Plant and Soil 118, 89-96, 1989. 9 KluwerAcademicPublishers. Printed in the Netherlands.

PLSO FA-12

Dinitrogen fixation in a water-stressed Alnus clone is limited by host xerotolerance

T. C. HENNESSEY 1'3, H. S. VISHNIAC 2, E. M. LORENZP and J. C. WILLIAMS2 1Department of Forestry and "Department of Botany and Microbiology, Oklahoma State University, Stillwater, OK 74078, USA. 3Address for correspondence

Key words:

actinorhizae, Alnus, Frankia, nitrogen-fixation, xerotolerance

Abstract
The osmotolerance, rather than the halotolerance, of the endosymbiont predicted the xerotolerance of acetylene reduction by Alnus nodulated with Frankia ARgP5 Ac. Cloned plants of Alnus glutinosa (L.) Gaertn. AG8022-16 were subjected to water stress under controlled conditions in an environmental growth chamber. Transpiration, stomatal conductance, and leaf water potential had decreased after successive l0 day periods of moderate (75% of water demand) and severe (50% of water demand) water stress. After severe stress had wilted the plants, reducing leaf water potential to -2.10MPa, nitrogenase activity had fallen to 2.51 #M per plant per hour. The reported rapid turnover of nitrogenase implies that Frankia mycelium was metabolically active at this low water potential, a water potential at which no Alnus-derived Frankia has been reported active. Although ARgP5 AGwas similar to other such strains in halotolerance (lower limit ca. -1.25 MPa), the low water potential limit for growth with glucose (a non-assimilated osmoticum) was ca. -2.53 MPa. Nitrogenase activity was apparently more limited by host xerotolerance than by endophyte xerotolerance.

Introduction
Investigations of the effects of water stress on actinorhizal dinitrogen fixation in the field (Dalton and Zobel, 1977; McNabb et al., 1979) and in the laboratory (Seiler and Johnson, 1984; Sundstr6m and Huss-Danell, 1987) have indicated that nitrogenase activity (acetylene reduction) is rapidly and markedly reduced at critical water potentials specific to the symbiotic partnerships. The xerotolerance of the endophyte has typically been determined as halotolerance. The in vitro halotolerance of Frankia isolates from Allocasuarina, Casuarina, and Purshia tridentata was reportedly greater than that of isolates from hosts not normally growing under sodic conditions (Dawson and Gibson, 1987; Shipton and Burggraaf, 1982), sugJournal articleJ-5400 of the OklahomaAgricultureExperiment Station, Oklahoma State University, Stillwater, OK 74078, USA. 89

gesfing that host and endophyte had been co-selected for xerotolerance. Halotolerance may be important in the spread of nodulation within and between host plants and in the survival of freeliving Frankia in saline soils. It is certainly important in utilization of actinorhizal plants, since salinity is a major problem in many arid or irrigated soils. Sodium chloride is, however, neither a neutral osmoticum nor one likely to be a major contributor to lowering the internal water potential of actinorhizal plants. In the course of investigating the effect of nodulation with Frankia strains of varying osmotolerance on growth and dinitrogen fixation of Alnus clones of varying xerotolerance, we have found that nodulated Alnus glutinosa will fix small but significant amounts of dinitrogen at internal water potentials lower than was suggested by the halotolerance of its Frankia endosymbiont, but within the range of water potentials allowing Frankia growth when glucose was used as osmoticum.

90

Hennessey et al. van der Crones solution (Dawson and Gordon, 1979) as needed. Approximately seven weeks after inoculation, nine nodulated plants were selected and transferred to a Percival model PGW132 controlled environment growth chamber for acclimatization. Previous experience had indicated that when the root systems of plants with dark green foliage were excavated the roots would prove to be well nodulated. During acclimatization and the subsequent experimental periods, ambient air temperature was 25 ___ 2~ for the 16h photoperiod and night temperature was 15 + 2~ Relative humidity during the light period, measured with a hygrothermograph, varied from 62 to 70%. Photosynthetic photon flux density at pot level averaged 760#molm-2s ~ as measured with a Licor model 190S quantum sensor. One week prior to the start of the experiment, individual pots were enclosed in plastic bags loosely secured around the base of the stem with twist-ties to prevent evaporation from the soil surface. During this preparatory phase, the daily water usage was quantified for each plant by weighing the potted plants prior to, and following, watering. The average daily volume of water used by each plant over a three day period determined the initial wellwatered treatment level.

