Acetylene reduction by roots and associated soil of New Zealand conifers

Soil Biol. Biochem.Vol. 5, pp. 171-179.PergamonPress 1973.Printedin Great Britain

ACETYLENE REDUCTION BY ROOTS AND ASSOCIATED SOIL OF NEW ZEALAND CONIFERS W. B. SILVESTER and K. J. Department

of Botany, University

BENNETT

of Auckland, Auckland, New Zealand

(Accepted 15 March 1972) extensive survey of native conifers (Dacrydium, Podocarpus, Libocedrus, Phylloreduction was often associated with their mycorrhizal short roots (‘nodules’). C,H, reduction was associated with roots only if it also occurred in surrounding soil, but it could be found in soils and not in the root region. Over 50 per cent of the C2H2 reduction activity could be removed by washing roots in distilled water while complete loss of activity occurred when they were surface sterilized with hypochlorite solution. CzH2 reduction may also be associated with long roots of podocarps and roots of non-podocarp species. Fermenting and humifying horizons of forest soils showed much greater rates of C2H2 reduction than either mineral soil or roots. Results suggest that previous claims for nitrogen fixation by podocarp roots can be attributed to nitrogen fixation by bacteria in the root region rather than by endophytic organisms. Summary-An

cludus and Agathis spp.) showed that C,H,

INTRODUCTION THE NATIVE conifer

species of New Zealand constitute the bulk of the country’s indigenous timber resources, and all species bear the characteristic short roots (Yeates, 1924; Baylis et al., 1963) commonly known as nodules. The determination of significant nitrogen fixation by these roots may therefore be of considerable economic significance. Allen and Allen (1965) have reviewed information about the widespread occurrence of this root type which superficially resemble legume nodules. The use of the term ‘nodule’ in relation to conifers is considered to be misleading (B. N. Richards, personal communication) as it implies analogy with legume nodules. These structures are in fact modified lateral roots (short roots) formed during the normal development of the conifer; the term short root will be adopted in this paper. Short roots have been grown in aseptic culture (Kahn, 1967) independently of endophytic organisms. It is normal for short roots to become infected by a phycomycete to produce endotrophic mycorrhizas (Baylis, et al., 1963) although under some conditions they may remain uninfected. Baylis (1969a) has synthesized the endotrophic mycorrhizas of Podocarpus and Agathis using spores of Endogone. There are also two reports (Uemura, 1964; Morrison and English, 1967) of Streptomyces occurring within short roots of conifers. Bond (1967) reviewed the evidence for nitrogen fixation by conifer short roots much of which is inconclusive. The current position is that while positive results have been reported by some workers (Bergersen and Costin, 1964; Becking, 1965; Morrison and English, 1961) others have reported negative or equivocal results (Bond, 1959; 1967; Baylis, 1969b; Furman, 1970). Current evidence for nitrogen fixation by conifer roots is based therefore on a limited number of isolated experiments with single species, mostly made on laboratory material. Our study was an attempt to clarify the issue and involved taking a large number of field samples of many species and assaying for nitrogenase using the C,H, reduction technique. 171

