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Acetylene reduction activity associated with tree roots in cypress wetlands
Soil Biol. Biochem Vol. 13, pp. 555 lo 557, 1981 Punted in Great Britain. All rights reserved
CopyrIght
0038.0717/81/060555-03$02.00/O 0 1981 Pergamon Press Ltd
SHORT COMMUNICATION Acetylene reduction activity associated with tree roots in cypress wetlands FORRESTE. DIERBERG*and PATRICK L. BREZONIK
Department of Environmental and Engineering Sciences, University of Florida, Gainesville, FL 32611, U.S.A.
(Accepted
10 May 1981)
Acetylene reduction activity (as evidence of N, fixation) has been found associated with non-nodulated roots of many plants including forage grasses, cereals, and several non-woody wetland plants (Dtibereiner et al. 1972; von Biilow and Diibereiner 1975; Patriquin and Keddy 1978; Zuberer and Silver 1978). The possibility of N, fixation in the rhizosphere and endorhizosphere of dominant woody plants in forested wetlands does not appear to have been investigated. Forested wetlands encompass large areas within the United States, especially in the Southeast (e.g. the Big Cypress Swamp in Florida, the Okefenokee Swamp on the Georgia-Florida border). Small areas of forested wetlands are common throughout this region, and are known as cypress domes, cypress strands, hydric hammocks and swamps. Such wetlands cover 25-60% of the land area in many Florida counties. Cypress trees (Taxodium distichum var. nutans) and swamp black gum (Nyssa sylvatica var. &flora) comprise the dominant overstory vegetation in these forested wetlands; buttonbush (Cephulanthus occidentab) is a common shrub in these systems. We measured nitrogenase activity by the C,H2 reduction method on washed excised roots (~0.4 cm dia) from the above-mentioned major woody species in cypress wetlands near Gainesville. Three sires were tested for rootassociated N2 fixation between August 1978 and September 1979. The major site was a 4.2-ha cypress dome about 17 km northeast of Gainesville in the Austin Cary Memorial Forest. We also sampled a l.OS-ha cypress dome just north of Gainesville that has received secondary sewage effluent for 4 yr and the cypress-lined margin of a soft water, colored lake (Newnans Lake) east of Gainesville. Small pond cypress trees (2-6cm dia at breast height; height 18&470 cm) with intact roots were transported in a bucket containing in situ substrate to the laboratory where the roots were excised. Roots were excised in the field for all other trees. Two to four hours elapsed between the time samples were taken and C2H2 was added to the excised roots in assay containers. Acetylene was generated from calcium carbide and water. Excised roots were washed free of soil with deionized water before being inserted (0.7-4.0 g fresh wt) into assay containers (9.5%70ml). Each experiment consisted of three to eight replicates. Incubations were carried out either aerobically (ambient air) or anaerobically (He) in the dark at room temperature (2426°C) with deionized water or in situ water added (filling 10% of the container volume). Pressure within the incubation bottles was maintained at 1 atm by bleeding excess pressure with a needle after adding C,H, (0.1 atm) and by replacing each 0.5 ml of gas phase withdrawn for analysis with 0.5 ml * Present address: Department of Science and Engineering, Florida Institute Melbourne, FL 32901, U.S.A.
c
500
loo-
T
(a)
(b)
z E
8o-
b B
natural
dome
6o-
2 I” 0 z
N 40-
g
1 -+ISE zo-
0
0
24
52 Time,
70
96
h
Fig. 1. Acetylene reduction activity in excised trees roots. (a) N. syluatica and surrounding peat sampled 30 August 1978, from Austin Cary dome and T. distichum sampled 23 October 1978, from Newnans Lake. (b) T. distichum sampled 5 April 1979, from Austin Cary cypress dome and sewage-enriched cypress dome. Each point represents the mean of 3-8 replications.
Environmental of Technology, 555
Short communications
556
z E
200-
0
20
42
68
Time,
92
h
Fig. 2. Acetylene reduction activity in surface-sterilized excised roots of Taxodium distichum sampled 22 September 1979. Each point represents the mean of 5 replicates.