Although no osmoticum yet employed is demonstrably neutral, this suggests that the xerotolerance of nodulated Alnus glutinosa was limited primarily by the physiology of the host rather than that of the endophyte.

Materials and methods

Plant materials Alnus glutinosa (L.) Gaertn. clone AG8022-16 was used as the host. Although Alnus is not generally regarded as drought resistant, both inter- and intraspecific variation in water-stress tolerances have been shown for Alnus (Hennessey et al., 1985; Hennessey et al., 1988). Clone AG8022-16 fell within the range of xerotolerance exhibited by 5 _previously examined clones of A. glutinosa (Hennessey et al., 1988). These 5 clones showed wide variation in withstanding moderate stress, although it was not possible to rank them consistently. Alnus maritima, however exhibited notably greater resistance to moderate stress than A. glutinosa or A. serrulata (Hennessey et al., 1985). Stock plants of clone AG8022-16 were grown in a greenhouse in a 2:1 (v/v) mixture of Jiffy Mix TM (a 1:1 mixture of shredded sphagnum peat moss and horticultural-grade vermiculite) and commercially available Oil Dry TM (Moltan Safety Absorbant, ground clay). Plants were fertilized weekly and watered with tap water as necessary. Replicated clones were produced by taking 15 cm apical softwood cuttings from the stock plants. The bases of the cuttings were dipped for 10 seconds in a solution containing 8000 ppm indol-3-butyric acid (IBA) in 2% ethanol, dusted with 5% benomyl, and placed in a large heated perlite-vermiculite propagation bed under intermittent mist. Approximately 6 weeks were required for the establishment of roots, after which misting was reduced. Unnodulated, rooted cuttings were selected for top and root uniformity, and were inoculated with Frankia strain ARgP5 A~ by pipetting 0.5 mL (ca. 50 #g protein) of homogenized mycelium (see below) onto the bare roots of each cutting. Inoculated clones were then transplanted into 2 L pots containing a sterilized 2:1 mixture of Jiffy Mix and Oil Dry. Potted plants were watered with N-free

Experimental conditions and measurements After the period of acclimatization, the plants were subjected to a series of water regimes applied for three consecutive 10-day periods. For the first 10-day period, each plant received 100% of the previously calculated daily addition needed to keep the soil near field capacity. For days 10-20 of the experiment, each plant was moderately stressed by the application of 75% of the initial well-watered value, and for days 20-30, each plant was severely stressed by applying only 50% of the initial wellwatered amount. Leaf stomatal conductance (gs) and transpiration (E,) were measured with a Licor model LI1600 steady state porometer, applied at the side of the midrib near the tip of the fourth fully expanded leaf on the main stem. (The fourth fully expanded leaf was, of course, a different leaf at each sam-

