Microb Ecol (1984) 10:107-114
MICROBIAL ECOLOGY 9 1984 Springer-Verlag
Nitrous Oxide Production by Nitrogen-Fixing, Fast-Growing Rhizobia S. Casella,~ C. Leporini,~ and M. P. Nuti 2 tIstituto di Microbiologia agraria, Universith d! Pisa, 56100 Pisa; 2Istituto di Chimica e Industrie agrarie, Universith di Padova, 35100 Padova, Italy Abstract. Rhizobium trifolii, R. leguminosarum, and R. "hedysarum, "" grown ex planta u n d e r anoxic conditions in a chemically defined medium, evolve NzO from NO3-, NO2-, and (NH4)2NO3. The a m o u n t o f nitrous oxide f o r m e d after 96 hours is about 0.2 # M x m g - ' cells d.w. Large availability o f organic m a t t e r enhances the p r o d u c t i o n o f N 2 0 f r o m nitrate by free-living R. trif01ii in peat/sand mixtures. Denitrification o f the above species and R. meliloti was detected also in planta. Nitrous oxide production increases almost linearly from 10-45 # M x mg -~ nodules d.w. when nitrogen-fixing plants are exposed to increasing concentrations o f nitrate (1-12 ~tM).
Introduction Early investigations by Rajagopalan [16] and Wilson [20] have shown that rhizobia are able to liberate gaseous nitrogen from nitrate. Subsequent work has unequivocally indicated that Rhizobium can use nitrate as an alternative electron acceptor to oxygen to generate A T P for nitrogenase activity [18]. Particularly R. japonicum and cow-pea strains can be grown anaerobically [4, 22] and exhibit substantial rates o f denitrification as either free-living or bacteroid cells. Fast-growing rhizobia are less studied, and the few strains screened [21] were described as unable to reduce nitrate by dissimilatory means. H o w ever, denitrification was suggested by Lepidi and Picci [9] for a strain o f R. leguminosarum, and for R. meliloti by Ishizawa [8]. Here experimental conditions are described, which are suitable to detect NEO f o r m a t i o n ex planta by R. trifolii, R. leguminosarum, and R. "hedysarum'" grown anoxically in the presence o f nitrate, nitrite, and a m m o n i u m nitrate. Denitrification by the above fast-growing rhizobia and R. meliloti is detectable also in planta by using N20-reductase inhibition test. Materials and M e t h o d s
Microorganisms and Plants Table 1 summarizes the bacterial strains used in this study. Strain 8ST-M7 was a spontaneous mutant of the wild-type R. trifolff 8ST; strain 8ST-M55 was obtained by thermal treatment as
S. Casella et al.
108 Table 1.
Bacterial strains and their properties
Bacterial species
Strain
R. trifolii
8ST
R. trifolii R. trifolii R. leguminosarum
8ST-M7 8ST-M55 LPR 1
R. "'hedysarum'"
RH19-R14
R. "'hedysarum'"
CC 1337
R. meliloti
L5-30
Phenotype Nod + Fix + on T. pratense T. incarnatum Nod + Fix- on T. pratense Nodon T. pratense Nod + Fix + on P. sativum V. hirsuta Nod + Fix + on Hedysarum coronarium Nod + Fix + on Hedysarum coronarium Nod + Fix + on M. sativa
Markers
Origin/reference
str r rif"
[1]
str ~ rifr str" rif" rif"
This study This study [7]
str" nf"
[ 12]
wild-type
[2]
str"
[ 15]
described previously [10]; strain LPR-1 is a rift derivative ofR. leguminosarum RCR 1105 reported elsewhere [ 13]. Stock cultures, derived from freeze-dried ampoules, were kept at room temperature in yeastmannitol-agar (YMA) slants [ 19]. Seeds of Trifolium pratense, T. incarnatum, Medicago sativa, and Hedysarum coronarium were kindly provided by Dr. M. Macchia (Istituto di Agronomia, Universit~ di Pisa), Pisum sativurn by Dr. D. Ceci (Centro Ricerche CO.VAL.PA., Medolla), and Vicia hirsuta was a generous gift o f Dr. J. Beringer (Rothamsted Exptl. Station, Harpeden, U.K.).
Media
and Growth Conditions
R. "'hedysarum, "' R. leguminosarum and R. trifolii were grown aerobically and anaerobically in flasks filled with yeast-mannitol broth (YMB; 19) or mannitol broth without yeast extract (MB) or M M D (minimal medium) having the following composition: sol. A (basal, g • 480 ml) KNO3 0.6, CaC12 x 2 H20 0.1, MgSO4 x 7 H20 0.25, FeCI3 • 6 H20 0.01; sol. B (phosphate) from stock solutions 50 g • 1-~, K2HPO4 • 3 HzO 20 ml, K H 2 P O 4 • 3 H20 20 ml; sol. C (minor elements) 1 ml from stock solution 20 mg • 1-~ of each MnSO4 • H20, ZnSO4 x 7 H20, CuSO4 • 5 H20, n3BOa, NazMoO4, and 0.1 ml from stock solution 20 ml • 1-~ COC12; sol. D (vitamins) 0.5 ml from stock solution, 20 mg • 1-~ of biotin, thiamine-HCl, pyridoxine, riboflavin, nicotinic acid, PA-BA, Ca-pantothenate; sol. E (sugars) 25 ml from stock solution 20% mannitol. The above solutions A to E were prepared and mixed when sterile. Sterile water was added up to 1,000 ml before inoculation. In some tests, nitrate was replaced by other combined nitrogen sources; equimolar amounts of nitrogen were always used. When anoxic growth was required culture flasks were layered with about 0.5 cm o f liquid paraffin and cultures were flushed with argon and sealed with Subba-seal prior to the incubation at 25-27~ Growth was assessed turbidimetrically at 660 nm, and by measurement of dry weight. Viable cell numbers of both aerobically- and anaerobicallygrown rhizobia were determined on aerobically incubated YMA plates. To assess the influence of organic matter on nitrous oxide production, tests were performed by inoculating 125 ml of log-cultures, grown in M M D with NO3- 6 m M as nitrogen source as described before, into flasks filled with the following mixtures: mix A contained neutral peat (68% organic matter content) 9 g, quartz sand 691 g, mannitol 1%. Mix B contained neutral peat 90 g, quartz sand 610 g, mannitol 10%. N O 3- was added to final concentration o f 6 mM. Peat was sterilized apart by tyndalization, and sand by autoclaving 45 min at 121~ Water content o f the 2 mixes, before inoculation, was adjusted to 15%. Flasks were incubated at 25-27~ after replacement of the gas phase with an atmosphere of 95% H2/5% CO2 (v/v).
