty Development grant at Hampshire College in 1979 and 1980, most biologists, it is useful to compare the two types of among other fellowships and awards. Dr. Van Raalte is a member of the American Society of Limnologists and Oceanographers, American symbioses. Legumes have a worldwide distributionand, Women in Science, the New England Estuarine Research Society, and among the angiosperms, rank second or third in number AAAS. Her research has focused on in plants since of species contained. Most of these species are trees, 1975, and she is currently studying plant ecology of riverine marshes. She has published extensively in several publications, including ABT shrubs, or vines in inaccessible tropical habitats and are, [See ABT 39(5)]. therefore, little studied. -Vincent (1974) lists 12,880

NITROGEN FIXATION 229 species in the Leguminosae, of which only about 10% nodules or even in culture, look more like fungal fila- have been examined for presence of root nodules. Of ments than bacteria. Actinomycetes comprise a very course, the species which are cultivated for food, forage, common group of bacteria, members of which are pri- and pasture in temperate habitats have been much better marily responsible for decomposition of composts and studied. These plants include peas, beans, , soy- man ures. beans, and lupins. The total global importance of - fixed nitrogen, clearly great, can only be estimated. TABLE2. Plantsin NorthAmerica Known to FormRoot According to Delwiche (1970), about 15-35 x 106 metric Noduleswith Actinomycete Bacteria* tons of nitrogen are fixed annually by the world's le- gumes. Reliable estimates exist for annual fixation rates Genus Family EcologicalSites by legumes in pastures or agriculturalfields. , Alnus Poorsoils, glacial till, , fix about 20-200 for example, can kg/ha (18-180 lbs./ gravel acre) of nitrogen annually-about equivalent to the Sand dunes,salt marshes, amount of nitrogen a farmer would add as fertilizer. tropicalforests, deserts, In all legumes, the bacteria found in the root nodules (introducedto N. America) belong to one genus, . are gram- Dryforest, chaparral, sub- negative rods occurring singly or in pairs and usually alpine,disturbed sandy Downloaded from http://online.ucpress.edu/abt/article-pdf/44/4/229/339852/4447478.pdf by guest on 03 October 2021 motile when young. Inside the nodules these bacteria soils become deformed and are called "bacteroids."There are Comptonia Myricaceae Disturbedor gravellyareas many species of rhizobia;some only form the association Eleagnus Eleagnaceae Poorsoils with one species of legume, whereas others are more Myrica Myricaceae Acidicbogs, sand dunes, promiscuous in their affiliations.Rhizobium sp. were first mine wastes isolated in culture in the 1880s. They could not be shown Shepherdia Eleagnaceae Sandysoils, disturbed to fix N -nitrogen in culture and require media contain- sites ing either oxidized or reduced forms of nitrogen; there- * fore, microbiologists have long assumed that these bac- since Torrey (1978). teria could fix only in the presence of the host plant, requiring genetic input from the plant. However, Keister For many years, the could be easily seen in and Evans (1976) have recently demonstrated, under nodules of the non-leguminous fixers, but could not be stringent laboratory conditions, that these bacteria can fix isolated in pure culture in the laboratory.Since the micro- N2 and therefore must possess the so-called "nif" (nitro- biologists' main tool in the study of bacterialmorphology, gen-fixing) genes after all. metabolism, etc., is isolation and growth of the organism The process by which rhizobia invade the plant ex- in defined media, the lack of success with these actinomy- emplifies the complexities of such symbioses. The first cetes was very frustrating.Finally, three years ago Calla- discernible event-seen even with an ordinary stereo- ham, Del Tredici, and Torrey (1978) were able to isolate microscope-is "curling" of the host's root hairs. The the Frankia sp. from sweet-fern (Comptonia peregrina). bacteria enter the tip of the curled hair, become enclosed In addition to using careful techniques and the proper in a polysaccharide membrane or thread, and invade medium, these researchers were probably successful, the root cortical cells. Here they multiply and eventually in contrast to others, for one simple reason: they were fill the cells. Nodules result from the bacterial and root patient. Frankia sp. are extremely slow-growing organ- . Inside the nodules, gaseous N2 is reduced to isms. Whereas most bacteria will cloud a nutrient broth the ammonium ion; the reduced nitrogen is then incor- culture overnight, the actinomycete nitrogen-fixer from porated into amino acids such as asparagine and trans- sweet-ferns requires some months to come to sizeable ported through the plant in the xylem. biomass. Quite possibly, past researchers had isolated The non-leguminous nitrogen-fixing plants are similar the organism but had thrown it away after a week or two! to the legumes in some ways and in many other ways Several species of Frankia have now been isolated are very different. The non-legumes do not belong to a from Alnus and Eleagnus sp.; thus, their growth patterns single family; they are a diverse group of woody dicoty- in culture can be examined, their morphology can be lendous angiosperms comprised of at least 160 species, studied with light and electron microscopes, and the spanning fifteen genera and seven families (Torrey1978). process of plant invasion can be compared with that of These plants typically grow in sites with poor soils- the legumes. The last, for example, is best done by grow- burned areas, dry forests, sandy habitats, and bogs (table ing the plants from seed axenically, i.e., in the absence of 2). Like the legumes, all of the few genera so far studied bacterial or other contaminants. The axenic condition harbor in their root nodules bacteria belonging to a single is important because all events occurring are then known genus. These bacteria are not in the genus Rhizobium, but to be caused by either the host plant or the single bac- rather in the genus Frankia, one of the actinomycetes. terium, and by no other agent. A few drops of the iso- These are long, filamentous bacteria which, inside the lated bacterial culture are dripped onto axenic young