N2 fixation and xerotolerance in Alnus

91

pling.) Transpiration was calculated internally by the porometer. Leaf water potential (psi) was measured using Merrill model 76-11 leaf cutter psychrometers connected to a Wescor model PR-55 microvoltmeter (see Brown and Van Haveren, 1971). Measurements of gs, Et, and psi were made between 12:00 and 13:00 hours (the photoperiod was initiated at 06:00 hours). Dinitrogen fixation was measured prior to any stress being imposed (at days 0 and 10) and again at the conclusion of each 10 day stress period (at days 20 and 30). Nitrogenase activity was measured using the acetylene reduction method (Gordon and Wheeler, 1978) and was expressed as micromoles of acetylene reduced per plant per hour. The acetylene reduction method required that each sampled plant and its pot be enclosed in a fresh, nonreactive, plastic bag below the first branch. This was done for each plant immediately after porometer and psychrometer sampling. To ensure a gas-tight seal and allow gas sampling from the bag, a small amount of plasticine (nonreactive) clay was fitted at the neck of the bag and fastened with a twist-tie and a septum was fitted through the side wall of the bag. Acetylene gas was generated each measurement day by the addition of calcium carbide to water. The gas was collected in a rubber bladder fitted with plastic tubing and a gas-tight septum. Ten to fifteen per cent (by bag volume) of the air enclosed around each plant was removed and replaced with an equal volume of acetylene. Each plant was then returned to the growth chamber for an hour of incubation at 25~ The bag was then agitated and two gas samples were withdrawn via the septum into 16mL VacutainersTM. Unnodulated plants, empty bags, and evacuated Vacutainers were used as controls. The l mL gas samples were later analyzed using a Tracor 565 gas-liquid chromatograph equipped with a flame ionization detector. The aluminum column (3.18mm i.d. x 1.82m) containing Poropak R was held at 150~ The carrier gas was helium flowing at 40 mL/min. Data were processed using a Hewlett-Packard 3390A integrator. Sample peak heights of ethylene were compared with ethylene standards. Fractional acetylene reduction values were multiplied by the factor bag volume/sample volume to give per plant ethylene production.

Frankia stocks
Frankia strain ARgP5 A~, gift of Dr. Lalonde (Universit6 Laval, Qu6bec), was grown and maintained on QMOD medium (Lalonde and Calvert, 1979) modified by reducing peptone to 1.0gL t, yeast extract to 0.25 g L ~, and omitting CaCO3, glucose, and lipid supplement, while adding 20/~M CaC12, 0.5 mM NH4CI, 0.2raM Nail.glutamate, and 5.0 mM Na.acetate, pH 6.7 (sterilized by filtration and added aseptically as substrate). Preliminary experiments established the inability of this strain to grow on 1% glucose or mannitol without the addition of acetate. This strain also failed to grow when Tween 80 (_+glucose), aspartate, citrate, fructose, glutamate, malate, pyruvate, succinate, or sucrose were supplied as sole substrates and grew very poorly when propionate was provided.

Determination of Frankia osmo- and halotolerance

Osmo- and halotolerance experiments were conducted for 20 days at 28~ (Precision Scientific Co., Model 815 refrigerated incubator) in 250 mL Erlenmeyer flasks containing 100mL of modified QMOD. The osmoticum (NaCI, glucose, or mannitol) was autoclaved separately in 90 mL of glass distilled water, then combined with aseptic additions of the medium ingredients so concentrated as to add only 10mL of volume. Each flask was inoculated with 0.2#g protein mL ~ of homogenized (Potter-Elvehjem tissue grinder) Frankia taken from a growing culture and washed three times in phosphate buffer-saline (PBS; per liter: 0.3g K2HPO4, 0.2g NaH2PO4, 9g NaCI) before homogenization. Protein content was determined, after digestion with NaOH, with acidic Coomassie Brilliant Blue G-250 (Biorad Laboratories), following the Biorad microassay procedure. The volume of homogenate was then adjusted to ca. 100~lg protein mL -~ . After incubation the entire contents of each flask were harvested by centrifugation, and washed with PBS before digestion and protein determination. The water potential of the medium (without glucose) was determined by freezing point depression (Precision Systems, Inc.,

92

Hennessey et al.