He. Gas samples were analysed for C2H, with a Varian Aerograph 600D g.c. with f.i.d. using a 2.7 m x 0.3 cm column at 55°C packed with Poropak R and N, as the carrier gas. Samples were withdrawn from the bottles four or more times during incubations periods of 3-5 days. Incubations of roots without C2Hz and of water with C2H2 but without roots to determine background CZH4 production consistently yielded negligible quantities of CZH~. In all experiments, there was a lag before C2H, was produced. The duration of the lag was variable but generally lasted less than 24 h and was independent of O2 conditions. Similar results have been obtained for excised roots of grasses (e.g. Dabereiner et al. 1972). The cause of the delay has been the subject of controversy, but van Berkum (1980) concluded that damage of nitrogenase by O2 is an unlikely cause. He suggested that N,-fixing bacteria grew on carbon substrates released from excised sorghum roots and that prolonged incubations overestimate NZ fixation associated with plants in the field. Reduction of CZH2 was found in washed, excised, nonnodulated roots of two dominant trees (black gum and pond cypress) from the Austin Cary cypress dome and pond cypress roots from Newnans Lake; insignificant rates were found in peat associated with black gum roots (Fig. 1). The results shown for each species in Fig. 1 represents averages of several samples of a single tree and indicate that fixation does not occur uniformly in or on the roots. Reduction of C,H2 was not observed during a 96 h incubation of cypress roots from the sewage-enriched cypress dome, but reduction occurred in a root sample obtained at the same time from the Austin Cary dome (Fig. lb), suggesting that NZ fixation is repressed by the concentrations of combined N in pore water of the sewage-enriched dome. Rates observed in cypress roots from the Austin Cary dome when leaves were starting to emerge in April (Fig. lb) were only one-tenth of those measured in cypress roots from the same dome near the end of the growing season (but substantially before leaf fall) in September (untreated controls in Fig. 2). suggesting a lmk between photosynthetic activity and root nitrogenase activity. An experiment was done to determine whether the N,-fixing agents are associated with root surfaces or with the interior root tissue. Excised roots of a 3.7 m cypress tree were divided into three groups and immersed for 1.5 h in either l”,:, or 10’4 chioramine-T (except the proximal cut
end) or in sterile phosphate buffer (pH 7). All assay bottles (70 ml capacity) contained 25 ml of dome water in addition to the roots. The occurrence of C,H2 reduction activity in chloramine-T treated roots (Fig. 2) indicates that N,-fixing organisms invade the root tissues. Most of the activity was associated with the root surface; however, activity was only 43 and 25% of the untreated controls in 1 and 10% chloramine-T treated roots, respectively. Patriquin and DGbereiner (1978) and Day and DGbereiner (1976) have reported that bacteria are the N,-fixing agents in the rhizosphere and roots of non-woody, nonnodulated plants. On this basis, bacteria would seem to be the most likely agents responsible for nitrogenase activity associated with the roots of wetlands trees. Given the location of fixation and the possibility that the trees studied here oxidize their rhizosphere, as Hook and Brown (1973) found for other wetland tree species, it is probable that the agents of fixation are microaerophiles, but the agents have not yet been isolated nor have their growth requirements been established. The maximum CzH2 reduction rate measured on cypress trees roots [17.3 nmol C,_H,g-’ fresh wt hi’] is about one-tenth of the rates found in nodules of nonleguminous trees but 10 times that reported for sediments and the rice root zone (Burns and Hardy, 1975). Data obtained by the procedures used here should be referred to as “potential nitrogenase activity” (Patriquin and Denike, 1978) since (i) transfer of fixed N to tree roots has not been demonstrated; (ii) NZ fixation was assayed by an indirect technique: and (iii) a lag was observed in the incubations of excised roots before C2H2 reduction began (van Berkum 1980). Nonetheless, these results and those of others, e.g. Patriquin and Keddy (1978), Smith and Patriquin (1978), suggest that N2 fixation associated with roots of wetland vegetation may be widespread. The occurrence of fixation in natural cypress domes is consonant with the low concentrations of available N and the low rates of mineralization of N in litterfall in cypress domes (F. E. Dierberg, unpublished Ph.D. dissertation, University of Florida, 1980). Nz fixation also was found by Dierberg in other areas of cypress domes, including surface litter, Azol!a floating on the water surface, and the nodulated shrub Myrica.
Acknowledgements~We thank Shirley Kooijman and Walter Judd for assistance in botanical identifications. This work was supported in part by a grant from the U.S. National Science Foundation.
