related to Purshia warrant closer study. Plummer (8) stated that organisms similar to thesymbiotic nodule-forming organism found on the roots of antelope ...
Plant Physiol. (1980) 65, 411-412 0032-0889/80/65/041 1/02/$00.50/0
Short Communication
Nodulation and Nitrogen Fixation in Cliffrose (Cowania mexicana var. stansburiana (Torr.) Jeps.)' Received for publication June 26, 1979 and in revised form September 10, 1979
TIMOTHY L. RIGHETTI AND DONALD N. MUNNS Department of Land, Air and Water Resources, University of California, Davis, California 95616 ABSTRACT Nodulation and acetylene reduction were demonstrated for cliffrose (Cowania mexicana var. stansburia ITorr.1 Jeps.) in a greenhouse trial. Nitrogen fixation was also verified with "N. Seedlings were grown in a soil known to nodulate both bitterbrush species (Psahia tridentata (Pursh) D.C. and P. glandulsa ICurranl) to which a suspension of crushed bitterbrush nodules had been added. Bitterbrush seedlings grew faster and reduced more acetylene than clffrose seedngs planted in the same pots, but the specific activity of the nodules and the nodule mass to root mass ratios were approximately the same.
Seeds came from three Southern California cliffrose populations. Five pregerminated seeds ofantelope bitterbrush were also planted in the sand layer of each pot at the same time. Cliffrose seedlings emerged approximately 12 days after bitterbrush seedlings. After 3 weeks each pot was thinned, leaving two plants of each genus. Pots were watered by weight to maintain a moisture content slightly below field capacity and were kept in the greenhouse under natural light. Fifteen weeks after planting, seedlings were separated from the soil and roots were gently washed. Acetylene-dependent ethylene production was measured. Plants were incubated for I h in 425-ml jars after which a 10-ml gas sample was taken as control. During the next hour the plants were exposed to a 10o carbide-generated acetylene atmosphere injected into the same container, and then another 10-ml gas sample was collected for nitrogen fixation estimation by the acetylene reduction assay. Gas samples were stored in 25-ml evacuated vials and ethylene was measured with a gas chromatograph. Shoot dry weights, root dry weights, nodule fresh weights, and nodule number were measured. Nodule mass to root mass ratios were estimated by using a moisture content of 90%o found in other cliffrose nodules. Nitrogen fixation in cliffrose nodules from comparable seedlings was also verified with "5N. For this experiment nodulated and unnodulated portions of roots were excised from plants and immediately transferred into incubation chambers which were evacuated to the vapor pressure of water. Chambers were refilled with an atmosphere that was 69%o Ar, 18% 02, 3% 15N2, and l1o0 '4N2. These samples were incubated 3 h and then subjected to Kjeldahl digestion and isotope analysis according to standard procedures (3).
Nitrogen-fixing root nodules are known to occur on rosaceous plants of the genera Dryas, Cercocarpus, Purshia, and perhaps Rubus (2). Species of other genera genetically capable of nitrogen fixation may have escaped detection because soil and environmental constraints or insufficient numbers of the appropriate endophyte reduce nodulation in the field. Some soils collected at Purshia sites fail to nodulate Purshia seedlings even under favorable greenhouse conditions (11), and in the field, drought may restrict nodulation on otherwise favorable soils (5). Genera closely related to Purshia warrant closer study. Plummer (8) stated that organisms similar to the symbiotic nodule-forming organism found on the roots of antelope bitterbrush (Purshia tridentata [Purshl D.C.) have been found on cliffrose (Cowania mexicana var. stansburiana [Torr.] Jeps.). McArthur et aL (7) also stated that nodules have been observed on cliffrose. There has been no RESULTS AND DISCUSSION confirmation, further description of the nodules, or evidence that they fix nitrogen. Cliffrose hybridizes with antelope bitterbrush Although there was considerable variation, all cliffrose seedlings and desert bitterbrush (Purshia glandulosa Curran) (1, 9, 10) and indistinintrogression occurs between the two genera (9, 10). This genetic were nodulated. Nodules were coralloid and visuallyrevealed a from those on bitterbrush. Light microscopy guishable exchange suggested that nitrogen fixation could occur in both genera, and that it might be possible to demonstrate nitrogen nodule and endophyte structure similar to that reported in bitterfixation in cliffrose through the use of a bitterbrush endophyte. brush nodules (6). Direct evidence for nitrogen fixation was apparent from the 0.286 atom excess 15N observed in "5N-incubated cliffrose nodules. No 15N enrichment was detectable in MATERIALS AND METHODS unnodulated portions of excised roots. Negligible ethylene was A surface soil sample in which seedlings of both Purshia species detected in the carbide-generated acetylene, and seedlings pronodulated was collected north of Bishop, California. Fifty ml of duced ethylene only in the presence of acetylene. Table I shows nodulation and acetylene reduction data for each an inoculum suspension containing 3.5 g crushed nodules in 500 ml distilled H20 was added to 1.5 kg soil in sealed plastic pots cliffrose seedling, means for the cliffrose seedlings, and means for before planting. For the inoculum, nodules were removed from the bitterbrush seedlings. Acetylene reduction rates were substangreenhouse seedlings of both Purshia species that had been grown tially smaller in cliffrose than in bitterbrush, but this may be due in five naturally nodulating soils. Ten unhulled cliffrose seeds per to cliffrose's slower growth or the slight age difference rather than pot were planted in a layer of sterile sand placed over the soil. a difference in nitrogen fixation capability. Relative efficiencies and nodule mass to root mass ratios were similar for both genera. The acetylene reduction rate of approximately 4 ,umol/g fresh nodule weight. h is similar to values reported for other greenhouse'This work was supported by an Earle C. Anthony Fellowship. 411
RIGHETTI AND MUNNS
412
Species
Plant Physiol. Vol. 65, 1980
Table I. Nodsdation and Rates of Acetylene Reduction for Cl4ffrose and Bitterbrush Seedlings Nodule Nodule Shoot Dry Root Dry Mass Nodule Seed Source Fresh Weight Weight Weight No.