Osmette) to be -0.033 MPa. The water potential of media with added osmotica was calculated from added osmolality (Weast, 1978). The range of useful concentrations of osmotica was determined in preliminary experiments. Preliminary experiments also led to the choice of this Frankia strain, as typical of Alnus-derived strains growing well in modified Lalonde's medium. ArMP-1 (an isolate of this laboratory from Alnus rubra); AGNIExo,A~ ACN1 1lm, ACNI 1 lq, and ARbN4/~i (gifts of Dr. Lalonde, Universit6 Laval, Quebec) all made significant (though not necessarily equal) growth at water potentials ~ < - 2 M P a provided by the addition of non-assimilated sugars. Halotolerance was determined in a modified synthetic medium, derived from BAP (Tjepkema et al., 1980; 1981; Murry et al., 1984), as well as in modified QMOD. Modified BAP contained 5 m M K2HPO4, 5 m M KH2PO4, 0.2mM MgSO4 septahydrate, 2.0 mM NH 4C1, trace elements as specified below, and (filter sterilized and aseptically added) 10.0mM acetate, pH 7.0. (No vitamin requirement by Frankia ARgP5 AGcould be detected). The Frankia trace element solution (FTMS) provided (per liter) 100 #M EDTA, 20/~M B (as boric acid), 40/2M Ca (as CaCO3 -k- HC1), 0.01 #M Co (as COC12hexahydrate), 0.05 #M Cu (as CuSO4 pentahydrate), 30 #M Fe (as FeSO 4 septahydrate), 10/zM Mn (as MnC12 tetrahydrate), 10/tM Mo (as Na2MoO4 dihydrate), 10/~M Ni (as NiC12 hexahydrate), and 2.0/zM Zn (as ZnSO 4 septahydrate). FTMS allowed growth equal to that obtained when Tjepkema's or Lalonde's micronutrients were used. The advantage of FTMS was that its use permitted all of the inorganic constituents of the medium to be autoclaved together at 20 strength, yielding a clear solution upon cooling which could then be combined with osmotica contained in the remaining medium volume. The 0smolality and derived

water potential of these media were calculated from data in Weast (1978).

Results
Alnus response to water stress

The response of the host plants to stress is shown in Table 1. Leaf water potential was stable at days 0 and 10 of the experiment under well-watered control conditions, but then declined linearly in response to the water-stress treatments. By day 30, mean leaf water potential had declined to - 2 . 1 0 M P a . Values of g~ and Et increased somewhat during the well-watered phase of the experiment, but then also decreased with increasing water stress, reaching minimal values at day 30. Near the end of the experiment, plants were visibly wilted prior to the daily application of a measured amount of water. However, neither leaf necrosis nor chlorosis were noted at any time. The nitrogenase activity increased during the first 10 days of the experiment, but then decreased as leaf water potential, gs, and E t decreased. When water potential had declined from -1.21 to - 1 . 6 9 M P a at day 20, nitrogenase activity was only one-fifth of the control level. Yet at day 30, when water potential was - 2 . 1 0 MPa and gs was 0.03 cm.s -~ , low but significant nitrogenase activity (2.51/~m.plant-'.h -~) was still observed.

Frankia response to osmotica The yield of Frankia mycelium when NaCI, glucose, or mannitol osmotica were added to modified QMOD is shown in Table 2. When NaC1 was used as osmoticum, a water potential of - 1 . 2 5 MPa

Table 1. Effect of water-stress on Alnus A G 8022-16 (Plants were well-watered up to the time of day-10 measurements, moderately stressed from then to the time of day-20 measurements, and severely stressed between day-20 and day-30) Day 0 Leaf water potential (MPa) Stomatal conductance (cms - I ) Transpiration ( # g c m - 2 s i) Acetylene reduction b ( # M p l a n t - ~ h -~) - 1.20 0.18 2.66 11.37 + 0.18 __+ 0.06 _+ 0.81 + 4.15 Day 10 - 1.21 0.28 3.79 20.27 + 0.23 __+ 0.13 _+ 1.45 + 8.01 Day 20 - 1.69 0.10 1.60 4.32 + 0.35 + 0.03 _+ 0.47 ___ 3.07 Day 30 a -2.I0 0.03 0.46 2.51 + 0.38 _ 0.01 __+ 0.15 ___ 2.11

a plants were wilted. b Three uninoculated plants, empty bags, and Vacutainers produced 0.00 ~_ 0.00 # M ethylene.

