i^T'*. D i v i s i o Agricultural Sciences l\ \; UNIVERSITY OF CALIFORN "0 c I < •) L 14* A * * hili HI^BHBHIHHI^H HHHH^I HHBll CALIFORNIA AGRICULTURAL EXPERIMENT STATION BULLETIN 842 Xhe great importance of legumes in agriculture is that they add nitrogen to the soil and thereby save the costs of nitrogen fertilizer, both for the legume crop and for the associated nonlegume crop. Leg- umes obtain nitrogen from the air, which contains uncombined nitro- gen—nearly 80 per cent—along with oxygen and other gases. Very few plants can utilize atmospheric nitrogen in its free form. However, if root-nodule bacteria of an effective strain have formed nodules on the roots of a legume, atmospheric nitrogen may be fixed within the nodules and converted to a utilizable compound. The successful establishment of legumes, particularly in a pasture mix for grazing, depends on effective nodulation. This can be obtained by inoculating the seed with an appropriate strain of root-nodule bac- teria. Methods of inoculating seed and measures that help to avoid inoculation failure are given in boxes on the following pages. All of the recommendations given here are of critical importance. There is no economical way to inoculate a field after planting. Faulty inoculation usually results in failure or partial failure of the legume stand. From this cause alone, California growers waste many thousands of dollars' worth of range-legume seed every year and waste also the labor and the fertilizer used. The Authors A. A. Holland was Lecturer in Agronomy and Assistant Research Agronomist in the Experiment Station, Davis, at the time this work was done. He is now at Monash Uni- versity, Clayton, Victoria, Australia. Specialist, Agricultural Extension Service, Davis. J. E. Street is Range Improvement W. A. Williams is Professor of Agronomy and Agronomist in the Experiment Station, Davis. AUGUST 1969 An abstract appears on page 19 RANGE-LEGUME INOCULATION AND NITROGEN FIXATION BY ROOT-NODULE BACTERIA INTRODUCTION nitrogen is an essential constituent of the fixed in a legume-grass pasture in North amino acids and proteins and is the key America. element in the structure of all living cells. Under California dryland conditions, Legumes are specialized plants because of effectively nodulated legumes are able to their faculty for fixing atmospheric nitro- fix significant amounts of nitrogen, though gen into compounds that can be utilized in the amounts do not approach Russell's plant metabolism. Actually, the legumes estimate. A reasonable average for a good alone cannot accomplish this. The process stand of range legumes might be at least takes place only in nitrogen-fixing nodules, 52 pounds per acre during one growing which are formed on legume roots in sym- season—equivalent to 250 pounds per acre biotic association with root-nodule bac- of ammonium sulfate. The cost of the teria of the genus Rhizobium. Both mem- inoculant is about 15 cents an acre. The bers of the symbiosis have their particular following tabulation gives data from Cali- functions, and each step of the process in- fornia. The amounts of atmospheric nitro- volves a close interaction between them. gen fixed in the clover roots were esti- In this association both members derive mated by comparing the yields from well- advantages. The rhizobia live within the nodulated, thrifty legume stands with root tissues, where they are protected those from unthrifty stands in the same from competitors and receive nutrients, area. The amount of nitrogen in purple such as carbohydrates. The legumes receive vetch was compared with that in barley, amino acids, which they build into pro- a nonlegume. teins. However, neither the legumes nor LEGUME, COUNTY, NITROGEN GAIN the rhizobia have any nutrient advantage AND REFERENCE LB./ACRE without the symbiosis. Together they can Subterranean clover, fix surprisingly large quantities of atmo- Hum- spheric nitrogen. boldt (Williams, Lenz, and Murphy, 1954) 45 Donald (1960) estimated that the world's annual income of biologically fixed nitro- Rose clover, Stanislaus gen, mainly from symbiotic sources, was of (Williams, Love, and the order of 100 million tons. Russell Berry, 1957) 50 (1950) estimated that well-nodulated leg- Purple vetch, Santa Barbara umes could fix up to 500 pounds of nitro- (Williams, Ririe, et al., gen per acre per year—the equivalent of 1954) 53 2,352 pounds per acre of ammonium sul- Rose clover, Placer fate. Erdman (1959) gave 106 pounds per (Martin, Williams, and acre as the average amount of nitrogen Johnson, 1957) 60 1 Submitted for publication September 29, 1964. Cover photo: Kondinin rose clover plants 105 days old. Vigorous plant at left grown from pellet-inoculated seed; small plant at right from uninoculated seed, planted at the same time and only a few feet away. Yolo County, March 1, 1967. 