Nitrous Oxide Production
109
Nodulation Test Nodulation by the strains used throughout this study was assessed by conventional methods [19], and nitrogen fixation estimated as ARA (acetylene reduction activity), and described below.
Acetylene Reduction Test Intact plants, 25 days old, were assayed for C2H2 reducing activity associated with their roots. The tests were performed at room temperature in the dark, in Erlenmeyr flasks fitted with silicone rubber septa. 5% (v/v) of the air was replaced with C:H2 and after 1 and 2 hours the C2H4 formed was measured by injecting 0.5 ml gas samples onto a Packard 419 gas-chromatograph as described [3].
Nitrous Oxide Detection For aerobic and anaerobic cultures of rhizobia, gas samples were transferred directly from flasks to the gas-chromatograph. For in planta assays, plants were grown in half-strength Jensen's [19] semisoft agar (5g x 1-1 of Bacto Special Agar Noble Difco), and kept up to 25 days for nodulation. When nodulated, the plants were aseptically transferred into tubes containing the same medium and 1-12 mM KNO 3. In the sealed (Subba-seal) tubes, 0.1% of the atmosphere (v/v) was replaced with C2Hz to stop N20-reduction. Roots with nodules were washed 3 times with sterile water before transfer onto nitrate agar. Viable cell numbers of contaminants and rhizobia were routinely checked by plating 0.2 ml of the last wash on Nutrient Agar (Difco) and YMA plates, respectively. N20 evolution was monitored by injecting 1 ml of gas sample in the gas-chromatograph fitted with an electron capture detector and 2m x 0.4 cm column packed with Porapak Q, using He as carrier gas.
Results and Discussion N i t r a t e h a s b e e n s h o w n to s e r v e a s a n e l e c t r o n a c c e p t o r d u r i n g c h e m o - o r g a n o t r o p h i c g r o w t h o f R . japonicum a n d s t r a i n s o f t h e c o w - p e a g r o u p [4]. W e w e r e a b l e t o g r o w R. trifolii, R. leguminosarum, a n d R. "hedysarum'" u n d e r a n o x i c c o n d i t i o n s i n a c h e m i c a l l y d e f i n e d m e d i u m a n d in t h e p r e s e n c e o f n i t r a t e , nitrite, and ammonium nitrate. Nitrous oxide formation from nitrate was det e c t a b l e (Fig. 1) f o r R. trifolii a n d R. "'hedysarum'" s o o n a f t e r 24 h o u r s i n c u b a t i o n , a n d r e a c h e d a b o u t 0.2 u M x m g -~ ceils d . w . a f t e r 3--4 d a y s i n c u b a t i o n a t 26~ i n M M D ; R. leguminosarum h a d a b e h a v i o r s i m i l a r t o R. trifolii 8 S T ( T a b l e 2). N 2 0 f o r m a t i o n r e m a i n e d u n d e t e c t a b l e f o r all s t r a i n s o f t h e a b o v e 3 s p e c i e s (a) i n t h e a b s e n c e o f n i t r a t e , (b) w h e n c u l t u r e s w e r e g r o w n a e r o b i c a l l y , a n d (c) w h e n r i c h m e d i a w e r e u s e d i n s t e a d o f M M D . T h e l a t t e r c o n d i t i o n c o n f i r m s t h e r e s u l t s o b t a i n e d b y Z a b l o t o w i c z et al. [21] w h o d e s c r i b e d fastgrowing rhizobia as unable to use NO3- by dissimilatory pathway, when grown anoxically in rich media. When different symbiotic phenotypes are compared, the pattern of N20 f o r m a t i o n r e m a i n s a l m o s t t h e s a m e : in t h e p r e s e n c e o f 6 m M KN03, R. trifolii p r o d u c e d i n 8 d a y s 0 . 4 2 ~ M N 2 0 ( s t r a i n 8ST, N o d + Fix+), 0 . 3 0 t~M N 2 0 ( s t r a i n 8 S T - M 7 , N o d + F i x - ) , a n d 0 . 3 2 / ~ M N 2 0 ( s t r a i n 8 S T - M 5 5 , N o d - ) x m g -~ o f
110
S. Casella et al. o
viabte call counts, R hedys|rum R H l g - R I 4
9
viable cell 9
R tri/olll
aST
N2O produced, strain R H l g - R 1 4 9 N20 produced, strain BST []
0 D 6 6 0 changes, strain RH19 R14 9 OD6~ 0 changes)strain 8ST
E 9 'o
E
t~ =
= o 7. | O
Z
2
t
~
~
4
5
g
7
Fig. I. Viable cell counts, OD660 changes, and N20 evolution during anoxic growth of R. trifolii 8ST and R. "hedysarum" RH19-R14 in the presence of 6 mM KNO3.
days
Table 2. N20 formation by different rhizobia grown anoxically in batch cultures in the presence of different nitrogen sources
Strain R. trifolii 8ST
Nitrogen source
Nitrous oxide formed
NO3NO2NH4* NH4+NO3NO3NO2-
0.30 0.42
R. trifolii 8ST-M55
NH4+ NH4+NO3NO3-
0.15 0.32
R. "hedysarum"RHl9-R14 R, leguminosarum LPR 1
NO2NH4 + NH4*NO3NO3NO3-
R. trifolii 8ST-M7
Data are expressed in g M x m g of anaerobic growth in MMD
-t cells d.w.