172

W. B. SILVESTER

AND

K. J. BENNETT

MATERIALS

AND

METHODS

The C2H, reduction assay for nitrogenase activity (Dilworth, 1966; Schollhorn and Burris, 1967) was modified in the following way. A gas mixture of Ar/O, : 712 was prepared by water displacement in a 3 1. polythene aspirator with a similar vessel for C,H, both maintained a 30 cm water pressure. Samples of roots or soil were placed in 30 ml McCartney bottles or 100 ml flasks fitted with serum liners, evacuated with a hand vacuum pump on a manifold, flushed three times with Ar/O,, 10 per cent of gas phase was then removed with a syringe and replaced with C2H,. During early survey work using this method it was necessary to ‘fix’ samples in the field by injecting O-5 ml 50 % (v/v) trichloracetic acid (TCA) (Stewart et al., 1968). This method sometimes led to greatly enhanced CzH4 peaks in soil samples so then we always returned the bottles to the laboratory and sampled from the living system. In all cases duplicate 0.1 cm3 gas samples were injected into a Hewlett-Packard gas chromatograph using a 130 cm Porapak T column, with N2 as the Carrie1 gas and a hydrogen flame ionization detector. Peak heights were measured and C,H, production estimated using C,H, as the internal standard. A check was kept on spontaneous C,H, production, known to be considerable from anaerobic soils (Smith and Scott-Russell, 1969), and on the amount of C,H, contamination of the C*H,. Spontaneous C,H4 production was insignificant in all tests and background C,H, contamination was routinely subtracted from C,H, peaks. Whenever possible samples were taken after various times to establish continuous production of CzH4. Results are expressed as nmoles C,H, g-’ dry wt. of tissue or soil. Roots were obtained by removing litter (L) and fermenting-humifying layers (F/H) of forest floor beneath specimen trees and cutting sample pieces from the root system which was concentrated at the interface between F/H and mineral soil. Roots were washed by shaking in a closed 200 ml bottle containing 50 ml distilled water or alternatively in the laboratory by brushing gently with a soft nylon brush on a glass plate under running deionized water. Surface sterilization was performed for 35 s in contantly stirred 2 % (w/v) sodium hypochlorite, soil being wrapped in muslin bags, followed by washing in running water for 4 min. RESULTS

Field survey for C,H, reduction A field survey was made of 14 species of indigenous conifer. The data (Table 1) show that almost all species tested were capable of C,H, reduction and that, when tested, soil also gave positive results. Most samples were taken from local lowland mixed forest at Swanson and Titirangi and the results (Table 1) for these areas represent a small proportion of tests made. On the other hand only one trip was made to Tongariro National Park therefore the results for these species represent one sampling period. Further, samples from Tongariro were fixed in TCA and the results, at least for soil samples, are open to criticism (see Materials and Methods section). All subsequent assays were performed on unfixed material. As samples were gassed and incubated in the field without control of temperature the values for C2H, reduction given should not be taken to indicate the relative performance of each species. Further, a zero result does not necessarily indicate that the species is incapable of C,H, reduction but probably indicates that insufficient trees were sampled. Despite the obvious limitations of this survey it does represent an extensive confirmation of nitrogenase activity associated with conifer roots.

ACETYLENE TABLE

REDUCTION

toturu G. Berm. ex D. Don Podocarpus totara soil 2. P. ferrugineus G. Benn. ex D. Don P. ferrugineus soil

1. Podocurous

Soil distant from 2

7. 8. 9. 10. 11. 12. 13.

14.

ROOTS

173

1. RESULTSOF FIELDSURVEYS FORC2Hz REDUCTION IN CONIFERSPECIES.ALL SAMPLES INCUBATED IN THE FIELDIN Ar/0&2H2 (V/V) (0.70/0.20/0.10) No. of samples

3. 4. 5. 6.

BY CONIFER

P. ferrugineus G. Benn. ex D. Don P. dacrydioides A. Rich P. hallii Kirk P. spicatus R. Br. ex Mirbel P. niualis Hook P. nivalis Hook soil Dacrydium biforme Pilger D. bidwillii Hook. f. ex Kirk D. laxifolium Hook. f. D. cupressinum Lamb. D. cupressinum Lamb. soil Libocedrus bidwillii Hook. f. Phyllocladus alpinus Hook. f. P. trichomanoides D. Don P. trichomanoides soil P. trichomanoides D. Don P. trichomanoides soil Agathis australis Salisb. Aguthis australis soil

Soil distant from 14 australis Salisb.

Agathis

6 2 6 2 2 1 8 1 1 1 1 3 4 2 7 2 2 1 6 2 2 2 8 2 2 4

Source*

S S S S S TNP S TNP TNP TNP TNP TNP TNP TNP S S TNP TNP S S UA UA T T T S

C2H4 production range (nmoles g-r h-l)

0.24-0.58 0.29-0.49 0.68-l .68 1.06-l .09 0.30-0.32 0.00 0.56-1.96 0.38 0.00 0.00 0.90 0.09-0.12 0.21-0.61 0.17-0.17 O.lCO.39 0.20-0.22 0.43-0.47 0.00 0.16-0.31 0~05-0~07 0.00 0.00 l-84-2.93 0.55-0.64

0.60-l *OO 1.00-1.24

* Sampling sites; S, Swanson; TNP, Tongariro National Park; T, Titirangi; UA, University of Auckland gardens.