REFERENCES VAN
BERKUM P. (1980) Evaluation of acetylene reduction by excised roots for the determination of nitrogen fixation in grasses. Soil Biology & Biochemistry 12, 141-145. VON Bii~ow J. W. P. and D~~BEREINER.I. (1975) Potential for nitrogen fixation in maize genotypes in Brazil. Proceedings of the Nutionul Academy of Sciences of the U.S.A. 72, 2389-2393. BURNS R. C. and HARDY R. W. F. (1975) Nitrogen Fixation in Bacteria and Higher Plants. Springer-Verlag. New York. DAY J. M. and D~~BEREINERJ. (1976) Physiological aspects of No-fixation by a Spirillum from Digiraria roots, Soil Biology
& Biochemistry
8, 45-50.
DC~BEREINER J., DAY J. M. and DART P. J. (1972) Nitrogenase activity and oxygen sensitivity of the Paspalum notatum-Azotohactrr paspali association. Journal of Generrrl Microbiology 71, 103%116. HOOK D. D. and BROWN C. L. (1973) Root adaptations and relative flood tolerance of five hardwood species. Forest Science 19, 225-229.
Short communications PATRIQUIND. G. and DENIKED. (1978) In situ acetylene reduction assays of nitrogenase activity associated with the emergent halophyte Spartina alternij7ora Loisel: methodological problems. Aquatic Botany 4, 21 l-226. PATRIQUIND. G. and D~BEREINERJ. (1979) Light microscopy observations of tetrazolium-reducing bacteria in the endorhtzosphere of maize and other grasses in Brazil. Canadian Journal of Microbiology 24, 734742.
PATRIQUIND. G. and KEDDYC. (1978) Nitrogen activity (acetylene reduction) in a Nova Scotian salt marsh; its
557
association with angiosperms and the influence of some edaphic factors. Aquatic Botany 4, 227-244. SMITHD. and PATRIQUIND. G. (1978) A survey of the angiosperms in Nova Scotia for rhizosphere nitrogenase (acetylene reduction) activity. Canadian Journal of Botany 56, 2218-2223.
ZUBERERD. A. and SILVERW. S. (1978) Biological dinitrogen fixation (acetylene reductton) associated with Florida mangroves. Applied and Environmental Microbzorogy 35, 567-575.
CopyrIght
0038.0717/81/060555-03$02.00/O 0 1981 Pergamon Press Ltd
SHORT COMMUNICATION Acetylene reduction activity associated with tree roots in cypress wetlands FORRESTE. DIERBERG*and PATRICK L. BREZONIK
Department of Environmental and Engineering Sciences, University of Florida, Gainesville, FL 32611, U.S.A.
(Accepted
10 May 1981)
Acetylene reduction activity (as evidence of N, fixation) has been found associated with non-nodulated roots of many plants including forage grasses, cereals, and several non-woody wetland plants (Dtibereiner et al. 1972; von Biilow and Diibereiner 1975; Patriquin and Keddy 1978; Zuberer and Silver 1978). The possibility of N, fixation in the rhizosphere and endorhizosphere of dominant woody plants in forested wetlands does not appear to have been investigated. Forested wetlands encompass large areas within the United States, especially in the Southeast (e.g. the Big Cypress Swamp in Florida, the Okefenokee Swamp on the Georgia-Florida border). Small areas of forested wetlands are common throughout this region, and are known as cypress domes, cypress strands, hydric hammocks and swamps. Such wetlands cover 25-60% of the land area in many Florida counties. Cypress trees (Taxodium distichum var. nutans) and swamp black gum (Nyssa sylvatica var. &flora) comprise the dominant overstory vegetation in these forested wetlands; buttonbush (Cephulanthus occidentab) is a common shrub in these systems. We measured nitrogenase activity by the C,H2 reduction method on washed excised roots (~0.4 cm dia) from the above-mentioned major woody species in cypress wetlands near Gainesville. Three sires were tested for rootassociated N2 fixation between August 1978 and September 1979. The major site was a 4.2-ha cypress dome about 17 km northeast of Gainesville in the Austin Cary Memorial Forest. We also sampled a l.OS-ha cypress dome just north of Gainesville that has received secondary sewage effluent for 4 yr and the cypress-lined margin of a soft water, colored lake (Newnans Lake) east of Gainesville. Small pond cypress trees (2-6cm dia at breast height; height 18&470 cm) with intact roots were transported in a bucket containing in situ substrate to the laboratory where the roots were excised. Roots were excised in the field for all other trees. Two to four hours elapsed between the time samples were taken and C2H2 was added to the excised roots in assay containers. Acetylene was generated from calcium carbide and water. Excised roots were washed free of soil with deionized water before being inserted (0.7-4.0 g fresh wt) into assay containers (9.5%70ml). Each experiment consisted of three to eight replicates. Incubations were carried out either aerobically (ambient air) or anaerobically (He) in the dark at room temperature (2426°C) with deionized water or in situ water added (filling 10% of the container volume). Pressure within the incubation bottles was maintained at 1 atm by bleeding excess pressure with a needle after adding C,H, (0.1 atm) and by replacing each 0.5 ml of gas phase withdrawn for analysis with 0.5 ml * Present address: Department of Science and Engineering, Florida Institute Melbourne, FL 32901, U.S.A.