0.42 0.09
0.35 0.09 0.55 0.40 after 8 days
cells, d . w . S o m e d i f f e r e n c e s w e r e d e t e c t e d w h e n t h e 3 s t r a i n s w e r e g r o w n i n MMD in the presence of various nitrogen sources: Table 2 shows that symbiotically defective strains evolve N20 from either NOz or NO3-, while strain 8ST produces more N20 from nitrate than from nitrite; this could be related to a transient accumulation of the latter compound during the assay, after about 3 days of cultivation in the presence of NO3-. Such an accumulation has been
Nitrous Oxide Production
111
Table 3. Effectsof the organic matter on the production of nitrous oxide by R. trifolii, grown ex planta in the presence of 6 mM KNO3 in peat/sand mixtures
Strain
R. trifolii 8ST R. trifolii 8ST-M7 R. trifolii 8ST-M55
Organic matter
N20 (~M x 100 g-i d.w.) after 8 days
1% 10% 1% 10% 1% 10%
0.75 1.16 0.17 1.60 0.16 2.05
described by Daniel et al. [4] for R. japonicum. W h e t h e r the b e h a v i o r o f defective strains is due to i n d e p e n d e n t m u t a t i o n a l events occurring during isolation, or linked to the loss o f symbiotic properties, awaits further investigations. T h e small a m o u n t s o f NzO f o r m e d in the presence o f (NH4)NO3 are probably p r o d u c e d f r o m the nitrate-nitrogen, since separate controls, obtained with washed cells pregrown in rich m e d i a or M M D plus nitrate or nitrite, and then exposed to a m m o n i u m nitrogen, gave undetectable levels o f nitrous oxide formation. T h e influence o f organic m a t t e r on N 2 0 f o r m a t i o n is shown in Table 3. All R. trifolii evolve nitrous oxide at increasing rates when grown in the presence o f increasing concentrations o f organic matter. T h e above results clearly indicate that fast-growing rhizobia, when cultivated anoxically ex planta, can use N O 3- and NO2- as electron acceptors to evolve gaseous N 2 0 . Furthermore, the process is enhanced when there is a large availability o f electron donors. Therefore, our data support previous suggestions that free-living rhizobia m a y r e m o v e fixed nitrogen from soil by dissimilatory pathways [4]. In order to verify whether N - N z O is p r o d u c e d in a n u m b e r o f forage and grain legumes nodulated by the previously studied and other fast-growing rhizobia, reduction o f added 6 m M nitrate was m o n i t o r e d during in planta assays. Well-nodulated, nitrogen-fixing Trifoliurn incarnatum, Hedysarum coronarium, Pisum sativum, and Medicago sativa were used, and b o t h A R A and N 2 0 evolution were measured as described in Methods. Figure 2a shows the f o r m a t i o n o f N 2 0 f r o m the nodules o f Pisurn sativurn and Medicago sativa, inoculated with their specific rhizobia, at increasing rates o f nitrate present in the m e d i u m ; in the same 2 systems, the specific A R A was m e a s u r e d and the data are reported in Fig. 2b. It appears that dissimilatory nitrate reduction increases with increasing concentrations o f a d d e d NO3-, while in the same conditions the specific A R A is reduced. T h e latter results are in agreement with findings in soybean [18] Phaseolus vulgaris [ 17], Lupinus angustifolius [11], M. sativa [6], and T. subterraneum [5]. By plotting the N 2 0 lost as a percentage o f a d d e d N O 3- versus increasing concentrations o f nitrate present in the m e d i u m , the relative loss decreases, as depicted in Fig. 2c. In other terms, nodule dissimilatory activity tends to reach a plateau when the
112
S. Casella et al.
M sat]va -R.rmzliloti
40-
0
41,
R SQ t ivu rn- R.legum inosaru m
.
~6
="E
~_
2o-
q.
.yi
ol
j
o
j
10-
~
c
~
E
100-
o u z,-,z :k'~
5o-
25-
KNO 3 concentration (mM)
Fig. 2. Evolution o f nitrous oxide (a) ARA, (b) percent o f N-NO3 lost, (c) as measured in planta in M. sativa and Pisum sativum at increasing concentrations of nitrate supplied to the system. Rates shown (2e) on the y axis represent the values of N 2 0 accumulation after 50 hours.
NOa- available to the system is between 9 and 12 mM. This trend towards saturation is better seen for alfalfa than pea nodules. In the case of alfalfa nodules, the N - N 2 0 evolution increases in time, at least during the first 48 hours of incubation, as shown in Fig. 3. For H. coronarium and T. incarnatum, an average o f 10-45 #M N 2 0 x mg -1 nodules d.w. were formed during the first 48 hours o f incubation in the presence of 12 m M NO3-. Particularly for R. "hedysarum" it was found that the 2 strains studied produced significantly different amounts o f N=O: strain R H 1 9 - R 1 4 evolved 15 #M, and strain CC1337 45 IzM x mg-' nodules d.w., after 48 hours incubation. It seems worth noting that the strain that evolves major amounts o f N 2 0 is also more effective for symbiotic nitrogen fixation than the other on H. coronarium [2].
Nitrous Oxide Production
~12
0
nre~
o/o___
o 9 mM
i1--1
6
113
mM
o
/
O~--
o o
g
~'o
ig
2b z'~ 3'0 a's 20 4'5
5'o
s~
Fig. 3. Gaseousnitrogen losses in well-nodulated, nitrogen fixing M. sativa inoculated with R. meliloti L5-30, after exposure to different concentrations of nitrate.