C,H,

reduction by Dacrydium cupressinum

Local material of this species was shown to have reproducible rates of &Hz reduction. A long term C,H4 production experiment was carried out to test sustained activity of the material. All material for this and subsequent experiments was obtained from areas of natural forest in the Waitakere Ranges, west of Auckland city. The following material was sampled: Humifying organic matter immediately above mineral soil (F/H). Soil from the upper A mineral horizon. Detached root systems including many short roots. As (c) but washed in water. Results presented in Fig. 1 show an almost linear production of C2H, over the 24 h period. The washing, which consisted simply of shaking roots in a jar of water, brought about a 50 per cent reduction in C2H, reduction activity which was sustained over 24 h. The results (Fig. 1) are means of two replicate bottles for each treatment. The agreement between replicates for this and most other experiments was very good. S.B.B. 5/1-M

174

W. B. SILVESTER AND K. J. BENNETT

* F/H .

/

h

FIG. 1. Ethylene production by roots of Dacrydium cupressinum and associated soil over 24 h (means of two replicates).

Locus of C,H,

reduction

The finding that at least some of the C2H, reduction activity could be removed by simple washing and that this activity was not regenerated in 24 h led us to investigate further the possible locus of C2H, reduction. Roots were brought to the laboratory, washed with a brush in running water combined with mild hypochlorite surface sterilization. The results (Table 2.) show that washing soil from D. cupressinum roots reduces their ability to reduce C,H, by 82 per cent while surface sterilization eliminates activity whether preceded by washing or not. Similar experiments have been made on other species and in all cases the effect of washing or surface sterilization with hypochlorite was to remove all or most of the C2H, reduction potential. Further, it has been found that: (a) Roots of P. trichomanoides lacking short lateral roots were capable of C,H, 1.20 nmoles CzH4 g-’ in 30 h compared with I.67 nmoles g-l for roots short shoots. (b) Roots of D. cuppressinum from which short roots had been removed were C,H, reduction; 2.95 nmoles C,H4 g-’ in 30 h (cf. Table 2). (c) Roots of Collospermum hastatum, produced l-15 nmoles C,H, g-l in 24 roots of Coprosma lucida and Cyathea medullaris from the same area showed Rhizosphere

reduction; possessing capable

of

h although no activity.

effect

Our results suggest that any nitrogen fixation activity (as indicated C,H,) resides in the soil not within the plant. The fact that roots activity on a weight basis than does the surface layer of mineral soil in strongly suggests enhanced activity of the nitrogen-fixing organisms root. To test this hypothesis, eight replicate samples of roots from sociated soil and overlying F/H material were collected and incubated

by ability to show greater which they lie in the region

reduce specific (Fig. 1) of the

Agathis australis, as-

in C2H2. Soil from

ACETYLENE TABLE 2. C,H,PRODUCTION

REDUCTION

BY CONIFER

BY F/H AND ROOTS OF D. cupressinum

Treatment

Material Roots (n = 4)

Unwashed Washed Surface sterilized Washed and surface sterilized

F/H (n = 2)

Unsterilized Sterilized

175

ROOTS INCUBATEDFOR

24 h

CZH4 production (nmoles g - ‘)

Reduction (%)

26.7 4,8 0 0

82 100 100

632.8 176.3

72

each root sample was then washed in 1 per cent calgon (sodium hexametaphosphate), to facilitate removal of all soil particles, and weighed in order to express results in terms of soil weight. The results (Table 3) showed poor replication probably due to the generally low specific activity of the material. Nevertheless the result is clear and confirms our previous finding. A specific enhancement of C,H, reduction in the rhizosphere soil compared with non-rhizosphere soil from the same horizon. TABLE~.C~H~PRODUCTION(~~ SOIL, RHIZOSPHERE SOIL AND australis

h) FROMMINERAL F/H OF &his

Material (n = 8)

CzH4 production (nmoles g- r)

Mineral soil Rhizosphere soil E/H

1.77 f 1.70* 5.16 f 2.10 37.00 rt 10.0

* Standard error.