c
500
loo-
T
(a)
(b)
z E
8o-
b B
natural
dome
6o-
2 I” 0 z
N 40-
g
1 -+ISE zo-
0
0
24
52 Time,
70
96
h
Fig. 1. Acetylene reduction activity in excised trees roots. (a) N. syluatica and surrounding peat sampled 30 August 1978, from Austin Cary dome and T. distichum sampled 23 October 1978, from Newnans Lake. (b) T. distichum sampled 5 April 1979, from Austin Cary cypress dome and sewage-enriched cypress dome. Each point represents the mean of 3-8 replications.
Environmental of Technology, 555
Short communications
556
z E
200-
0
20
42
68
Time,
92
h
Fig. 2. Acetylene reduction activity in surface-sterilized excised roots of Taxodium distichum sampled 22 September 1979. Each point represents the mean of 5 replicates.
He. Gas samples were analysed for C2H, with a Varian Aerograph 600D g.c. with f.i.d. using a 2.7 m x 0.3 cm column at 55°C packed with Poropak R and N, as the carrier gas. Samples were withdrawn from the bottles four or more times during incubations periods of 3-5 days. Incubations of roots without C2Hz and of water with C2H2 but without roots to determine background CZH4 production consistently yielded negligible quantities of CZH~. In all experiments, there was a lag before C2H, was produced. The duration of the lag was variable but generally lasted less than 24 h and was independent of O2 conditions. Similar results have been obtained for excised roots of grasses (e.g. Dabereiner et al. 1972). The cause of the delay has been the subject of controversy, but van Berkum (1980) concluded that damage of nitrogenase by O2 is an unlikely cause. He suggested that N,-fixing bacteria grew on carbon substrates released from excised sorghum roots and that prolonged incubations overestimate NZ fixation associated with plants in the field. Reduction of CZH2 was found in washed, excised, nonnodulated roots of two dominant trees (black gum and pond cypress) from the Austin Cary cypress dome and pond cypress roots from Newnans Lake; insignificant rates were found in peat associated with black gum roots (Fig. 1). The results shown for each species in Fig. 1 represents averages of several samples of a single tree and indicate that fixation does not occur uniformly in or on the roots. Reduction of C,H2 was not observed during a 96 h incubation of cypress roots from the sewage-enriched cypress dome, but reduction occurred in a root sample obtained at the same time from the Austin Cary dome (Fig. lb), suggesting that NZ fixation is repressed by the concentrations of combined N in pore water of the sewage-enriched dome. Rates observed in cypress roots from the Austin Cary dome when leaves were starting to emerge in April (Fig. lb) were only one-tenth of those measured in cypress roots from the same dome near the end of the growing season (but substantially before leaf fall) in September (untreated controls in Fig. 2). suggesting a lmk between photosynthetic activity and root nitrogenase activity. An experiment was done to determine whether the N,-fixing agents are associated with root surfaces or with the interior root tissue. Excised roots of a 3.7 m cypress tree were divided into three groups and immersed for 1.5 h in either l”,:, or 10’4 chioramine-T (except the proximal cut
end) or in sterile phosphate buffer (pH 7). All assay bottles (70 ml capacity) contained 25 ml of dome water in addition to the roots. The occurrence of C,H2 reduction activity in chloramine-T treated roots (Fig. 2) indicates that N,-fixing organisms invade the root tissues. Most of the activity was associated with the root surface; however, activity was only 43 and 25% of the untreated controls in 1 and 10% chloramine-T treated roots, respectively. Patriquin and DGbereiner (1978) and Day and DGbereiner (1976) have reported that bacteria are the N,-fixing agents in the rhizosphere and roots of non-woody, nonnodulated plants. On this