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Ethylene formation in some coniferous forest soils
ETHYLENE FORMATION CONIFEROUS FOREST T. Department
of
LINIXERG,
LJ. GRANHALL
IN SOME SOILS
and B.
BERG
Microbiology, Swedish University of Agricultural Sciences. S-750 07 Uppsala 7. Sweden
Summary--Ethylene formation in different litter components and soil layers from a Scats pine forest (site I) and a mixed Scats pine and Norway spruce forest (site 2) is reported. The effects of temperature. moisture, length of incubation and anaerobic incubation on CZH4 formation in humus are also reported. The highest concentrations of CtH, that accumulated in 24 h under ambient conditions in the laboratory were 3 and 17 parts/IO6 in humus from sites I and 2 respectively and 153 parts/lo’ in pine-needle litter. The most frequent microfungi from site I were tested for C,H, formation on different media. A Mwor .sikn~icus isolate was found to form C’?H., in the presence of methionine. The origin and significance
of the CzH4 formed
INTRODUCTION
is discussed.
When N, fixation in coniferous forests in central Sweden was investigated by the C,H,-reduction technique (Granhall and Lindberg, 1978), control samples of soil without C,H, were often found to release C,H,. Investigations were therefore carried out in order to study microbial C,H, formation to see whether the amounts formed in such soils were high enough to be able to affect root growth or activities of soil organisms.
Ethylene is formed in soils as a result of microbial activity (Smith and Restall, 1971) and concentrations varying up to 75 parts/IO’ (in waterlogged soil) have been reported (Smith and Dowdell, 1974). A wide range of fungi (Lynch, 1972) and bacteria (Primrose, 1976) with the ability to form CzH4 in pure culture have been isolated and identified. It has been suggested that spore-forming anaerobic bacteria (Smith and Cook, 1974) and the fungus Mucor hiemulis MATERIALS AND METHODS (Lynch, 1975) constitute the most important groups of Tat sites C>H,-forming microorganisms in soils. The formaSamples were collected from two coniferous forests tion of C,H, is also common in plant tissues, the C,H, functioning as an important plant-growth reguin central Sweden, a Scats pine (Pinus s~~~~s~ris L.) lator (Abeles, 1973). Plants. fungi and bacteria active forest on glaciofluvial sand (Ivantj~rnsheden, site I ; 60”49’N; 16”3O’E; altitude 185 m above mean sea in this respect generally require methionine to form C,H, (Abeles. 1972; Lynch. 1972; Primrose. 1976). level). and a mixed Scats pine and Norway spruce (Picea shies L.) forest on sandy moraine (Siljansfors, but ethanol (Abeles, 1972) or phenolic acids (Considine and Patching, 1975) can also be used by some site 2; 60”SS’N; l4”23’E; altitude 28&320m above fungal species. mean sea level). Details about the sites and their C2H4 in soil can be metabolized by certain aerobic climatologi~l characteristics are given by Granhall microorganisms (De Bont, 1976), can become chemiand Lindberg (1978). cally-adsorbed on organic matter (Witt and Weber, The shrub- and field-layer at site 1 consists of 1975) or can be liberated into the atmosphere (Abeles heather (Ccrlluna t’u&ris (L.) Hull.), cowberries (Vacc’t N/.. 197 I ). therefore, the C,H, concentrations found cinium ciris-idaea L.), feather mosses (Dicranunl, WJ/Oin soil are a result of synthesis and removal, the procorn&m, Plrurozium and Polytrichum spp) and cup cesses being regulated by environmental factors, parlichens (Cladonin spp). The soil profile is a weak iron ticularly O2 availability (Smith and Russel, 1969; podsol. The shrub layer at site 2 consists of sparsely-distiiSmith and Restall, 1971; Smith and Dowdell, 1974), C2H, in soils affects plants and microorganisms. buted junipers (Juniperus communis L.). The field layer Root growth of cereals, conifers and other plants is is composed of three main strata, I: feather mosses adversely affected at concentrations above 1 part/lo’ alone, II: feather mosses and dwarf shrubs, mainly blueberries (Varciniutn mq’rti/lus L.). heather and cow(Smith and Russel, 1969; Lill and McWha, 1976). Soil fungistasis has often been attributed to the presence of berries, and III: Sphagnum mosses and dwarf shrubs. Scattered, smaller areas (stratum IV) are composed of C,H, (Smith, 1973), and it has also been claimed that Sphffgnu~z mosses alone. The soil profile is a we& the growth and activity of bacteria and nematodes are developed iron podsol. affected (Smith, 1976). The effect of C,H, depends on the nutrient conditions (Smith, 1976) and CO2 concentrations in the soil (Radin and Loomis, 1969), Sampling procedure Six experimental plots (1 x 1 m) were chosen at higher amounts of both counteracting the inhibition random in a 120-yr-o!d pine stand (site l), three in a caused by C,H*. ‘,H,%,Ih P 637
T.