3] . ' ROOT NODULES AND NITROGEN FIXATION Nodule development Brockwell, and Zwar, 1960), which causes curling of certain root hairs—a prelude to on a legume root the invasion of these root hairs by the rhi- The first stage in the symbiosis between zobia. This process occurs only at certain rhizobia and legumes is the multiplication specialized infection sites — foci — on a of rod-shaped rhizobia in the soil in the young root (Nutman, 1948 and 1949; Dart region of a legume root. The multipli- and Pate, 1959). The infection foci remain cation is initiated and stimulated by sub- available for only a limited time. As the stances secreted by the root and exuded root elongates, the new infection foci are from it. One of these exudates is trypto- progressively more distant from the seed. phan (Rovira, 1959). The rhizobia convert Many strains of rhizobia are able to tryptophan to indoleacetic acid (Kefford, multiply in the rhizosphere of any legume SEED INOCULATION 1 Select a good commercial inoculant, pre- Guard against drying, both before and after pared from a strain of root-nodule bacteria planting. Always remember that drying kills ' that is specific for the legume to be planted. these bacteria quickly. Make sure the legume species is named on the 7. Plant as soon as possible. Reinoculate label; no other inoculant will do. Mixtures seed if it has been held longer that 24 hours of bacterial strains suitable for a great num- after inoculating. The cost of inoculant is ber of different species and varieties of leg- small in comparison with the costs of seed, ., umes are not satisfactory. fertilizer, soil preparation, and planting. Pel- 2. Check the freshness of the inoculant by let the seed (see next paragraph) if planting the expiration date stamped on the con- must be delayed. tainer. The inoculant is a living culture of 8. Pellet the inoculant on the seed to pro- root-nodule bacteria, which are killed by dry- tect the bacteria from drying. Directions for ing and also by high temperatures. Make sure pelleting are in the box on page 6. Always use this culture has been stored continuously un- pellet-inoculated seed if it must be sown in a ^ der refrigeration. Poor storage conditions can dry seedbed, or broadcast without a soil cover, cause nodulation failure by reducing the or sown from the air. Even with pelleting the * number of viable bacteria in the inoculant. protection is temporary, and the inoculant 3. Use the inoculant before the expiration becomes valueless in three weeks under dry i date. Do not carry it over to another year. The conditions. 1 number of bacteria that survive will be too 9. Always use pellet-inoculated seed where' low for effective nodulation. native legumes are abundant. This includes 4. Use the inoculant at the rate recom- many areas of California. Pellet with four mended by the manufacturer and stamped on times the amount of inoculant recommended ^ the container, or else at a higher rate—up to by the manufacturer. four times this amount (see paragraph 9, be- The root-nodule bacteria already in the . low). Never use a lower amount. soil in such areas often act as parasites on in- 5. Mix inoculant with seed thoroughly. troduced legumes. These bacteria are ineffec- Follow closely the printed directions on the tive nitrogen fixers with introduced legumes, container for the wet-mix method, but do but they do invade the young roots in great not use excess moisture, Make sure the inocu- numbers and thus prevent adequate nodu- lant sticks to all the seeds. lation of a new legume by bacteria of the ef- re- 6. Hold inoculated seed in a cool, shady fective strain supplied in the inoculant. It 4 place, and plant it in a moist seedbed. quires a large amount of inoculant to compete [4 — and can initiate the preliminary stages of secrete polygalacturonase (Fahraeus and root-hair curling described above. How- Ljunggren, 1959). The function of poly- ever, because of the specific relationship galacturonase has not been fully deter- between a host legume and a compatible mined. It may act with indoleacetic acid strain of rhizobia, the bacteria that cause to increase the plasticity of the cellulose curling of root hairs may belong to a strain wall of a root hair and enable it to invagi- that cannot nodulate the available legume. nate (fold inward). The root hair responds If so, these rhizobia do not enter the root to the presence of invasive rhizobia by hairs. folding inward at some point near its tip The following reactions proceed only if to form a very fine tube, the infection the strain of Rhizobium is invasive, i.e., thread, which extends inside the root hair capable of stimulating nodule formation (Nutman, 1963). in a legume root. Rhizobia of an invasive Concurrently with the formation of an strain produce an extracellular polysaccha- infection thread, a specialized tetraploid ride slime, which induces the legume to cell forms in the adjacent root-cortex AND FIELD PROBLEMS with a large population of ineffective bacteria.