basis, bacteria would seem to be the most likely agents responsible for nitrogenase activity associated with the roots of wetlands trees. Given the location of fixation and the possibility that the trees studied here oxidize their rhizosphere, as Hook and Brown (1973) found for other wetland tree species, it is probable that the agents of fixation are microaerophiles, but the agents have not yet been isolated nor have their growth requirements been established. The maximum CzH2 reduction rate measured on cypress trees roots [17.3 nmol C,_H,g-’ fresh wt hi’] is about one-tenth of the rates found in nodules of nonleguminous trees but 10 times that reported for sediments and the rice root zone (Burns and Hardy, 1975). Data obtained by the procedures used here should be referred to as “potential nitrogenase activity” (Patriquin and Denike, 1978) since (i) transfer of fixed N to tree roots has not been demonstrated; (ii) NZ fixation was assayed by an indirect technique: and (iii) a lag was observed in the incubations of excised roots before C2H2 reduction began (van Berkum 1980). Nonetheless, these results and those of others, e.g. Patriquin and Keddy (1978), Smith and Patriquin (1978), suggest that N2 fixation associated with roots of wetland vegetation may be widespread. The occurrence of fixation in natural cypress domes is consonant with the low concentrations of available N and the low rates of mineralization of N in litterfall in cypress domes (F. E. Dierberg, unpublished Ph.D. dissertation, University of Florida, 1980). Nz fixation also was found by Dierberg in other areas of cypress domes, including surface litter, Azol!a floating on the water surface, and the nodulated shrub Myrica.
Acknowledgements~We thank Shirley Kooijman and Walter Judd for assistance in botanical identifications. This work was supported in part by a grant from the U.S. National Science Foundation.
REFERENCES VAN
BERKUM P. (1980) Evaluation of acetylene reduction by excised roots for the determination of nitrogen fixation in grasses. Soil Biology & Biochemistry 12, 141-145. VON Bii~ow J. W. P. and D~~BEREINER.I. (1975) Potential for nitrogen fixation in maize genotypes in Brazil. Proceedings of the Nutionul Academy of Sciences of the U.S.A. 72, 2389-2393. BURNS R. C. and HARDY R. W. F. (1975) Nitrogen Fixation in Bacteria and Higher Plants. Springer-Verlag. New York. DAY J. M. and D~~BEREINERJ. (1976) Physiological aspects of No-fixation by a Spirillum from Digiraria roots, Soil Biology
& Biochemistry
8, 45-50.
DC~BEREINER J., DAY J. M. and DART P. J. (1972) Nitrogenase activity and oxygen sensitivity of the Paspalum notatum-Azotohactrr paspali association. Journal of Generrrl Microbiology 71, 103%116. HOOK D. D. and BROWN C. L. (1973) Root adaptations and relative flood tolerance of five hardwood species. Forest Science 19, 225-229.
Short communications PATRIQUIND. G. and DENIKED. (1978) In situ acetylene reduction assays of nitrogenase activity associated with the emergent halophyte Spartina alternij7ora Loisel: methodological problems. Aquatic Botany 4, 21 l-226. PATRIQUIND. G. and D~BEREINERJ. (1979) Light microscopy observations of tetrazolium-reducing bacteria in the endorhtzosphere of maize and other grasses in Brazil. Canadian Journal of Microbiology 24, 734742.
PATRIQUIND. G. and KEDDYC. (1978) Nitrogen activity (acetylene reduction) in a Nova Scotian salt marsh; its
557
association with angiosperms and the influence of some edaphic factors. Aquatic Botany 4, 227-244. SMITHD. and PATRIQUIND. G. (1978) A survey of the angiosperms in Nova Scotia for rhizosphere nitrogenase (acetylene reduction) activity. Canadian Journal of Botany 56, 2218-2223.
ZUBERERD. A. and SILVERW. S. (1978) Biological dinitrogen fixation (acetylene reductton) associated with Florida mangroves. Applied and Environmental Microbzorogy 35, 567-575.