638
LINDRERG.U. GRANHAM and
I5-yr-old pine stand (site 1) and ten in a 160-yr-old mixed pine and spruce stand (site 2). Samples were collected on various occasions during the field seasons of 1974 and 1975. Soil blocks, 20cm to the deep x 4dm2* were dug up and transported laboratory in plastic bags for subsampling of humus (A,-layer) and mineral soil down to 5 cm below the A,-layer. Mineral soil samples down to 1Ocm below the A,,-layer were sieved to collect root samples (root dia 1-4mm). Humus material from the old stand at site I was also collected at random and freed of roots for use in laboratory ex~riments. Various litter samples were also collected. Some litter samples (pine-needle litter and pine-root litter) and pieces of pulp cellulose were placed in the field in litter bags (Berg and Staaf, 1979) and collected after 0.5, 1, 1.5, 2, 3.5 and 4 yr. Fungal
ix&es
isolates of some of the most frequent microfungi at site 1 (Soderstrom and Baath, 1978) were grown on agar media containing mineral salts (Lynch and Harper. 1974) and supplemented with glucose (5 g 1-i). or with ethanol (5 g l- ‘), or with glucose (10 g I- i) and methionin~ (I g l- ‘). The following isolates were tested for C2H, formation: ~~reo~usj~ju~7 pullulans (de Bary) Arnaud. A., sp., Ceutospora pinastri (Fr.) Hohnel, CIudosporium ciadosporioides (Fres.) de Vries, C. herharum (Pers) Link ex. Fr., Hormonema sp., Morteriella isahellina Oudem, M. nana Linnemann, M. ra~nannjanu (MBlIer) Linnemann, M_rceliurn radicis atro~~re}~.~Melin, O~diade~dron n7a~us Barron, Peniciliium ‘spinuiosl~m Thorn, two Pen~cij~~u~n spp, Trichoderma polysporum (Link ex. Pers.) Rifai, T. uiride Pers. ex. S. F. Gray and Verticillium bulhillosum W. Gams 62 Malla. Tests were also made using the known C,H,-forming fungus Mucor hiemaiis Wehmer (ATCC 26035) and a related Mucor .~~~~uticusHagem.. isolated from site I. All fungi were grown on 5 ml medium in 25 ml testtubes with porous caps at 24°C for 6-12 days until the agar. surface was covered with mycelium (approximately the same amount in all tubes), after which the porous caps were replaced by screw-caps with rubber stoppers. Exprriments
Soil and litter samples were incubated in 30-ml glass bottles with screw-caps and rubber stoppers under ambient gas conditions and tested for C,H, formation. Root-free humus was incubated for 3-10 days under air or N, and was assayed and gushed daily to avoid accumulation of inhibitory volatile substances (Smith and Restall, 1971). Experiments with root-free humus were also performed where no flushing was carried out, or where gas conditions were changed from ambient to anaerobic during incubation by flushing with N,. In these experiments, C2H4 and CO, were assayed. The effects of temperature and moisture on C,H, formation were tested on the root-free humus after it had been dried at room temperature and moisture conditions adjusted with distilled water. Moisture contents are expressed as 7” H,O on a dry weight basis. The moisture content used for temperature ex-
B.
BERG
periments was 160% I-l,0 (437; of WHC), and the material was adapted to various temperatures for 24 h before incubation. Soil and fungal samples were incubated in the laboratory at 24°C. Gas samples (I ml) were withdrawn by means of a gastight syringe and were submitted to g.c. analysis. The rates of C2H, formation given (pmol C,H, h-’ g dw- ‘) refer to specific periods or in some cases to the highest rate. The gas volumes of tested bottles and tubes were determined by filling with water (relative volumes of gas: solid materials varied between 225: I depending on moisture content). Dry weights were determined after heating to constant weight at 105°C. Gas-chromatographic
analysis
C2H, formation was determined by means of a Varian 600 D g.c. (Granhall and Lundgren. 1971). CO, was measured by means of a Perkin-Elmer 3920 g.c. with a Porapak Q column (3 m x 3.2 mm) and a hot wire detector with He (30 ml mini) as carrier gas. The detector temperature was 200°C and the oven temperature 45°C. RESULTS
Soil and iitter samples The A,-layer of the two stands at site 1 showed different rates of CtH4 formation. In the young pine stand activities were always low (April, May, July and September 1974) and close to the limit of detection (I pmol C2H4). In the old stand the highest rate (61 pmol C,H, h-i gdw-‘) was found in August (Table 1). This rate is equivalent to an accumulation of 3 parts/lo6 CzH4 in the bottle in 24 h. No obvious difference in rates was found in this case between O-20 and 2&40 h incubations. The pH of the A,-layer at the investigated stands at site 1 varied between 3.5 and 4.0. The activities in the A,-layer at site 2 (Table 1) were similar to those at site 1 (July and August. @40 h incubations). The highest rate found (stratum II in July) was 184 pmol C2H, hh’ g dw-’ (40-70 h incubation) which is equivalent to an accumulation of I7 parts/to’ in 24 h. Samples of slightly decomposed peat from the ~p~~~~~u~)~ strat~~m (IV) gave lower rates (July and October, O-40 h incubation) than other strata. Incubation for longer than 40 h gave in most cases (7 out of 8) when C2H, formation occurred, considerably higher rates (2-l l-fold increase). Thus ambient incubations for more than 40 h had a marked stimulating effect on accumulation of C’,H,. The pH of the A,-layer at site 2 varied between 5.6 and 6.4. Due to difficulties in separating microbial C,H,-formation from that of living plants in the surlace layer (S-layer) such results are not reported. A very low rate of C,H, evolution (~5 pmoles h - ’ g dw _ ‘) occurred in mineral soil samples from all sites investigated. However, pine roots sieved from this soil layer showed significant activity (Table 2A). The maximum rates (potentials) for CzH4 formation of various litter components are presented in Table 2A. All samples tested except pure cellulose and pine root litter evolved C2H4 when tested under ambient gas conditions at 24°C. Rates were generally higher at moisture contents above 100% Hz0 than below.