EfSct of pOz

Roots of D. cupressinum and overlying F/H were incubated at pOZ of 0.0, O-05, 0.10, 0.20 and 0.40 atm, pCzHz 0.1 atm, Ar to 1 atm. &Hz production was determined at intervals during the 27 h incubation and results for two periods are presented in Fig. 2. The optimum p0, for C2H, reduction is at 0.2 atm for F/H but is nearer 0 * 1 atm for roots. Inhibition of CzHz reduction occurred at high p0, and there was a marked increase in rate after 17 h which was especially marked in root samples incubated without oxygen (Table 4). C,H4 production rate increased with time at all concentrations of oxygen, possibly due to the uptake in oxygen by respiration in all bottles, those bottles at high p0, showing the smallest increase in C,H, reduction. Identity of organisms Effect of light on C,H, reduction. Duplicate samples of soil associated with roots of D. cupressinum were incubated under acetylene in the light (3000 lux) and dark at 25°C in

30 ml McCartney bottles. The results (Table 5) showed no significant difference in C,H, reduction between light and dark samples even after 21 h. We conclude therefore that blue-green algae were not active in the soil associated with conifer roots.

176

W. B. SILVESTER AND K. J. BENNETT

r

I

0

I

01

0.2

I

I

03

0.4

POP

FIG. 2. Rates of ethylene production by roots and F/H of Dacrydium cupressinum for initial 17 h (broken line) and 17-27 h (continuous line) at varying initial oxygen tensions. TABLE 4. EFFECTOF pOz ON C,H, PRODUCTIONBY ROOTSAND F/H OF D. cupressinum RATE DURING 17-27 h INCUBATIONAS PERCENTAGEOF INITIALRATE Initial pOz (atm)

Roots F/H

0.0

0.05

0.10

0.20

0.40

830 370

100 70

61 50

50 35

11 50

Isolation of nitrogen-fixing bacteria. Sixty samples of soils and roots of various conifer species were inoculated either directly onto nitrogen-free agar for isolation of nitrogen fixing bacteria (Bremner and Shaw, 1958) or initially onto high-nitrogen media such as yeast extract agar and subsequently onto nitrogen-free agar. All isolates obtained were tested for C2H, reduction over 24 h. One sample from soil showed C,H, reduction. This was subsequently found to be a mixed culture, one component of which has been tentatively identified as Azotobacter.

DISCUSSION

The present survey of mixed forest species showed that low rates of C,H, reduction are commonly associated with short roots of conifers. This activity is irregular in distribution with low and variable rates reaching a maximum of ca. 3 nmoles C,H4 g- ’ h-l. The theoretical ratio of C,H, : NH, produced by nitrogenase is 3 : 2 and values close to this have been obtained (Schollhorn and Burris, 1967; Stewart et al., 1968; Bergersen, 1970). Bergersen (1970) has shown that this ratio varies widely with different organisms and under different conditions. However, assuming this ratio to hold, the calculated rate of nitrogen fixation

ACETYLENE

REDUCTION

BY CONIFER

ROOTS

177

TABLE 5. C&H, PRODUCTION BY SOIL SAMPLESASSOCIATEDWITH ROOTS OF D. CUpreSSinUm INCUBATED IN THE LIGHT (3000 LUX) AND

DARK

C,H, production (nmoles g- ‘) (n = Time (h)

Light

5

19 21

would

be

nmoles

NH3 g-l

Dark

(i)

(ii)

(i)

(ii)

1.22 4.50 5.99

0.75 4.38 5.18

0.63 4.24 6.07

0.88 3.80 4.61

(28 ng N g-l h- ‘), a value considerably lower than 6). Thus we feel that the general failure to find nitrogen fixation associated with roots, is not because fixation is not occurring but that the rate is extremely low and that those results obtained reflect variable high populations of active organisms in the root region. previously

2

2)

published

TABLE

6.

data

h-l

(Table

COMPARISON OF NITROGEN FIXATION RATES WITH OTHER POSITIVE REPORTS Max atm %

Ref.

Species Podocarpus lawrencii P. rospigliosii Agathis australis Dacrydium cupressinum

Bergersen and Costin, 1964 Becking, 1965 Morrison and English, 1966 This work

lSN excess

nmoles N g-l (fresh wt) h-’

0.078

66

0.48 0.048

184 * 8.4t

* Could not be calculated as total nitrogen in sample not given. t Calculated on basis of dry weight = 30 per cent of fresh weight, C2H4:NH3 theoretical ratio 3 : 2.