Time from K N O 3 addition (hours)
Other studies, to be published elsewhere, c a r d e d out with plants grown in the presence o f :SN as KNO3 ( 1 - 1 2 raM) have confirmed the a b o v e gas-chrom a t o g r a p h i c data: n o d u l a t i o n and A R A are inhibited by increasing (3-12 m M , according to the legume) c o n c e n t r a t i o n o f nitrate; 15N e n r i c h m e n t tests indicate that nitrogen losses increase almost linearly with increasing a m o u n t s o f nitrate supplied. As it appears that N 2 0 evolution a n d nitrogen fixation (acetylene reduction) m a y run as c o n t e m p o r a r y activities in planta, though with different reciprocal rates according to the symbiotic system, these studies suggest that a broader knowledge is needed on the relationships between the two processes inside the nodule before an adequate a p p r o a c h to o v e r c o m i n g nitrate inhibition effects in the field is established. Acknowledgments. This investigation was supported by the National Research Council, Italy (Spe-
cial grant IPRA, sub-project 1, paper n. 71). The skillful assistance of Dr. C. Robba and Mrs. M. Bonfanti-Casella is gratefully acknowledged.
References 1. Casella S, Ceci D, Bonari E, Lepidi AA, Nuti MP (1981) Batterizzazione di leguminose da foraggio e da granella: un triennio di ricerche in Italia. Rivista di Agronomia 3-4:173-182
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2. Casella S, Reynolds KC, Dyson JR, Gault RR, Brockwell J (1984) Nodulation studies on legumes exotic to Australia: Hedysarum coronariurn. FEMS Microbiol Letters, in press. 3. Citernesi U, Neglia R, Seritti A, Lepidi AA, Bagnoli G, Galluzzi R, Nuti MP (1977) Nitrogenfixing bacteria in the gastroenteric cavity of soil animals. Soil Biol Biochem 9:71-72. 4. Daniel RM, Smith IM, Phillip JAD, Ratcliffe HD, Drozd JW, Bull AT (1980) Anaerobic growth and denitrification by Rhizobiumjaponicum and other Rhizobia. J Gen Microbiol 120: 517-521 5. Gibson AH (1976) Recovery and compensation by nodulated legumes to environmental stress. In: Nutman PS (ed) Symbiotic nitrogen fixation in plants, Cambridge Univ Press, London pp 385-405 6. Heichel GH, Vance CP (1979) Nitrate N and Rhizobium strain roles in alfalfa seedling nodulation and growth. Crop Sci 19:512-518 7. Hooykaas P J J, Snijdewint FGM, Schilperoort RA (1983) Identification of the sym plasmids of Rhizobium leguminosarum strain 1001 and its transfer to and expression in other rhizobia and Agrobacterium tumefaciens (in press) 8. Ishizawa S (1980) Note on nitrate reduction in Rhizobium. Soil Sci Plant Nutr 26:447-450 9. Lepidi AA, Picci G (1967) Nitrato-riduzione e denitrificazione ad opera di Rhizobium leguminosarum (Frank) in terreni cobalto-molibdeno-carenti. Agric Ital pp 223-236 10. Lepidi AA, Nuti MP, Bagnoli G, Filippi C, Galluzzi R (1979) Further researches on the involvement of Rhizobium large plasmids in legume nodulation. In: Bond DA, ScarasciaMugnozza GT, Poulsen MH (eds) Some current research on Vicia faba in Western Europe, EEC Publ Off, Luxemburg pp 436-460 11. Manhart J, Wong PP (1980) Nitrate effect on nitrogen fixation (acetylene reduction). Plant Physiol 65:502-505 12. Nuti MP, Casella S, Filippi C, Lepidi AA, Galluzzi R (1981) Rhizobia as inoculant for field trials in marginal soils of middle and northern Italy. In: Gibson AH, Newton WE (eds) Current Perspectives in Nitrogen Fixation, Austr Acad Sci Canberra p 514 13. Nuti MP, Lepidi AA, Schilperoort RA, Hooykaas PJJ, Prakash RK (1982) The plasmids of Rhizobium and symbiotic nitrogen fixation. In: Kahl G, Schell J (eds) Molecular Biology of Plant Tumors, Academic Press pp 561-588 14. Pedrazzini F, Nannipieri P (1982) Evolution of nitrous oxide in a nitrate-treated soil at a low partial pressure of acetylene. Plant and Soil 66:429-431 15. Prakash RK, Hooykaas PJJ, Lebedoer AM, Nuti MP, Lepidi AA, Juliot JS, Denari6 J (1980) Detection, isolation, and characterization of large plasmids in Rhizobium. In: Newton WE, Orme-Johnson N H (eds) Nitrogen Fixation, vol II. Univ Park Press, Baltimore, pp 136-163 16. Rajagopalan T (1938) Studies on groundnut nodule organism. IV. Physiology of the organism: intermediary metabolism. Ind J Agric Sci 8:379-382 17. Rigaud J (1976) Effet des nitrates sur la fixation d'azote par les nodules de haricot (Phaseolus vulgaris L.). Physiol Veg 14:297-308 18. Rigaud J, Bergersen FJ, Turner GL, Daniel RM (1973) Nitrate-dependent anaerobic acetylene reduction and nitrogen fixation by soybean bacteroids. J Gen Mierobiol 77:137-144 19. Vincent J (1970) A manual for the practical study of root-nodules bacteria. IBP Handbook n 15, Blackwell Sci Publ, Oxford, pp 164 20. Wilson JK (1947) The legume bacteria liberate gaseous nitrogen from nitrate. Proc Soil Sci Soc Am 12:215 21. Zablotowicz RM, Eskew DL, Focht DD (1978) Denitrification in Rhizobium. Can J Microbiol 24:757-760 22. Zablotowicz RM, Focht DD (1979) Denitrification and anerobic nitrate-dependent acetylene reduction in cowpea Rhizobium. J Gen Microbiol I 11:445-448
MICROBIAL ECOLOGY 9 1984 Springer-Verlag
Nitrous Oxide Production by Nitrogen-Fixing, Fast-Growing Rhizobia S. Casella,~ C. Leporini,~ and M. P. Nuti 2 tIstituto di Microbiologia agraria, Universith d! Pisa, 56100 Pisa; 2Istituto di Chimica e Industrie agrarie, Universith di Padova, 35100 Padova, Italy Abstract. Rhizobium trifolii, R. leguminosarum, and R. "hedysarum, "" grown ex planta u n d e r anoxic conditions in a chemically defined medium, evolve NzO from NO3-, NO2-, and (NH4)2NO3. The a m o u n t o f nitrous oxide f o r m e d after 96 hours is about 0.2 # M x m g - ' cells d.w. Large availability o f organic m a t t e r enhances the p r o d u c t i o n o f N 2 0 f r o m nitrate by free-living R. trif01ii in peat/sand mixtures. Denitrification o f the above species and R. meliloti was detected also in planta. Nitrous oxide production increases almost linearly from 10-45 # M x mg -~ nodules d.w. when nitrogen-fixing plants are exposed to increasing concentrations o f nitrate (1-12 ~tM).