Ethylene
formation
in some coniferous
forest soils
639
Table 1. Ethylene formation and moisture in the A,-layer of different vegetational strata at sites 1 (old stand) and 2 during 1974 and 1975 in relation to different incubation periods. Number of samples (n). Direct comparison between sites and at different vegetational periods could be made for samples incubated &40 h (marked with frames). Mean values k SE
Site 1
Stratum II (feather mosses . and dwarf shrubs)
Year
Date
1974
22 April 27 May
22 July
20 August
25 September
2
II (feather mosses and dwarf shrubs)
1975
23 September
1974
13 August
1975
14 July 7 October
I (feather
mosses)
III (Sphagnum mosses and dwarf shrubs)
1974
13 August
1975
14 July 7 October
1975
14 July 7 October
IV (Sphagnum mosses)
1975
14 July 7 October
Incubation period (h)
n
G-70 (X20 2&40 &40 c-20 2WO O-40 O-20 2&40 O-40 O-20 20-40 &40 f&40 4&90 &20 2&70 &40 40-70 O-40 40-60 t&20 2&70 O-40 O-40 4&70 g-40 4&60 O-40 4&70 &40 40-60 &40 40-70 O-40 4&60
10 4 4 4 20 20 20 40 40 40 15 15 15 25 25 16 16 5 5 4 4 4 4 4 23 23 8 8 15 15 20 20 5 5 8 8
Ethylene formation (pmol C2H4 hh’ gdw-‘) 15 f 4 36 k 10 18 k4 27 f 7 22 f 6 25 + 4 24 + 4 61 k9 15*4 38 + 5 I&O 2+0 2*0 23 k 8 47 + 10 25 + 7 71 f 36 50 f 16 184 If: 65 15 + 10 159 + 54 0 0 0 33 * 9 99 * 31 69 f 38 127 + 63 34 + 5 141 f 54 4&l 2+1 16 f 3 48 k 5 0 0
Moisture (% H,O) 296 147 147 147 185 185 185 130 130 130 55 55 55 229 229 190 190 68 68 422 422 176 176 176 268 268 326 326 448 448 463 463 400 400 308 308
Table 2A. Ethylene formation potentials (maximum rates during 3 days incubations) of different litter components and roots from site 1 and 2 in relation to different moisture ranges. Number of samples (n). Not determined (ND). Mean values + SE
Pine twigs, branches and logs Pine and spruce twigs, branches Pine bark Pine and spruce bark Spruce bark Pine stumps Pine needles Spruce needles Pine roots (sieved from mineral Litter bag material Pine root-litter (I yr old) Cellulose (0.4/l yr old) Pine needles (1.5 yr old) Pine bark (1 yr old)
and logs
soil)
Site
n
1 2
22 3 10
I 2 2 1 2 1 2
3 5 5 6
I
10
1 1 1 1
9 20
Ethylene formation (pmol C,H, h-’ g dw-‘) 100% H,O
8*2 18 + 5 3+1 ND 22+ 11 0 61 + 14 179 f 57 ND 22 * 10 0 0 ND ND
42 15 25 4 10
38 k 4 82 k 21 25 + 6 26 f 3 ND 2+1 9+3 647 + 56 85 &- 38 54+ 15
20 15 9
ND 0 1652 & 139 43 + 4
28 14 38 8
+ 8 f 7 f 7 + 7 +4 +4 +4 * 5 * 5 f 5 f 2 + 2 + 2 k 7 + 7 + 14 * 14 * 4 + 4 + 13 + 13 + 17 + 17 + 17 f 14 + 14 k 5 + 5 + 14 + 14 f 22 f 22 + 26 k 26 + 45 -f 45
T.
640
and B. BERG
LINDBERG,U.GRANHALL
Table 2B. Ethylene formation rates (C-50 h incubations) of pine-needle litter in litter bags at site 1 in relation to different stages of decomposition (weight loss). Number of samples (n = 20). Mean values f SE Time in the field (days)
Ethylene formation (pmolC,H, h-’ gdw-‘) 976 + 323 + 180 * 153 +
185 734 1228 1480
Decomposition stage f% remaining weight)
Moisture level (% H,O)
82.2 zf 0.9 55.7 f 1.5 37.0 If: 1.3 33.5 & 1.3
164 f 13 270 & 18 109 rt 26 190 & 17
124 78 49 13
Table 3. The effect of temperature on ethylene formation in root-free humus from site 1, when incubated at 160% H,O content for O-92 or O-48 h under ambient gas conditions; n = 20. Mean values k SE Temperature (“C)
Incubation period (h)
Ethylene formation (pmol h-r gdw-‘)
O-92 o-92 O-92 c-92 O-48 O-48 O-48
0 0 8+1 27 ;f: 1 25 + 7 203 + 4 384 + 11
5
10 15 20 24 28 37
Highest rates of C2H, accumulation occurred in pine needle litter, up to 153 parts/IO’ in 24 h. Pine-needle litter is the most abundant litter component at all sites investigated (Staaf and Berg, 1977). C,H, accumulation rates of pine needle litter in litterbags (O-50 h incubation) decreased in proportion to the degree of decomposition (weight loss) (Table 2B), at non-limiting moisture contents (> 100% H,O). When root-free humus from site 1 was tested for C,H, formation at various moisture contents a marked increase in activity (Fig. IA) was found at moisture contents over 340% H,O up to the water holding capacity (384% H20) during short incubations (O-24 h), whereas a marked optimum (Fig. 1B) at lower moisture contents (200-300%) was found during longer incubations (2448 h). When the humus was incubated under N, or air and flushed daily, the rates of CzH4 formation under N, was initially (O-24 h) 6 times that under air, but the rates declined as the incubation continued (Fig. 2). Ethylene
The same trends were found in a shorter incubation experiment without flushing (Fig. 3A), and when gas conditions were changed from ambient to anaerobic (Fig. 3B). COz release was less affected than CZHJ formation and was only slightly lower under anaerobic conditions. ‘The temperature experiment (Table 3) showed that no C,H, was formed at and below 10°C. Incubations above 24°C had a strong stimulating effect on C2H4 formation, and the rate at 37°C was about 8 times that at 24°C. Fungul isolates All isolates tested grew on media containing methionine or ethanol, but only the two Mucor species formed C,H., and only on the methionine medium. M. sihticus formed C,H, aerobically at a rate that was approximately half of that of M. ~i~~~~is on the same medium after 12 days growth (38 and 79 pmol C,H, h-i tube-’ respectively. O-24 h incubations).
fwmrtlon
lpmole. C2HL h“gdw-‘)Mean’S
E
I
Fig. 1A. The effect of moisture on CsH, formation in root-free humus from site 1 at 24”C, when incubated under ambient gas conditions for O-24 h; n = 20.