230 THE AMERICAN BIOLOGY TEACHER, VOLUME 44, NO.4, APRIL 1982 seedlings, and the roots examined daily. As in legumes, poplar trees (Populus tremuloides) in plots with black the bacteria in the actinomycete-nodulated plants stim- alder (Alnus glutinosa). We hope in several years to see ulate curling; they enter via the root hairs, increased poplar biomass owing to the fertilizing effect eventually fill the host's cortical cells, and induce nodules of the . These trees could be used for pulp or wood to form. In the few cases studied so far, it has been shown chips for heat production. that nitrogen is transported from the nodules to the host Alders are being used at the New England Farm Center system in the form of amino acids, as is characteristic in a unique and imaginative way: as a -enriching for- of the legumes. Despite physiological similarities,the root age for sheep (figs. 2 and 3). Researchers at the nodules induced by the actinomycetes are morphologi- Center are determining how large-scale sheep farming cally distinctfrom legume nodules. There are two general might be reintroduced to New England where there were types: the A1nus-type in which knobby, coralloid struc- three million sheep in the mid-1880s. There are several tures are formed (fig. 1) and the Myrica-type which look aspects to their research; see, for example, an article on like small clusters of upward-growingroots. sheep-guarding dogs by Coppinger and Coppinger Another important difference between the Rhizobium (1980). fixers and the Frankia fixers is the soil they and their host The image of sheep feeding on alder trees appears at plants inhabit. Legumes have exacting requirements. first glance to be an odd one, but there are several rea- For example, alfalfa requires a rich soil of pH above 5.8, sons why this idea may work. Most importantly,sheep do Downloaded from http://online.ucpress.edu/abt/article-pdf/44/4/229/339852/4447478.pdf by guest on 03 October 2021 careful management with regular lime additions, and eat alders, especially when the leaves are tender, and frequent mowing. Another legume used for hay, silage, these leaves have a high leaf protein content, comparable or pasture is , which also requires good, well- in amount to that of alfalfa. In addition, ruminants such as drained soils and fertilizerapplications. As seen in table 2, sheep can be pastured on marginal lands where the non-legume fixers will grow in a variety of habitats,in- such as corn do not develop well. Alders will grow on cluding forests and disturbed sites. Because of this and these marginal soils; they do so with little maintenance, because they can contribute substantialamounts of nitro- and they even enrich the nutrient-poor soil with nitro- gen to these habitats, these plants appear to play an im- gen. Finally, alders are perennials. Once planted, they portant role in habitats where legumes will have difficulty continue production for many years (unlike alfalfa, for growing. example, which must be replanted every two to three years). Uses for the Actinomycete-nodulated Plants Two of the primary uses to date of actinomycete- nodulated plants are in forestry and agriculture. For example, in the Pacific Northwest, red alder (Alnus rubra) has been planted with black cottonwood (Populus tri- chocarpa) for fast production of wood or "biomass." 7~~~~~~~~ Red alder has also been planted as a nurse crop for Douglas fir (Pseudotsuga menziesii). Similar plantings are underway in the Northeast. At the New England Farm Center of Hampshire College in Amherst, Massachusetts, and at the Cabot Foundation for Botanical Research, Harvard Forest, in Petersham, Massachusetts, Dr. John Torrey and I recently planted

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FIGURE1. The so-calledAlnus type of nodule. This is a Ceanathus FIGURE 2 At the New England Farm Center, Hampshire College, a americanus (New Jersey tea) root. sheep feeds on a small alder tree.