Root Mass
Acetylenedependent Specific Ethylene Activity Production
p,mol/g mg
Cowania mexicana C. mexicana C. mexicana C. mexicana C. mexicana C. mexicana C. mexicana C. mexicana C. mexicana C. mexicana C. mexicana mean
mg
mg
ratio
Kingston Mtn' Kingston Mtn White Mtnb White Mtn White Mtn White Mtn White Mtn Clark Mtnc Clark Mtn Clark Mtn
nmol/plant * h fresh
260 150 60 0.040 32 383 100 17 70 0.024 4 38 70 70 30 0.042 7 99 90 80 28 0.035 17 115 50 40 32 0.080 19 115 70 70 28 0.040 14 76 90 80 22 0.028 12 107 110 90 71 0.078 16 214 40 30 10 0.033 11 31 130 100 40 0.040 20 270 100 78 34 0.044 15 145 Truckee 430** 410** 200** 0.048 Purshia tridentata meand 58** 870** a Collected 6 Aug 78, Excelsior Mine Road, one-quarter mile beyond recent mining activity. b Collected 8 Aug 78, White Mtn-Bristlecone access, one mile beyond Pinon Picnic Area. 'Provided by Rancho Santa Ana Botanic Garden, collected 2 Aug 77 northeast side of Clark Mtn San Bernardino County. d Means from 14 P. tridentata seedlings are marked with a double asterisk when significantly different from C. mexicana mean (P = 0.01). e Collected 10 Aug 77, Hwy 89 north of Truckee, California, at Prosser Dam Road.
grown antelope bitterbrush seedlings (5). The data indicate that cliffrose has a nitrogen-fixing potential. A field study verifying nodulation in some cliffrose populations has recently been completed (D. Nelson, personal communication). A better understanding ofsoil and environmental constraints is required to determine if nodulation regularly occurs and if it has any advantage or ecological impact in the marginal environment where cliffrose grows. Other arid adapted rosaceous shrubs may also have a nitrogen-fixing capability. The simplest and most promising technique for new discoveries is probably greenhouse trials in compatible soils with a broad spectrum crushed nodule inoculum from related and co-habitated species. A multiple-strain inoculum with endophytes isolated from a variety of sources is also feasible (4). Apache plume (Fallugia paradoxa [D. Don] Endl.) is a prime candidate. A small population of putative cliffrose-Apache plume hybrids has been reported in Arizona and artificial crossings have produced viable seedlings (1). Apache plume may fix nitrogen, but if it does not, a thorough study of chifrose-Apache plume hybrids would be interesting. Acknowledgments-We thank C. C. Delwiche for assistance with "N incubation and analysis, Lisa Morrison-Baird for assistance with microscopy, and M. A. Righetti for assistance with field work.
wt-h 6.4 2.2 3.3 4.1 3.6 2.7 4.9 3.0 3.1 6.8 4.0 4.4
LITERATURE CITED 1. BLAUER AC, AP PLUMMER, ED McARTHUR, R STEVENS, BC GUINTA 1975 Characteristics and hybridization of important intermountain shrubs. I. Rose family. USDA Forest Service Research Paper INT-169. 2. BOND G 1976 Root nodule symbioses with actinomycete-like organisms. In PS Nutman, ed, Symbiotic Nitrogen Fixation in Plants. Cambridge University Press, Cambridge, pp 443-474 3. BREMNER JM 1965 Isotope-ratio analysis of nitrogen in nitrogen-15 tracer investigations. In CA Black, ed, Methods of Soil Analysis, Part 2. American Society of Agronomy, Madison, Wis, pp 1256-1286 4. CALLAHAM D, PD TREDICI, JG ToRREY 1978 Isolation and cultivation in vitro of the actinomycete causing root nodulation in Comptonia Science 199: 899-902 5. DALTON DA, DB ZOBEL 1977 Ecological aspects of nitrogen fLxation by Purshia tridentata. Plant Soil 48: 57-80 6. KREBILL RG, JM MUIR 1974 Morphological characterization of Frankiapurshiae, the endophyte in root nodules of bitterbrush. Northwest Sci 48: 266-268
7. McARTHUR ED, BE GIUNTA, AP PLUMMER 1974 Shrubs for restoration of depleted ranges and disturbed areas. Utah Sci 35: 28-33 8. PLUMMER AP 1976 Revegetation of disturbed intermountain area sites. In JL Thomas, ed, Reclamation and Use of Disturbed Land in the Southwest. Univ Arizona Press, Tucson, pp 302-339 9. STEBBINs GL 1959 The role of hybridization in evolution. Am Phil Soc Proc 103: 231-251 10. STUTz HC, LK THOMAS 1964 Hybridization and introgression in Cowania and Purshia. Evolution 18: 183-195 11. WAGLE RF, J VLAMIS 1961 Nutrient deficiencies in two bitterbrush soils. Ecology 42: 745-752