Ethylene formation in some coniferous forest soils Ethylene
fwmatian
[ pmol:s
C2H,, h-’ g dw’)
0
Mean?
641
S E,
-* 0%
5l
92
139
173
191
2L7
3L7
38L
Moisture
(%t$O)
Fig. 1s. Same as Fig. lA, but incubated for another 24 h (24-48 h). Ethylene
farmatmn
Inmoles
C_,Hc g d&l
Meant
S.E.
Aerobic
1
I
Fig. 2. Cumulative C,H, formation in root-free humus from site 1 at 245% H,O and 24°C. when flushed daily and incubated ufider aerobic or anaerobic gas conditions; n = 20.
DISCUSSION
Soil and fitter
samples
Low moisture content was found to be a factor limiting CzH4 accumulation in the decomposing litter (Table 2A). The rise in activity in very wet humus (Figs 1A and 1B) and during prolonged incubations (Table 1) indicates that 0, depletion stimulates C,H, formation and is not a direct effect of moisture content as such. When humus was incubated under N, it was evident that anaerobic conditions favoured C,H, formation and depressed CO, release (Figs 2, 3A, 3B). The decrease in the rate of C2H4 formation during prolonged anaerobic incubation is probably due to exhaustion of specific substrates, rather than to the development of a CzH,-consuming flora. Abeles et al.
(1‘971),Rovira and Vendrell (1972) and de Bont (1976) have shown that under anaerobic conditions CzH4 is not consumed by microorganisms. Our investigation (unpublished) showed a significant positive correlation between CsH., formation and moisture in the range 50-200~0 Hz0 in the A,-layer of site 1. But, we also found a significant positive correlation between C&H, formation and total amounts of C, N and P respectively. Litter bag experiments (Table 2B) also indicate that substrate quality influences C2H4 formation pH was not correlated with C2H4 formation at any of the sites investigated. The marked increase in &,I%, formation at temperatures above 24°C (Table 3) may be due to release of previously-formed C#, adsorbed on organic
T.
642
LINDBERG, U. GRANHALL and B. BERG
I’4 ,A I’/’amb:ent /I’
A
I
,
30
150
- 100
- 50
10 20
1 0
30
50
60
70
T,me
Ihl
Fig. 3A. Cumulative C,HI formation and CO, release in root-free humus from site 1 at 24-C. when incubated under ambient (258”:, H,O) or anaerobic gas conditions (273”/,, H,O); n = 10.
Since there was no clear evidence of the importance of bacteria in C,H, evolution in soils at the time of our investigations (1974-75) we did not look for C,H,-forming bacteria, but the marked stimulation of low 0, conditions on C,H, formation found by us indicates that anaerobic or facultative anaerobic bacteria were active in this respect. Several facultative anaerobic bacteria, such as Pseudomonas spp, have been shown to form C2H, at high rates (Primrose and Dilworth, 1976; Primrose, 1976) and are common in the soils we investigated (M. Clarholm, personal communication). The potentials for accumulation of C,H, in humus soil and various litter components reported here are well above 1 part/106, a concentration which have previously been reported as inhibiting root growth (Smith and Russel, 1969) and fungal spore germination (Smith, 1973). As our experiments were performed under laboratory conditions at 24°C and as other factors, such as COz, counteract the effect of C2H4 (Chadwick and Burg, 1967) it is difficult to draw any definite conclusions on the in situ effect of the observed CzH4 formation. The high potentials for accumulation of C2H4 commonly found in the organogenic layers of the coniferous forest soils we investigated, however, are likely to cause adverse effect on root growth, at least under wet conditions when soil temperatures exceed 10°C.
Acknowledgements-We thank Dr B. Soderstrom of the Department of Microbial Ecology, University of Lund. for providing us with fungal isolates. This investigation was Snanced by a grant from the Swedish Council for Forestry and Agricultural Research.
REFERENCES
Fig. 3B. Cumulative C,H, formation (solid line) and CO, release (dashed line) in root-free humus from site 1 (283% H,O) at 24-C when changing from ambient to anaerobic gas conditions after 17 h; n = 10.
matter pletion
(Witt and Weber, or cell lysis (Lynch
1975), increased 0, deand Harper, 1974). Such
effects are, however, not related to actual field conditions as soil temperatures seldom exceed 15°C. Regarding the origin of evolved C2H, it should be noted that living roots in untreated samples of A, soil could have contributed to C,H, formation, because Kays rr al. (1974) reported that roots when physically disturbed release C,H,. Significant C,H, formation in the mineral soil layer was in fact mainly found in the presence of roots (Table 2A). Both M. hiemnlis, which is a coloniser of decaying litter (Lynch, 1975), and the closely-related M. siluaticus are found at the sites investigated (B. Soderstrom, personal communication) and at moderate moisture contents may be responsible for shortterm (aerobic) C,H, formation in the litter and soil samples investigated. None of the Penicillium spp tested formed C,H,, although Considine and Patching (1975) found that some Penicillium spp can do so.