Removal of much of the C,H, reduction activity by washing roots, complete removal by mild surface sterilization and maintenance of constant rates of C2H, reduction for 24 h under these conditions suggests that endophytic organisms are not involved but that the C2H, reduction is essentially non-specific and located in the rhizosphere. There is, however, an enhanced activity in the root region over that found in the mineral soil due possibly to exudation from the mycorrhiza or the higher plant. Similar results have been indicated for Pinus radiata mycorrhizas (Richards and Voigt, 1964) and marram (Ammophila arenaria) (Hassouna and Wareing, 1964) and nitrogen-fixing organisms specific to the rhizosphere of Paspalum notatum have been isolated by Dorbereiner (1966). The identity of the nitrogen-fixing organism remains unsolved. Our single isolation of Azotobacter from soil must be treated with caution as podocarp soils are acknowledged to be acidic. pH

Our

records

(measured

with

glass electrode

on soil paste)

for these soils range

from

5 .O to 5.8. The lower limit for nitrogen fixation by Azotobacter is 6-O (Jensen, 1954) and as the rhizosphere pH is likely to be even lower than soil pH (Jurgensen and Davey, 1970)

178

W. B. SILVESTER AND K. J. BENNETT

it is unlikely that Azotobacter will play a significant role in either the rhizosphere or soils of these forests. The effect of varying p0, on C,H, reduction shows optima at near atmospheric concentrations for both F/H and roots. Inhibition at high pOz is a common feature of nitrogenase in microorganisms (Dalton and Postgate, 1969). However there is an obvious conditioning effect in the experiment described here whereby an increase of C,H4 production occurs with time at low ~0~. Dalton and Postgate (1969) have shown that C,H, reduction is increased at p0, levels below atmospheric and that there may be a reversible switching off mechanism induced by exposure to oxygen at levels above that in which they have been conditioned. In all our experiments material was exposed to gas mixtures by evacuation. The lag phase indicated in Fig. 1 may have been due to this reversible mechanism after exposure, by evacuation, to 20 per cent oxygen (oxygen being normally depleted in soil atmospheres). However the increase in reduction with time at low p0, is unlikely to be due to this phenomenon but probably reflects enhanced activity of facultative anaerobic organisms. The low rates of nitrogen fixation associated with podocarp roots are unlikely to have any significant effect on total soil nitrogen in forests. However, the rates of fixation recorded in F/H layers may be more than 10 times the maximum root rate. Jones (1970) has shown that soils and litter under Pseudotsuga may fix 3 *8 and 1 a9 pg N g-’ day-l respectively. Our figures when converted to nitrogen fixed would give a maximum F/H rate of 6.7 pg N g-’ day-‘, while the soil rate may be 0.10 pg N g-’ day-l. More work is required to assess the ecological importance of this possible nitrogen increment especially in terms of its distribution in the soil profile, seasonal fluctuation and identity of organisms. Finally it must be pointed out that these comments refer to forest species and conditions. Many species of podocarp can be regarded as pioneer plants and this has been attributed to their ability to absorb nutrients such as phosphorus from deficient soils via their mycorrhizal roots (Baylis et al., 1963; Morrison and English, 1967; Baylis, 1969b). Under these conditions nitrogen fixed in the rhizosphere before the accumulation of significant F/H may be important. Further, in the case of the extreme pioneers of high altitudes such as P. nivalis in New Zealand and P. lawrencei in Australia the mechanism may be even more important. Acknowledgements-Financial assistance from the Forest Research Institute of the New Zealand Forest Service and the New Zealand Universities Grants Committee is gratefully acknowledged. Mr. G. GRAYSTON rendered invaluable technical assistance and Miss P. BODLEY,Plant Diseases Division, Department of Scientific and Industrial Research identified the bacterial isolate.

REFERENCES ALLEN E. K. and ALLEN 0. N. (1965) Nonleguminous plant symbiosis. In Microbiology and Soil Fertility (C. M. Gilmore and 0. N. Allen, Eds.) pp, 77-106, Oregon State University Press, Corvallis, Oregon, BAYLISG. T. S. (1969a) Synthesis of mycorrhizas in Podocarpus and Agathis with Endogone spores. Nature, Land. 221, 1267-1268.