NITROGEN FIXATION 231 The steps leading to an alder pasture are many, and earlier. In fact, as scientists began to consider the fertiliz- improvements doubtless will be made at each phase as ing or forage possibilities of these plants, they learned this work continues. The emphasis is on development of that the idea is not a new one. In the Orient, alders have a planting regime that farmers can use: rapid and easy long been planted on farm land as a means of restoring planting of hundreds of alders that are healthy and well fertility, and it is reported that farmers in Nova Scotia nodulated. At this point, we collect the alder seeds in the use alders as forage. (Red alder is the most utilized hard- field. (There are likely to be genetic differences in seed wood in the Pacific northwest, but it is used primarily stock, but genetic selection must await a future project.) for lumber, wood chips, and furniture,and not yet for its Germinated seedlings are inoculated with the isolated fertilizingabilities per se.) But certainly in contrast to the actinomycetes; as more of the Frankia sp. are isolated- legumes, the importance of the actinomycete-nodulat!d and this emphasizes the importance of such isolations- angiosperms as fixers has been underestimated. In a the effectiveness of different strains in inducing nodula- fairly recent review of nitrogen fixation, Delwiche (1970) tion can be determined. The seedlings are grown in pot- claimed that legumes were the most significant fixers ting soil in the greenhouse. For fast planting, they are worldwide. Only ten years later, fixation by the non- grown in commercially available, easily opened, plastic legumes is generally assumed to be at least equal in im- containers. Once the seedlings are about 15-30 cm tall, portance to that of the legumes on a global basis. they are planted in a field in a small hole, cut with a soil Once a new field has been established, it is interesting Downloaded from http://online.ucpress.edu/abt/article-pdf/44/4/229/339852/4447478.pdf by guest on 03 October 2021 auger. The planting design should offer substantialforage to piece together the steps that led to that establishment. to the sheep, yet allow them to walk easily among the In the present case, the International Biological Pro- shrubs. An alder pasture could perhaps also be planted gramme (IBP) played an important role. In this approx- with an additional forage such as timothy, but that re- imately ten-year effort in the 1960s and 1970s scientists quires future study. Similar planting techniques for man- from many nations joined forces to study areas (whole aged forests are under development in other parts of the ecosystems, for instance) in a coordinated way. Nitrogen- United States, and the results of these efforts will provide fixation studies received considerable attention, and the an interesting comparison to our work at Hampshire first extensive surveys of the non-legumes were ac- College. complished. This entailed a great deal of difficult field work, since the nodules of actinomycete-infected plants The Development of a New Field are attached to extensive root systems. Such surveys were conducted on the diverse groupings of plants Readers may be wondering why the nitrogen fertiliz- shown in table 2 before a general picture could emerge. ing potential of plants such as alders was not recognized (Continued on p. 254)

FIGURE 3. Alder (Alnus glutinosa or Euro0 pean black alder) grown in a field before and after sheep grazed the field. The alders were a preferredfood item.

232.THE AMERICAN BIOLOGY TEACHER, VOLUME44 NO 4 APRIL1982

232 THE AMERICAN BIOLOGY TEACHER, VOLUME 44, NO. 4, APRIL 1982 Nitrogen Fixation ing system, the types and uses of the tree crop in the forest, the length of . . . from p. 232 time before harvest, and the location of the forest. Unfortunately, there is still little information In addition to the IBP, economic and ecological consid- available on nitrogen fixation that can be used by forest erations have stimulated research on the potential bene- managers. Various research projects are now under way fits of actinomycete-nodulated plants. They are already in different forest habitats;perhaps in a few years we will being used in strip-mined areas cleared of soil and vege- be better able to evaluate the opportunities for nitrogen- tation which must be re-vegetated under recent environ- fixing plants in these cases. mental laws. Three of these nitrogen-fixing species- Acknowledgment-The author thanks John Torrey for helpful autumn-olive and russian-olive (Eleagnus sp.) and black discussions and also Arthur Westing and Cliff Goodband for alder-are now used extensively in such reclamation reading the manuscript. programs. Economic considerations come into play in the increas- References ing numbers of our nation's managed forests, where CALLAHAM, D., DEL TREDICI,P., and TORREY, J.G. 1978. manipulation of the forests' nitrogen supply is becoming Isolation and cultivation in vitro of the actinomycete causing very important. But the opportunities for the non-legumes root nodulation in Comptonia. Science 199:899. in these forests is not as clear as in strip-minedsites. Some COPPINGER, L., and COPPINGER, R.P. 1980. So firm a Downloaded from http://online.ucpress.edu/abt/article-pdf/44/4/229/339852/4447478.pdf by guest on 03 October 2021 foresters feel that these plants are definitely useful; others friendship. Natural History 89:12. DELWICHE, C.C. 1970. The nitrogen cycle. Scientific Ameri- are much less certain. They disagree because the primary car 223:136. benefit of nitrogen-fixing plants-addition of nitrogen to KEISTER,D.L., and EVANS, W.R. 1976. Oxygen requirement the soil-can also be achieved by adding nitrogen as for acetylene reduction by pure culture of rhizobia. Journal fertilizer. Cost/benefit analyses must be compared for of Bacteriology 129:149. both fertilizerand fixing systems, but these analyses are TORREY, J.G. 1978. Nitrogen fixation by actinomycete-nodu- A lated angiosperms. BioScience 28:586. very complicated. forester would have to consider VINCENT, J.M. 1974. Root-nodule symbioses with Rhizobium. factors such as the cost of fertilizernow and in the future, In Quispel, A. (ed.), The biology of nitrogen fixation. New the cost of establishing and managing the nitrogen-fix- York: American Elsevier Publishing Company, Inc.

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254 THE AMERICAN BIOLOGY TEACHER, VOLUME 44, NO. 4, APRIL 1982