ABELES F. B. (1972) Biosynthesis and mechanism of action of ethylene. Annual Review of Plunf Physiology 23. 259-292. ABELES F. B. (1973) Ethylenein Plant Biology. Academic Press, New York. ABELES F. B., CRAKER L. E., FORRENCE L. E. and LEArma G. R. (1971) Fate or air pollutants: Removal of ethylene. sulfur dioxide, and nitrogen dioxide by soil. Science 173. 914916. BERG B. and STAAF H. (1979) Decomposition rate and chemical changes in decomposing needle litter of Scats pine 1. Influence of stand age. In Ecology of Conifrrous Forests. (T. Persson Ed.) Swedish Coniferous Forest Project 1. Ecological Bulletin (Stockholm) 30, In
press. DE BONT J. A. M. (1976)
Oxidation of ethylene by soil bacteria. Antonie van Leeuwenhoek 42, 59-7 I. CHADWICK A. V. and BURG S. P. (1967) An explanation of the inhibition of root growth caused by indole-3-acetic acid. Plant Physiology 42, 4 15-420. CONSIDINE P. J. and PATCHING J. W. (1975) Ethylene production by micro-organisms grown on phenolic acids. Annals OJ Applied Biology 81, I 15-l 19. GRANHALL U. and LUNDGREN A. (1971) Nitrogen fixation in Lake Erken. Limnoloyy and Oceanography 16, 71 I-719. GRANHALL U. and LINDBERG T. (1978) Nitrogen fixation in some coniferous forest ecosystems. In Environmental Role of Nitrogen-fixing Blue-green Algae and Asymhiotic
Ethylene
formation
in some coniferous
Bacteria. (U. Ciranhall, Ed.) Ecological Bulletin (Stockholm) 26, 178-192. KAYS S. J., NICKLOW C. W. and SIMONS D. H. (1974) Ethylene in relation to the response of roots to physical impedance. Plant & Soil 40, 565-571. LILL R. E. and MCWHA J. A. (1976) Production of ethylene by incubated litter of Pinus radiata. Soil Biology & Biochemistry 8, 61-63. LYNCH J. M. (1972) Identification of substrates and isolation of microorganisms responsible for ethylene production in the soil. Nature 240, 45-46. LYNCH J. M. (1975) Ethylene in soil. Nature 256, 57&577. LYNCH J. M. and HARPER S. H. T. (1974) Formation of ethylene by a soil fungus. Journal of General Microbiology 80, 187-195. PRIMROSE S. B. (1976) Ethylene-forming bacteria from soil and water. Journul of‘ General Microbiology 97, 343-346. PRIMROSE S. B. and DILWORTH M. J. (1976) Ethylene production by bacteria. Journal oj’ General Microbiology 93, 177-181. RADIN J. W. and Loo~ls R. S. (1969) Ethylene and carbon dioxide in the growth and development of cultured radish roots. Plant Physiology 44, 1584-l 589. ROVIRA A. D. and VENDRELL M. (1972) Ethylene in sterilized soil: Its significance in studies of interactions between microorganisms and plants. Soil Biology & Biochemistry 4, 63-69.
SMITH A. Narure SMITH A. reduced
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M. (1973) Ethylene as a cause of soil fungistasis. 246. 3 1l-3 13. M. (1976) Ethylene production by bacteria in microsites in soil and some implications to agriculture. Soil Biology & Biochemistry 8, 293-298. SMITH K. A. and RUSSEL R. S. (1969) Occurrence of ethylene, and its significance, in anaerobic soil. Narure 222, 769-771. SMITH K. A. and RESTALL S. W. F. (1971) The occurrence of ethylene in anaerobic soil. Jourtlal qf Soil Science 22, 43&443. SMITH A. M. and COOK R. J. (1974) Implications of ethylene production by bacteria for biological balance of soil. Nature 252, 703-705. SMITH K. A. and DOWDELL R. J. (1974) Field studies of the soil atmosphere. I. Relationships between ethylene. oxygen, soil moisture content, and temperature. Journal of Soil Science 25, 2 17-230. SBDERSTR~~M B. E. and BKKTH E. (1978) Soil microfungi in three Swedish coniferous forests. Holarcric Ecology 1, 62-72. STAAF H. and BERG B. (1977) Mobilization of plant nutrients in a Scats pine forest mor in Central Sweden. Sib Frnnica 11, 21G-217. WITT W. W. and WEBER J. B. (1975) Ethylene adsorption and movement in soils and adsorption by soil constituents. Weed Science 23. 302-307.
of
LINIXERG,
LJ. GRANHALL
IN SOME SOILS
and B.
BERG
Microbiology, Swedish University of Agricultural Sciences. S-750 07 Uppsala 7. Sweden
Summary--Ethylene formation in different litter components and soil layers from a Scats pine forest (site I) and a mixed Scats pine and Norway spruce forest (site 2) is reported. The effects of temperature. moisture, length of incubation and anaerobic incubation on CZH4 formation in humus are also reported. The highest concentrations of CtH, that accumulated in 24 h under ambient conditions in the laboratory were 3 and 17 parts/IO6 in humus from sites I and 2 respectively and 153 parts/lo’ in pine-needle litter. The most frequent microfungi from site I were tested for C,H, formation on different media. A Mwor .sikn~icus isolate was found to form C’?H., in the presence of methionine. The origin and significance
of the CzH4 formed
INTRODUCTION
is discussed.
When N, fixation in coniferous forests in central Sweden was investigated by the C,H,-reduction technique (Granhall and Lindberg, 1978), control samples of soil without C,H, were often found to release C,H,. Investigations were therefore carried out in order to study microbial C,H, formation to see whether the amounts formed in such soils were high enough to be able to affect root growth or activities of soil organisms.