N2 fixation and xerotolerance in Alnus

Table 2. Effect of osmotica added to modified Lalonde's medium on Frankia ARgP5 A~ yield
Osmoticum Concentration Osmolality" psi (MPa)

93

/~g protein SD

Experiment 1
NaC1 0.00% 0.30% 0.60% 1.20% 1.55% 1.80% 0.000 0.097 0.192 0.382 0.494 0.573 - 0.033 - 0.273 -0.507 -0.977 - 1.254 - 1.449 131.00 75.00 55.88 _+ 0.75 36,25 ~ 22.758 10,62 b

Experiment 2 D-glucose

0.0% 4.0% 8.0% 11.0% 15.0% 17.0% 20.0%

0.000 0.233 0.491 0.700 1.011 1.178 1.449

-0.033 -0.609 -1.246 -1.763 - 2.531 - 2.944 - 3.614

121.25 + 133,75 _+ 140.00 _+ 98.25 _+ 81.00 24.37 h 6.378 14.62 b

7.50 5.00 2.00 9.75 5.25

Experiment 3 Mannitol

0.0% 1.5% 2.0% 3.5%

0.000 0.084 0.112 0.200

-0.033 -0.241 -0.310 - 0.527

120.88 _+ 5,74 130.59 +_ 3.97 30.51 _+ 1.09 14.09 h

Experiment 4 None Glucose

NaCI
+ glucose

Mannitol
+ glucose

0.00% 4.0% 15.0% 0.30% 0.30% 4.0% 1.55% 2.0% 2.0% 4.0% 3.5%

0.000 0.233 1.011 0.097 0.097 +0.233 0.494 0.112 0.112 +0.233 0.200

-0.033 -0.609 -- 2.531 -0.273 -0.849 - 1.254 -0.310 -0.886 - 0.527

120.88 _+ 5,74 194.02 _+ 10.63 15.68 b 142.06 3.45 168.31 +_ 10.15 12.08 b 30.51 _+ 1.09 114.49 +_ 6.51 14,09 b

Weast, 1978; b average of two determinations. Cultures were incubated for 20 days at 28~ flask.

after inoculation with 20 #g protein per

(1.55% NaCl) resulted in no or insignificant (2 to 4 # g protein over that supplied as inoculum) growth. The inoculum was not quantitatively recovered at lower water potentials. In contrast, the lower limit of water potential allowing growth when glucose was used as osmoticum was ca. - 2 . 5 3 M P a (15.0% glucose), since this water potential allowed no or insignificant growth (ca. 4/tg protein over that supplied in the inoculum) both in the data shown and in preliminary experiments. The inoculum was not quantitatively recovered after exposure to water potentials approaching or less than - 3 MPa. With mannitol as osmoticum, growth declined abruptly between - 0 . 2 4 1 M P a (1.5% mannitol) and - 0 . 3 1 0 M P a (2.0% mannitol) or less. However, the addition of

4.0% glucose to media containing growth-limiting concentrations of either NaCI or mannitol increased yield. Halotolerance was markedly lower in the synthetic medium, but yield was again increased at 0.3% NaCI by the addition of 4% glucose (Table 3). It should be noted that the limit of halotolerance in this experiment was unchanged by the glucose addition.

Discussion Although the glucose tolerance of Frankia ARg5 AG was surprising, the effects of water-stress on nodulated Alnus and the halotolerance of the

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Hennessey et al.

Table 3. H a l o t o l e r a n c e o f Frankia A R g P 5 4~" in synthetic m e d i u m

% NaCI psi ( M P a ) Itg protein + SD '~ 153.14 + 92.42 _+ 53.01 h 28.13 _+ 11.04 + 6.77 4.71 3.64 0.50 0.25 +4%glucose psi (Mpa) pg protein +_ SD" 121.18 116.86 64.66 19.43 7.92 8.65 _+ 26.47 + 11.18 _+ 2.34 __+ 0.14 _+_ 0.13

0.00 0.3% 0.6% 0.9% 1.2% 1.5%

-0.109 -0.349 -0.584 -0.818 - 1.053 - 1.290

0.685 0.925 1.159 1.394 1.629 1.866

yields lower t h a n 2 0 # g protein indicate that the i n o c u l u m was not recovered. b average o f two d e t e r m i n a t i o n s .