Introduction Early investigations by Rajagopalan [16] and Wilson [20] have shown that rhizobia are able to liberate gaseous nitrogen from nitrate. Subsequent work has unequivocally indicated that Rhizobium can use nitrate as an alternative electron acceptor to oxygen to generate A T P for nitrogenase activity [18]. Particularly R. japonicum and cow-pea strains can be grown anaerobically [4, 22] and exhibit substantial rates o f denitrification as either free-living or bacteroid cells. Fast-growing rhizobia are less studied, and the few strains screened [21] were described as unable to reduce nitrate by dissimilatory means. H o w ever, denitrification was suggested by Lepidi and Picci [9] for a strain o f R. leguminosarum, and for R. meliloti by Ishizawa [8]. Here experimental conditions are described, which are suitable to detect NEO f o r m a t i o n ex planta by R. trifolii, R. leguminosarum, and R. "hedysarum'" grown anoxically in the presence o f nitrate, nitrite, and a m m o n i u m nitrate. Denitrification by the above fast-growing rhizobia and R. meliloti is detectable also in planta by using N20-reductase inhibition test. Materials and M e t h o d s
Microorganisms and Plants Table 1 summarizes the bacterial strains used in this study. Strain 8ST-M7 was a spontaneous mutant of the wild-type R. trifolff 8ST; strain 8ST-M55 was obtained by thermal treatment as
S. Casella et al.
108 Table 1.
Bacterial strains and their properties
Bacterial species
Strain
R. trifolii
8ST
R. trifolii R. trifolii R. leguminosarum
8ST-M7 8ST-M55 LPR 1
R. "'hedysarum'"
RH19-R14
R. "'hedysarum'"
CC 1337
R. meliloti
L5-30
Phenotype Nod + Fix + on T. pratense T. incarnatum Nod + Fix- on T. pratense Nodon T. pratense Nod + Fix + on P. sativum V. hirsuta Nod + Fix + on Hedysarum coronarium Nod + Fix + on Hedysarum coronarium Nod + Fix + on M. sativa
Markers
Origin/reference
str r rif"
[1]
str ~ rifr str" rif" rif"
This study This study [7]
str" nf"
[ 12]
wild-type
[2]
str"
[ 15]
described previously [10]; strain LPR-1 is a rift derivative ofR. leguminosarum RCR 1105 reported elsewhere [ 13]. Stock cultures, derived from freeze-dried ampoules, were kept at room temperature in yeastmannitol-agar (YMA) slants [ 19]. Seeds of Trifolium pratense, T. incarnatum, Medicago sativa, and Hedysarum coronarium were kindly provided by Dr. M. Macchia (Istituto di Agronomia, Universit~ di Pisa), Pisum sativurn by Dr. D. Ceci (Centro Ricerche CO.VAL.PA., Medolla), and Vicia hirsuta was a generous gift o f Dr. J. Beringer (Rothamsted Exptl. Station, Harpeden, U.K.).
Media
and Growth Conditions
R. "'hedysarum, "' R. leguminosarum and R. trifolii were grown aerobically and anaerobically in flasks filled with yeast-mannitol broth (YMB; 19) or mannitol broth without yeast extract (MB) or M M D (minimal medium) having the following composition: sol. A (basal, g • 480 ml) KNO3 0.6, CaC12 x 2 H20 0.1, MgSO4 x 7 H20 0.25, FeCI3 • 6 H20 0.01; sol. B (phosphate) from stock solutions 50 g • 1-~, K2HPO4 • 3 HzO 20 ml, K H 2 P O 4 • 3 H20 20 ml; sol. C (minor elements) 1 ml from stock solution 20 mg • 1-~ of each MnSO4 • H20, ZnSO4 x 7 H20, CuSO4 • 5 H20, n3BOa, NazMoO4, and 0.1 ml from stock solution 20 ml • 1-~ COC12; sol. D (vitamins) 0.5 ml from stock solution, 20 mg • 1-~ of biotin, thiamine-HCl, pyridoxine, riboflavin, nicotinic acid, PA-BA, Ca-pantothenate; sol. E (sugars) 25 ml from stock solution 20% mannitol. The above solutions A to E were prepared and mixed when sterile. Sterile water was added up to 1,000 ml before inoculation. In some tests, nitrate was replaced by other combined nitrogen sources; equimolar amounts of nitrogen were always used. When anoxic growth was required culture flasks were layered with about 0.5 cm o f liquid paraffin and cultures were flushed with argon and sealed with Subba-seal prior to the incubation at 25-27~ Growth was assessed turbidimetrically at 660 nm, and by measurement of dry weight. Viable cell numbers of both aerobically- and anaerobicallygrown rhizobia were determined on aerobically incubated YMA plates. To assess the influence of organic matter on nitrous oxide production, tests were performed by inoculating 125 ml of log-cultures, grown in M M D with NO3- 6 m M as nitrogen source as described before, into flasks filled with the following mixtures: mix A contained neutral peat (68% organic matter content) 9 g, quartz sand 691 g, mannitol 1%. Mix B contained neutral peat 90 g, quartz sand 610 g, mannitol 10%. N O 3- was added to final concentration o f 6 mM. Peat was sterilized apart by tyndalization, and sand by autoclaving 45 min at 121~ Water content o f the 2 mixes, before inoculation, was adjusted to 15%. Flasks were incubated at 25-27~ after replacement of the gas phase with an atmosphere of 95% H2/5% CO2 (v/v).