Ethylene is formed in soils as a result of microbial activity (Smith and Restall, 1971) and concentrations varying up to 75 parts/IO’ (in waterlogged soil) have been reported (Smith and Dowdell, 1974). A wide range of fungi (Lynch, 1972) and bacteria (Primrose, 1976) with the ability to form CzH4 in pure culture have been isolated and identified. It has been suggested that spore-forming anaerobic bacteria (Smith and Cook, 1974) and the fungus Mucor hiemulis MATERIALS AND METHODS (Lynch, 1975) constitute the most important groups of Tat sites C>H,-forming microorganisms in soils. The formaSamples were collected from two coniferous forests tion of C,H, is also common in plant tissues, the C,H, functioning as an important plant-growth reguin central Sweden, a Scats pine (Pinus s~~~~s~ris L.) lator (Abeles, 1973). Plants. fungi and bacteria active forest on glaciofluvial sand (Ivantj~rnsheden, site I ; 60”49’N; 16”3O’E; altitude 185 m above mean sea in this respect generally require methionine to form C,H, (Abeles. 1972; Lynch. 1972; Primrose. 1976). level). and a mixed Scats pine and Norway spruce (Picea shies L.) forest on sandy moraine (Siljansfors, but ethanol (Abeles, 1972) or phenolic acids (Considine and Patching, 1975) can also be used by some site 2; 60”SS’N; l4”23’E; altitude 28&320m above fungal species. mean sea level). Details about the sites and their C2H4 in soil can be metabolized by certain aerobic climatologi~l characteristics are given by Granhall microorganisms (De Bont, 1976), can become chemiand Lindberg (1978). cally-adsorbed on organic matter (Witt and Weber, The shrub- and field-layer at site 1 consists of 1975) or can be liberated into the atmosphere (Abeles heather (Ccrlluna t’u&ris (L.) Hull.), cowberries (Vacc’t N/.. 197 I ). therefore, the C,H, concentrations found cinium ciris-idaea L.), feather mosses (Dicranunl, WJ/Oin soil are a result of synthesis and removal, the procorn&m, Plrurozium and Polytrichum spp) and cup cesses being regulated by environmental factors, parlichens (Cladonin spp). The soil profile is a weak iron ticularly O2 availability (Smith and Russel, 1969; podsol. The shrub layer at site 2 consists of sparsely-distiiSmith and Restall, 1971; Smith and Dowdell, 1974), C2H, in soils affects plants and microorganisms. buted junipers (Juniperus communis L.). The field layer Root growth of cereals, conifers and other plants is is composed of three main strata, I: feather mosses adversely affected at concentrations above 1 part/lo’ alone, II: feather mosses and dwarf shrubs, mainly blueberries (Varciniutn mq’rti/lus L.). heather and cow(Smith and Russel, 1969; Lill and McWha, 1976). Soil fungistasis has often been attributed to the presence of berries, and III: Sphagnum mosses and dwarf shrubs. Scattered, smaller areas (stratum IV) are composed of C,H, (Smith, 1973), and it has also been claimed that Sphffgnu~z mosses alone. The soil profile is a we& the growth and activity of bacteria and nematodes are developed iron podsol. affected (Smith, 1976). The effect of C,H, depends on the nutrient conditions (Smith, 1976) and CO2 concentrations in the soil (Radin and Loomis, 1969), Sampling procedure Six experimental plots (1 x 1 m) were chosen at higher amounts of both counteracting the inhibition random in a 120-yr-o!d pine stand (site l), three in a caused by C,H*. ‘,H,%,Ih P 637
T.
638
LINDRERG.U. GRANHAM and
I5-yr-old pine stand (site 1) and ten in a 160-yr-old mixed pine and spruce stand (site 2). Samples were collected on various occasions during the field seasons of 1974 and 1975. Soil blocks, 20cm to the deep x 4dm2* were dug up and transported laboratory in plastic bags for subsampling of humus (A,-layer) and mineral soil down to 5 cm below the A,-layer. Mineral soil samples down to 1Ocm below the A,,-layer were sieved to collect root samples (root dia 1-4mm). Humus material from the old stand at site I was also collected at random and freed of roots for use in laboratory ex~riments. Various litter samples were also collected. Some litter samples (pine-needle litter and pine-root litter) and pieces of pulp cellulose were placed in the field in litter bags (Berg and Staaf, 1979) and collected after 0.5, 1, 1.5, 2, 3.5 and 4 yr. Fungal
ix&es
isolates of some of the most frequent microfungi at site 1 (Soderstrom and Baath, 1978) were grown on agar media containing mineral salts (Lynch and Harper. 1974) and supplemented with glucose (5 g 1-i). or with ethanol (5 g l- ‘), or with glucose (10 g I- i) and methionin~ (I g l- ‘). The following isolates were tested for C2H, formation: ~~reo~usj~ju~7 pullulans (de Bary) Arnaud. A., sp., Ceutospora pinastri (Fr.) Hohnel, CIudosporium ciadosporioides (Fres.) de Vries, C. herharum (Pers) Link ex. Fr., Hormonema sp., Morteriella isahellina Oudem, M. nana Linnemann, M. ra~nannjanu (MBlIer) Linnemann, M_rceliurn radicis atro~~re}~.~Melin, O~diade~dron n7a~us Barron, Peniciliium ‘spinuiosl~m Thorn, two Pen~cij~~u~n spp, Trichoderma polysporum (Link ex. Pers.) Rifai, T. uiride Pers. ex. S. F. Gray and Verticillium bulhillosum W. Gams 62 Malla. Tests were also made using the known C,H,-forming fungus Mucor hiemaiis Wehmer (ATCC 26035) and a related Mucor .~~~~uticusHagem.. isolated from site I. All fungi were grown on 5 ml medium in 25 ml testtubes with porous caps at 24°C for 6-12 days until the agar. surface was covered with mycelium (approximately the same amount in all tubes), after which the porous caps were replaced by screw-caps with rubber stoppers. Exprriments
Soil and litter samples were incubated in 30-ml glass bottles with screw-caps and rubber stoppers under ambient gas conditions and tested for C,H, formation. Root-free humus was incubated for 3-10 days under air or N, and was assayed and gushed daily to avoid accumulation of inhibitory volatile substances (Smith and Restall, 1971). Experiments with root-free humus were also performed where no flushing was carried out, or where gas conditions were changed from ambient to anaerobic during incubation by flushing with N,. In these experiments, C2H4 and CO, were assayed. The effects of temperature and moisture on C,H, formation were tested on the root-free humus after it had been dried at room temperature and moisture conditions adjusted with distilled water. Moisture contents are expressed as 7” H,O on a dry weight basis. The moisture content used for temperature ex-
B.