Alnus-derived Frank& strain which were observed in this study were to be expected from the literature. In the present study, at water potentials below -1.21 MPa, gradual stomatal closure occurred, transpiration was reduced, and nitrogenase activity decreased. It is unclear why g~ increased from 0.18 to 0.28cms J between day 0 and day 10 while water potentials remained constant. The magnitude of the values of gs reported in this paper, however, are consistent with other reports for A. glutinosa (Hennessey et al., 1985; Seiler, 1985; Sundstr6m and Huss-Daneli, 1987; Hennessey et al., 1988) and A. rubra (Pezeshki and Hinckley, 1982). The relationship between stomatal closure and nitrogenase activity has been demonstrated by others (Huang et al., 1975; Sundstr6m and HussDanell, 1987), and is believed to reflect the strong dependence of nitrogenase activity on photosynthesis (Gordon and Wheeler, 1978), although additional hypotheses have been suggested (Sundstr6m and Huss-Danell, 1987). The correlation between lowered water potential and decreased nitrogenase activity in A. glutinosa has been previously reported by Seiler and Johnson (1984), who found that at a shoot water potential of - 1 . 3 M P a , nitrogenase activity was only onefourth of controls, and by Sundstr6m and HussDanell, (1987), who found nitrogenase activity was reduced at moderate water potential below - 0 . 6 MPa and severely inhibited at water potentials in the range of - !.1 to - !.4 MPa, As these authors have suggested, comparisons among studies to determine a threshold value of water potential limiting nitrogenase activity are difficult, depending on whether or not water potential was measured on shoots that were equilibrated with the root system. Measurement of root or nodule water potential was considered impractical in the present

investigation, since it would have required disturbance of the system. While a steep water potential gradient may be assumed while transpiration was active, the virtually closed stomata and wilted condition of the severely stressed plants at day 30 implied root-shoot equilibration. Enclosed plants have been reported to equilibrate within 8 hours (Sundstr6m and Huss-Danell, 1987). The halotolerance of ARgP5 A~ in modified QMOD was similar to that recorded for other Alnus isolates, in spite of differences in media and length of incubation. Shipton and Burggraaf(1982) reported that isolates from Alnus glutinosa (LDAgpl) and Alnus viridis (Avcli) showed minimal growth after 35 days at - 1 . 6 MPa. Dawson and Gibson (1987) reported that an Alnus rubra isolate (Arl3) made only 0.87 generations in 21 days when grown in the presence of NaC1 contributing - 0 . 9 1 1 MPa to water potential. In contrast, isolates from Casuarina and Allocasuarina made 3.02 to 5.06 generations. Shipton and Burggraaf (1983) reported that isolates from Casuarina and HippophaE showed growth after 48 days at - 2.27 MPa in a medium containing NaC1 as osmoticum. The lower halotolerance observed in synthetic medium may be due to the absence of such amino acids as glutamic acid, glycine, and proline which were present in the complex medium. These amino acids are osmoprotectants which may be accumulated from the medium by other procaryotes (Larsen et al., 1987; Yancey et al., 1982). Other osmotica have been employed, most widely polyethylene glycol (PEG). Shipton and Burggraaf (1982) reported that PEG 4000 affected growth to essentially the same extent as NaCI at equivalent water potential. However, since PEG 4000 can be toxic to Frankia (Shipton and Burggraaf, 1983; data of this laboratory), it may be