Nitrous Oxide Production
109
Nodulation Test Nodulation by the strains used throughout this study was assessed by conventional methods [19], and nitrogen fixation estimated as ARA (acetylene reduction activity), and described below.
Acetylene Reduction Test Intact plants, 25 days old, were assayed for C2H2 reducing activity associated with their roots. The tests were performed at room temperature in the dark, in Erlenmeyr flasks fitted with silicone rubber septa. 5% (v/v) of the air was replaced with C:H2 and after 1 and 2 hours the C2H4 formed was measured by injecting 0.5 ml gas samples onto a Packard 419 gas-chromatograph as described [3].
Nitrous Oxide Detection For aerobic and anaerobic cultures of rhizobia, gas samples were transferred directly from flasks to the gas-chromatograph. For in planta assays, plants were grown in half-strength Jensen's [19] semisoft agar (5g x 1-1 of Bacto Special Agar Noble Difco), and kept up to 25 days for nodulation. When nodulated, the plants were aseptically transferred into tubes containing the same medium and 1-12 mM KNO 3. In the sealed (Subba-seal) tubes, 0.1% of the atmosphere (v/v) was replaced with C2Hz to stop N20-reduction. Roots with nodules were washed 3 times with sterile water before transfer onto nitrate agar. Viable cell numbers of contaminants and rhizobia were routinely checked by plating 0.2 ml of the last wash on Nutrient Agar (Difco) and YMA plates, respectively. N20 evolution was monitored by injecting 1 ml of gas sample in the gas-chromatograph fitted with an electron capture detector and 2m x 0.4 cm column packed with Porapak Q, using He as carrier gas.
Results and Discussion N i t r a t e h a s b e e n s h o w n to s e r v e a s a n e l e c t r o n a c c e p t o r d u r i n g c h e m o - o r g a n o t r o p h i c g r o w t h o f R . japonicum a n d s t r a i n s o f t h e c o w - p e a g r o u p [4]. W e w e r e a b l e t o g r o w R. trifolii, R. leguminosarum, a n d R. "hedysarum'" u n d e r a n o x i c c o n d i t i o n s i n a c h e m i c a l l y d e f i n e d m e d i u m a n d in t h e p r e s e n c e o f n i t r a t e , nitrite, and ammonium nitrate. Nitrous oxide formation from nitrate was det e c t a b l e (Fig. 1) f o r R. trifolii a n d R. "'hedysarum'" s o o n a f t e r 24 h o u r s i n c u b a t i o n , a n d r e a c h e d a b o u t 0.2 u M x m g -~ ceils d . w . a f t e r 3--4 d a y s i n c u b a t i o n a t 26~ i n M M D ; R. leguminosarum h a d a b e h a v i o r s i m i l a r t o R. trifolii 8 S T ( T a b l e 2). N 2 0 f o r m a t i o n r e m a i n e d u n d e t e c t a b l e f o r all s t r a i n s o f t h e a b o v e 3 s p e c i e s (a) i n t h e a b s e n c e o f n i t r a t e , (b) w h e n c u l t u r e s w e r e g r o w n a e r o b i c a l l y , a n d (c) w h e n r i c h m e d i a w e r e u s e d i n s t e a d o f M M D . T h e l a t t e r c o n d i t i o n c o n f i r m s t h e r e s u l t s o b t a i n e d b y Z a b l o t o w i c z et al. [21] w h o d e s c r i b e d fastgrowing rhizobia as unable to use NO3- by dissimilatory pathway, when grown anoxically in rich media. When different symbiotic phenotypes are compared, the pattern of N20 f o r m a t i o n r e m a i n s a l m o s t t h e s a m e : in t h e p r e s e n c e o f 6 m M KN03, R. trifolii p r o d u c e d i n 8 d a y s 0 . 4 2 ~ M N 2 0 ( s t r a i n 8ST, N o d + Fix+), 0 . 3 0 t~M N 2 0 ( s t r a i n 8 S T - M 7 , N o d + F i x - ) , a n d 0 . 3 2 / ~ M N 2 0 ( s t r a i n 8 S T - M 5 5 , N o d - ) x m g -~ o f
110
S. Casella et al. o
viabte call counts, R hedys|rum R H l g - R I 4
9
viable cell 9
R tri/olll
aST
N2O produced, strain R H l g - R 1 4 9 N20 produced, strain BST []
0 D 6 6 0 changes, strain RH19 R14 9 OD6~ 0 changes)strain 8ST
E 9 'o
E
t~ =
= o 7. | O
Z
2
t
~
~
4
5
g
7
Fig. I. Viable cell counts, OD660 changes, and N20 evolution during anoxic growth of R. trifolii 8ST and R. "hedysarum" RH19-R14 in the presence of 6 mM KNO3.
days
Table 2. N20 formation by different rhizobia grown anoxically in batch cultures in the presence of different nitrogen sources
Strain R. trifolii 8ST
Nitrogen source
Nitrous oxide formed
NO3NO2NH4* NH4+NO3NO3NO2-
0.30 0.42
R. trifolii 8ST-M55
NH4+ NH4+NO3NO3-
0.15 0.32
R. "hedysarum"RHl9-R14 R, leguminosarum LPR 1
NO2NH4 + NH4*NO3NO3NO3-
R. trifolii 8ST-M7
Data are expressed in g M x m g of anaerobic growth in MMD
-t cells d.w.