BERG
periments was 160% I-l,0 (437; of WHC), and the material was adapted to various temperatures for 24 h before incubation. Soil and fungal samples were incubated in the laboratory at 24°C. Gas samples (I ml) were withdrawn by means of a gastight syringe and were submitted to g.c. analysis. The rates of C2H, formation given (pmol C,H, h-’ g dw- ‘) refer to specific periods or in some cases to the highest rate. The gas volumes of tested bottles and tubes were determined by filling with water (relative volumes of gas: solid materials varied between 225: I depending on moisture content). Dry weights were determined after heating to constant weight at 105°C. Gas-chromatographic
analysis
C2H, formation was determined by means of a Varian 600 D g.c. (Granhall and Lundgren. 1971). CO, was measured by means of a Perkin-Elmer 3920 g.c. with a Porapak Q column (3 m x 3.2 mm) and a hot wire detector with He (30 ml mini) as carrier gas. The detector temperature was 200°C and the oven temperature 45°C. RESULTS
Soil and iitter samples The A,-layer of the two stands at site 1 showed different rates of CtH4 formation. In the young pine stand activities were always low (April, May, July and September 1974) and close to the limit of detection (I pmol C2H4). In the old stand the highest rate (61 pmol C,H, h-i gdw-‘) was found in August (Table 1). This rate is equivalent to an accumulation of 3 parts/lo6 CzH4 in the bottle in 24 h. No obvious difference in rates was found in this case between O-20 and 2&40 h incubations. The pH of the A,-layer at the investigated stands at site 1 varied between 3.5 and 4.0. The activities in the A,-layer at site 2 (Table 1) were similar to those at site 1 (July and August. @40 h incubations). The highest rate found (stratum II in July) was 184 pmol C2H, hh’ g dw-’ (40-70 h incubation) which is equivalent to an accumulation of I7 parts/to’ in 24 h. Samples of slightly decomposed peat from the ~p~~~~~u~)~ strat~~m (IV) gave lower rates (July and October, O-40 h incubation) than other strata. Incubation for longer than 40 h gave in most cases (7 out of 8) when C2H, formation occurred, considerably higher rates (2-l l-fold increase). Thus ambient incubations for more than 40 h had a marked stimulating effect on accumulation of C’,H,. The pH of the A,-layer at site 2 varied between 5.6 and 6.4. Due to difficulties in separating microbial C,H,-formation from that of living plants in the surlace layer (S-layer) such results are not reported. A very low rate of C,H, evolution (~5 pmoles h - ’ g dw _ ‘) occurred in mineral soil samples from all sites investigated. However, pine roots sieved from this soil layer showed significant activity (Table 2A). The maximum rates (potentials) for CzH4 formation of various litter components are presented in Table 2A. All samples tested except pure cellulose and pine root litter evolved C2H4 when tested under ambient gas conditions at 24°C. Rates were generally higher at moisture contents above 100% Hz0 than below.
Ethylene
formation
in some coniferous
forest soils
639
Table 1. Ethylene formation and moisture in the A,-layer of different vegetational strata at sites 1 (old stand) and 2 during 1974 and 1975 in relation to different incubation periods. Number of samples (n). Direct comparison between sites and at different vegetational periods could be made for samples incubated &40 h (marked with frames). Mean values k SE
Site 1
Stratum II (feather mosses . and dwarf shrubs)
Year
Date
1974
22 April 27 May
22 July
20 August
25 September
2
II (feather mosses and dwarf shrubs)
1975
23 September
1974
13 August
1975
14 July 7 October
I (feather
mosses)
III (Sphagnum mosses and dwarf shrubs)
1974
13 August
1975
14 July 7 October
1975
14 July 7 October
IV (Sphagnum mosses)
1975
14 July 7 October
Incubation period (h)
n
G-70 (X20 2&40 &40 c-20 2WO O-40 O-20 2&40 O-40 O-20 20-40 &40 f&40 4&90 &20 2&70 &40 40-70 O-40 40-60 t&20 2&70 O-40 O-40 4&70 g-40 4&60 O-40 4&70 &40 40-60 &40 40-70 O-40 4&60
10 4 4 4 20 20 20 40 40 40 15 15 15 25 25 16 16 5 5 4 4 4 4 4 23 23 8 8 15 15 20 20 5 5 8 8
Ethylene formation (pmol C2H4 hh’ gdw-‘) 15 f 4 36 k 10 18 k4 27 f 7 22 f 6 25 + 4 24 + 4 61 k9 15*4 38 + 5 I&O 2+0 2*0 23 k 8 47 + 10 25 + 7 71 f 36 50 f 16 184 If: 65 15 + 10 159 + 54 0 0 0 33 * 9 99 * 31 69 f 38 127 + 63 34 + 5 141 f 54 4&l 2+1 16 f 3 48 k 5 0 0
Moisture (% H,O) 296 147 147 147 185 185 185 130 130 130 55 55 55 229 229 190 190 68 68 422 422 176 176 176 268 268 326 326 448 448 463 463 400 400 308 308
Table 2A. Ethylene formation potentials (maximum rates during 3 days incubations) of different litter components and roots from site 1 and 2 in relation to different moisture ranges. Number of samples (n). Not determined (ND). Mean values + SE
Pine twigs, branches and logs Pine and spruce twigs, branches Pine bark Pine and spruce bark Spruce bark Pine stumps Pine needles Spruce needles Pine roots (sieved from mineral Litter bag material Pine root-litter (I yr old) Cellulose (0.4/l yr old) Pine needles (1.5 yr old) Pine bark (1 yr old)
and logs
soil)
Site
n
1 2
22 3 10
I 2 2 1 2 1 2
3 5 5 6
I
10
1 1 1 1
9 20
Ethylene formation (pmol C,H, h-’ g dw-‘)
8*2 18 + 5 3+1 ND 22+ 11 0 61 + 14 179 f 57 ND 22 * 10 0 0 ND ND
42 15 25 4 10
38 k 4 82 k 21 25 + 6 26 f 3 ND 2+1 9+3 647 + 56 85 &- 38 54+ 15
20 15 9
ND 0 1652 & 139 43 + 4
28 14 38 8
+ 8 f 7 f 7 + 7 +4 +4 +4 * 5 * 5 f 5 f 2 + 2 + 2 k 7 + 7 + 14 * 14 * 4 + 4 + 13 + 13 + 17 + 17 + 17 f 14 + 14 k 5 + 5 + 14 + 14 f 22 f 22 + 26 k 26 + 45 -f 45
T.