N, fixation and xerotolerance in Alnus doubted that PEG is useful in determining Frankia xerotolerance. We have used glucose and mannitol, osmotica which have been widely employed in adjusting water potential for microorganisms other than Frankia. Mannitol might also be considered toxic, from the data of Table 2. Glucose, in turn, does not appear to be a neutral osmoticum. The data of Tables 2 and 3 show a significant increase in yield when 4% glucose was added to 0.3% NaC1 or to 2% mannitol. Since ~4C-glucose was not admitted to the mycelium of non-assimilating Frankia strains (Blom and Harkink, 1981; personal communication from Drs. M D Stowers and D B Steele) and since no instance of growth consequent upon increasing concentration of sugars under these circumstances appears in the microbiological literature, the suggestion that glucose constituted an additional energy source was rejected. It appears likely that reagent grade glucose contains some other element or factor which was required for maximal yields, even in the complex medium used. The fact that the limit of halotolerance in synthetic medium was unaffected by glucose addition further indicates that the glucose factor did not operate directly on halotolerance. Since none of the osmotica used in any reports currently available is demonstrably neutral, that is, affects Frankia physiology in no other way than through its effect on water potential, it is not possible to assess the limits of osmotolerance with confidence. It is possible, from the data presented, to assume that the osmotolerance limits for Alnusderived Frankia strains occur at lower water potentials than do the limits of halotolerance. Dinitrogen fixing ability (acetylene reduction) roughly parallels growth in media of lowered water potential (Burggraaf and Shipton, 1983: Shipton and Burggraaf, 1982). The sensitivity of vesicles and the short half-life of nitrogenase (see Tjepkema et al., 1980; Lopez et al., 1986; Noridge and Benson, 1986; Baker and Huss-Danell, 1986) makes it unlikely that nitrogenase activity could persist over the two successive 10-day periods of water stress in the absence of healthy mycelium. If halotolerance were an appropriate indicator of osmotolerance, Frankia ARgP5 A~ should have been unable to fix dinitrogen in the severely stressed host plants. In fact, significant dinitrogen fixation occurred at - 2 . 1 MPa, a water potential well within the limit for ARgP5 "~ when glucose osmotica were em-

95

ployed but considerably lower than halotolerance limits for Alnus-derived Frankia strains. Dinitrogen fixation by nodulated Alnus was correlated with the osmotolerance of the Frankia symbiont rather than with its halotolerance. And since this Frankia strain was active at a water potential which wilted the Alnus host, host xerotolerance appears to be the primary limiting factor in the utilization of these actinorhizal plants.

Acknowledgments
We wish to thank J Leland Booth for expert technical assistance. This material is based on work supported in part by the National Science Foundation under grant PCM 8213804.

References
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N 2 f i x a t i o n and xerotolerance in A l n u s
Seiler J R 1985 Morphological and physiological changes in black alder induced by water stress. Plant Cell Environ. 8, 219-222. Seiler J R and Johnson J D 1984 Growth and acetylene reduction of black alder seedlings in response to water stress. Can. J. For. Res. 14, 477-480. Shipton W A and Burggraaf A J P 1982 Frankia growth and activity as influenced by water potential. Plant and Soil 69, 293-297. Shipton W A and Burggraaf A J P 1983 Aspects of the cultural behaviour of Frankia and possible ecological implications. Can. J. Bot. 61, 2783-2792. Sundstr6m K-R and Huss-Danell K 1987 Effects of water stress on nitrogenase activity in Alnus incana. Physiol. Plant 70+ 342-348. Tjepkema J D, Ormerod W and Torrey J G 1980 Vesicle formation and acetylene reduction in Frankia Cpll cultured in defined nutrient media. Nature (London) 287, 633-635. Tjepkema J D, Ormerod W and Torrey J G 1981 Factors affecting vesicle formation and acetylene reduction (nitrogenase activity) in Frankia sp. Cpll. Can. J. Microbiol. 27, 815-823. Weast R C 1978 CRC Handbook of Chemistry and Physics, 59th ed. CRC Press, Inc.; West Palm Beach, Florida. Yancey P H, Clark M E, Hand S C, Bowlus R D and Somero G N 1982 Living with water stress: Evolution of osmolyte systems. Science 217, 1214-1222.

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