0.42 0.09
0.35 0.09 0.55 0.40 after 8 days
cells, d . w . S o m e d i f f e r e n c e s w e r e d e t e c t e d w h e n t h e 3 s t r a i n s w e r e g r o w n i n MMD in the presence of various nitrogen sources: Table 2 shows that symbiotically defective strains evolve N20 from either NOz or NO3-, while strain 8ST produces more N20 from nitrate than from nitrite; this could be related to a transient accumulation of the latter compound during the assay, after about 3 days of cultivation in the presence of NO3-. Such an accumulation has been
Nitrous Oxide Production
111
Table 3. Effectsof the organic matter on the production of nitrous oxide by R. trifolii, grown ex planta in the presence of 6 mM KNO3 in peat/sand mixtures
Strain
R. trifolii 8ST R. trifolii 8ST-M7 R. trifolii 8ST-M55
Organic matter
N20 (~M x 100 g-i d.w.) after 8 days
1% 10% 1% 10% 1% 10%
0.75 1.16 0.17 1.60 0.16 2.05
described by Daniel et al. [4] for R. japonicum. W h e t h e r the b e h a v i o r o f defective strains is due to i n d e p e n d e n t m u t a t i o n a l events occurring during isolation, or linked to the loss o f symbiotic properties, awaits further investigations. T h e small a m o u n t s o f NzO f o r m e d in the presence o f (NH4)NO3 are probably p r o d u c e d f r o m the nitrate-nitrogen, since separate controls, obtained with washed cells pregrown in rich m e d i a or M M D plus nitrate or nitrite, and then exposed to a m m o n i u m nitrogen, gave undetectable levels o f nitrous oxide formation. T h e influence o f organic m a t t e r on N 2 0 f o r m a t i o n is shown in Table 3. All R. trifolii evolve nitrous oxide at increasing rates when grown in the presence o f increasing concentrations o f organic matter. T h e above results clearly indicate that fast-growing rhizobia, when cultivated anoxically ex planta, can use N O 3- and NO2- as electron acceptors to evolve gaseous N 2 0 . Furthermore, the process is enhanced when there is a large availability o f electron donors. Therefore, our data support previous suggestions that free-living rhizobia m a y r e m o v e fixed nitrogen from soil by dissimilatory pathways [4]. In order to verify whether N - N z O is p r o d u c e d in a n u m b e r o f forage and grain legumes nodulated by the previously studied and other fast-growing rhizobia, reduction o f added 6 m M nitrate was m o n i t o r e d during in planta assays. Well-nodulated, nitrogen-fixing Trifoliurn incarnatum, Hedysarum coronarium, Pisum sativum, and Medicago sativa were used, and b o t h A R A and N 2 0 evolution were measured as described in Methods. Figure 2a shows the f o r m a t i o n o f N 2 0 f r o m the nodules o f Pisurn sativurn and Medicago sativa, inoculated with their specific rhizobia, at increasing rates o f nitrate present in the m e d i u m ; in the same 2 systems, the specific A R A was m e a s u r e d and the data are reported in Fig. 2b. It appears that dissimilatory nitrate reduction increases with increasing concentrations o f a d d e d NO3-, while in the same conditions the specific A R A is reduced. T h e latter results are in agreement with findings in soybean [18] Phaseolus vulgaris [ 17], Lupinus angustifolius [11], M. sativa [6], and T. subterraneum [5]. By plotting the N 2 0 lost as a percentage o f a d d e d N O 3- versus increasing concentrations o f nitrate present in the m e d i u m , the relative loss decreases, as depicted in Fig. 2c. In other terms, nodule dissimilatory activity tends to reach a plateau when the
112
S. Casella et al.
M sat]va -R.rmzliloti
40-
0
41,
R SQ t ivu rn- R.legum inosaru m
.
~6
="E
~_
2o-
q.
.yi
ol
j
o
j
10-
~
c
~
E
100-
o u z,-,z :k'~
5o-
25-
KNO 3 concentration (mM)
Fig. 2. Evolution o f nitrous oxide (a) ARA, (b) percent o f N-NO3 lost, (c) as measured in planta in M. sativa and Pisum sativum at increasing concentrations of nitrate supplied to the system. Rates shown (2e) on the y axis represent the values of N 2 0 accumulation after 50 hours.
NOa- available to the system is between 9 and 12 mM. This trend towards saturation is better seen for alfalfa than pea nodules. In the case of alfalfa nodules, the N - N 2 0 evolution increases in time, at least during the first 48 hours of incubation, as shown in Fig. 3. For H. coronarium and T. incarnatum, an average o f 10-45 #M N 2 0 x mg -1 nodules d.w. were formed during the first 48 hours o f incubation in the presence of 12 m M NO3-. Particularly for R. "hedysarum" it was found that the 2 strains studied produced significantly different amounts o f N=O: strain R H 1 9 - R 1 4 evolved 15 #M, and strain CC1337 45 IzM x mg-' nodules d.w., after 48 hours incubation. It seems worth noting that the strain that evolves major amounts o f N 2 0 is also more effective for symbiotic nitrogen fixation than the other on H. coronarium [2].
Nitrous Oxide Production
~12
0
nre~
o/o___
o 9 mM
i1--1
6
113
mM
o
/
O~--
o o
g
~'o
ig
2b z'~ 3'0 a's 20 4'5
5'o
s~
Fig. 3. Gaseousnitrogen losses in well-nodulated, nitrogen fixing M. sativa inoculated with R. meliloti L5-30, after exposure to different concentrations of nitrate.