BAYLISG. T. S. (1969b) Mycorrhizal nodules and growth of Podocarpus

in nitrogen poor soils. Nature, Lond.

223, 1385-1386.

BAYLISG. T. S., MCNABB R. F. R. and MORRISONT. M. (1963) The mycorrhizal Trans. Br. Mycol. Sot. 46, 378-384. BECKINCJ. H. (1965) Nitrogen fixation and mycorrhiza

nodules

of podocarps.

in Podocarpus root nodules. PI. Soil 23,213-226. BERCIERSEN F. J. (1970) The quantitative relationship between nitrogen fixation and the acetylene reduction assay. Amt. J. biol. Sci. 23, 1015-1025. BERGERSENF. J. and COSTINA. B. (1964) Root nodules of Podocarpus lawrencei and their ecological significance. Aust. J. biol. Sci. 17, 44-48. BOND G. (1959) The incidence and importance of biological fixation of nitrogen. Adu. Sci. 15,382-386.

ACETYLENE

REDUCTION

BY CONIFER

ROOTS

179

BONDG. (1967) Fixation of nitrogen by higher plants other than legumes. Ann. Rev. PI. Physiol. 18, 107-126. BREMNERJ. M. and SHAW K. (1958) Denitrification in soil. J. ugric. Sci. Cumb. 51, 22-52. DALTONH. and POSTGATEJ. R. (1969) Effect of oxygen on growth of Azofobacter chroococcum in batch and continuous cultures. J. gen. Microbial. 54, 463. DILWORTHM. J. (1966) Acetylene reduction by nitrogen-fixing preparations from Clostridiumpusteuriunum. Biochim.

Biophys.

Acta 127, 285-294.

DOBEREINER J. (1969) Azotobacterpuspalli Pesq. ugropec.

sp. n., a nitrogen-fixing

bacterium in the rhizosphere

of Puspullum.

Bras. 1, 357-365.

FURMANT. E. (1970) The nodular mycorrhizae of Podocarpus rospigliosii. Am. J. Bot. 57, 91c-915. HASSOUNAM. G. and WAREINGP. F. (1964) Possible role of rhizosphere bacteria in nitrogen nutrition of Ammophila

urenaria.

Nature, Lond. 202, 467-469.

JENSENH. L. (1954) The Azotobacteriaceae. Bucteriol. Rev. 18, 195-214. JONESK. (1970) Nitrogen fixation in the phyllosphere of Douglas fir. Pseudotsagu

douglusii.

Ann. Bot. 34,

239-244.

JLJRGENSEN M. F. and DAVEY C. B. (1970) Non-symbiotic nitrogen-fixing microorganisms in acid soils and the rhizosphere. Soils Fert. 33, 435446. KAHN A. G. (1967) Podocurpus root nodules in sterile culture. Nature, Land. 215, 1170-1171. MORRISONT. M. and ENGLISHD. A. (1967) The significance of mycorrhizal nodules of Aguthis australis. New Phytol.

66, 245-250.

RICHARDSB. N. and VOIGT G. K. (1964) Role of mycorrhiza in nitrogen fixation. Nature, Lond. 201,310-311. SCHOLLHORNR. and BURRISR. H. (1967) Acetylene as a competitive inhibitor of nitrogen fixation. Proc. nutn. Acad. Sci. U.S. 58, 213-216.

SMITH E. A. and SCOTT-RUSSELLR. (1969) Occurrence of ethylene and its significance in anaerobic soil. Nature,

Land. 222, 769-771.

STEWARTW. D. P., FITZGERALDG. P. and BURRIS R. H. (1968) Acetylene reduction by nitrogen-fixing blue-green algae. Archiv. Mikrobiol. 62, 336-348. UEMURAS. (1964) Isolation and properties of microorganisms from root nodules of non leguminous plants. Bull. Govt. Forest. Exp. Stn. Meguro,

167, 59-91.

YEATESF. S. (1924) The root nodules of New Zealand pines. N.Z. J. Sci. Tech. 7, 121-124.