Time from K N O 3 addition (hours)
Other studies, to be published elsewhere, c a r d e d out with plants grown in the presence o f :SN as KNO3 ( 1 - 1 2 raM) have confirmed the a b o v e gas-chrom a t o g r a p h i c data: n o d u l a t i o n and A R A are inhibited by increasing (3-12 m M , according to the legume) c o n c e n t r a t i o n o f nitrate; 15N e n r i c h m e n t tests indicate that nitrogen losses increase almost linearly with increasing a m o u n t s o f nitrate supplied. As it appears that N 2 0 evolution a n d nitrogen fixation (acetylene reduction) m a y run as c o n t e m p o r a r y activities in planta, though with different reciprocal rates according to the symbiotic system, these studies suggest that a broader knowledge is needed on the relationships between the two processes inside the nodule before an adequate a p p r o a c h to o v e r c o m i n g nitrate inhibition effects in the field is established. Acknowledgments. This investigation was supported by the National Research Council, Italy (Spe-
cial grant IPRA, sub-project 1, paper n. 71). The skillful assistance of Dr. C. Robba and Mrs. M. Bonfanti-Casella is gratefully acknowledged.
References 1. Casella S, Ceci D, Bonari E, Lepidi AA, Nuti MP (1981) Batterizzazione di leguminose da foraggio e da granella: un triennio di ricerche in Italia. Rivista di Agronomia 3-4:173-182
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2. Casella S, Reynolds KC, Dyson JR, Gault RR, Brockwell J (1984) Nodulation studies on legumes exotic to Australia: Hedysarum coronariurn. FEMS Microbiol Letters, in press. 3. Citernesi U, Neglia R, Seritti A, Lepidi AA, Bagnoli G, Galluzzi R, Nuti MP (1977) Nitrogenfixing bacteria in the gastroenteric cavity of soil animals. Soil Biol Biochem 9:71-72. 4. Daniel RM, Smith IM, Phillip JAD, Ratcliffe HD, Drozd JW, Bull AT (1980) Anaerobic growth and denitrification by Rhizobiumjaponicum and other Rhizobia. J Gen Microbiol 120: 517-521 5. Gibson AH (1976) Recovery and compensation by nodulated legumes to environmental stress. In: Nutman PS (ed) Symbiotic nitrogen fixation in plants, Cambridge Univ Press, London pp 385-405 6. Heichel GH, Vance CP (1979) Nitrate N and Rhizobium strain roles in alfalfa seedling nodulation and growth. Crop Sci 19:512-518 7. Hooykaas P J J, Snijdewint FGM, Schilperoort RA (1983) Identification of the sym plasmids of Rhizobium leguminosarum strain 1001 and its transfer to and expression in other rhizobia and Agrobacterium tumefaciens (in press) 8. Ishizawa S (1980) Note on nitrate reduction in Rhizobium. Soil Sci Plant Nutr 26:447-450 9. Lepidi AA, Picci G (1967) Nitrato-riduzione e denitrificazione ad opera di Rhizobium leguminosarum (Frank) in terreni cobalto-molibdeno-carenti. Agric Ital pp 223-236 10. Lepidi AA, Nuti MP, Bagnoli G, Filippi C, Galluzzi R (1979) Further researches on the involvement of Rhizobium large plasmids in legume nodulation. In: Bond DA, ScarasciaMugnozza GT, Poulsen MH (eds) Some current research on Vicia faba in Western Europe, EEC Publ Off, Luxemburg pp 436-460 11. Manhart J, Wong PP (1980) Nitrate effect on nitrogen fixation (acetylene reduction). Plant Physiol 65:502-505 12. Nuti MP, Casella S, Filippi C, Lepidi AA, Galluzzi R (1981) Rhizobia as inoculant for field trials in marginal soils of middle and northern Italy. In: Gibson AH, Newton WE (eds) Current Perspectives in Nitrogen Fixation, Austr Acad Sci Canberra p 514 13. Nuti MP, Lepidi AA, Schilperoort RA, Hooykaas PJJ, Prakash RK (1982) The plasmids of Rhizobium and symbiotic nitrogen fixation. In: Kahl G, Schell J (eds) Molecular Biology of Plant Tumors, Academic Press pp 561-588 14. Pedrazzini F, Nannipieri P (1982) Evolution of nitrous oxide in a nitrate-treated soil at a low partial pressure of acetylene. Plant and Soil 66:429-431 15. Prakash RK, Hooykaas PJJ, Lebedoer AM, Nuti MP, Lepidi AA, Juliot JS, Denari6 J (1980) Detection, isolation, and characterization of large plasmids in Rhizobium. In: Newton WE, Orme-Johnson N H (eds) Nitrogen Fixation, vol II. Univ Park Press, Baltimore, pp 136-163 16. Rajagopalan T (1938) Studies on groundnut nodule organism. IV. Physiology of the organism: intermediary metabolism. Ind J Agric Sci 8:379-382 17. Rigaud J (1976) Effet des nitrates sur la fixation d'azote par les nodules de haricot (Phaseolus vulgaris L.). Physiol Veg 14:297-308 18. Rigaud J, Bergersen FJ, Turner GL, Daniel RM (1973) Nitrate-dependent anaerobic acetylene reduction and nitrogen fixation by soybean bacteroids. J Gen Mierobiol 77:137-144 19. Vincent J (1970) A manual for the practical study of root-nodules bacteria. IBP Handbook n 15, Blackwell Sci Publ, Oxford, pp 164 20. Wilson JK (1947) The legume bacteria liberate gaseous nitrogen from nitrate. Proc Soil Sci Soc Am 12:215 21. Zablotowicz RM, Eskew DL, Focht DD (1978) Denitrification in Rhizobium. Can J Microbiol 24:757-760 22. Zablotowicz RM, Focht DD (1979) Denitrification and anerobic nitrate-dependent acetylene reduction in cowpea Rhizobium. J Gen Microbiol I 11:445-448