640
and B. BERG
LINDBERG,U.GRANHALL
Table 2B. Ethylene formation rates (C-50 h incubations) of pine-needle litter in litter bags at site 1 in relation to different stages of decomposition (weight loss). Number of samples (n = 20). Mean values f SE Time in the field (days)
Ethylene formation (pmolC,H, h-’ gdw-‘) 976 + 323 + 180 * 153 +
185 734 1228 1480
Decomposition stage f% remaining weight)
Moisture level (% H,O)
82.2 zf 0.9 55.7 f 1.5 37.0 If: 1.3 33.5 & 1.3
164 f 13 270 & 18 109 rt 26 190 & 17
124 78 49 13
Table 3. The effect of temperature on ethylene formation in root-free humus from site 1, when incubated at 160% H,O content for O-92 or O-48 h under ambient gas conditions; n = 20. Mean values k SE Temperature (“C)
Incubation period (h)
Ethylene formation (pmol h-r gdw-‘)
O-92 o-92 O-92 c-92 O-48 O-48 O-48
0 0 8+1 27 ;f: 1 25 + 7 203 + 4 384 + 11
5
10 15 20 24 28 37
Highest rates of C2H, accumulation occurred in pine needle litter, up to 153 parts/IO’ in 24 h. Pine-needle litter is the most abundant litter component at all sites investigated (Staaf and Berg, 1977). C,H, accumulation rates of pine needle litter in litterbags (O-50 h incubation) decreased in proportion to the degree of decomposition (weight loss) (Table 2B), at non-limiting moisture contents (> 100% H,O). When root-free humus from site 1 was tested for C,H, formation at various moisture contents a marked increase in activity (Fig. IA) was found at moisture contents over 340% H,O up to the water holding capacity (384% H20) during short incubations (O-24 h), whereas a marked optimum (Fig. 1B) at lower moisture contents (200-300%) was found during longer incubations (2448 h). When the humus was incubated under N, or air and flushed daily, the rates of CzH4 formation under N, was initially (O-24 h) 6 times that under air, but the rates declined as the incubation continued (Fig. 2). Ethylene
The same trends were found in a shorter incubation experiment without flushing (Fig. 3A), and when gas conditions were changed from ambient to anaerobic (Fig. 3B). COz release was less affected than CZHJ formation and was only slightly lower under anaerobic conditions. ‘The temperature experiment (Table 3) showed that no C,H, was formed at and below 10°C. Incubations above 24°C had a strong stimulating effect on C2H4 formation, and the rate at 37°C was about 8 times that at 24°C. Fungul isolates All isolates tested grew on media containing methionine or ethanol, but only the two Mucor species formed C,H., and only on the methionine medium. M. sihticus formed C,H, aerobically at a rate that was approximately half of that of M. ~i~~~~is on the same medium after 12 days growth (38 and 79 pmol C,H, h-i tube-’ respectively. O-24 h incubations).
fwmrtlon
lpmole. C2HL h“gdw-‘)Mean’S
E
I
Fig. 1A. The effect of moisture on CsH, formation in root-free humus from site 1 at 24”C, when incubated under ambient gas conditions for O-24 h; n = 20.
Ethylene formation in some coniferous forest soils Ethylene
fwmatian
[ pmol:s
C2H,, h-’ g dw’)
0
Mean?
641
S E,
-* 0%
5l
92
139
173
191
2L7
3L7
38L
Moisture
(%t$O)
Fig. 1s. Same as Fig. lA, but incubated for another 24 h (24-48 h). Ethylene
farmatmn
Inmoles
C_,Hc g d&l
Meant
S.E.
Aerobic
1
I
Fig. 2. Cumulative C,H, formation in root-free humus from site 1 at 245% H,O and 24°C. when flushed daily and incubated ufider aerobic or anaerobic gas conditions; n = 20.
DISCUSSION
Soil and fitter
samples
Low moisture content was found to be a factor limiting CzH4 accumulation in the decomposing litter (Table 2A). The rise in activity in very wet humus (Figs 1A and 1B) and during prolonged incubations (Table 1) indicates that 0, depletion stimulates C,H, formation and is not a direct effect of moisture content as such. When humus was incubated under N, it was evident that anaerobic conditions favoured C,H, formation and depressed CO, release (Figs 2, 3A, 3B). The decrease in the rate of C2H4 formation during prolonged anaerobic incubation is probably due to exhaustion of specific substrates, rather than to the development of a CzH,-consuming flora. Abeles et al.
(1‘971),Rovira and Vendrell (1972) and de Bont (1976) have shown that under anaerobic conditions CzH4 is not consumed by microorganisms. Our investigation (unpublished) showed a significant positive correlation between CsH., formation and moisture in the range 50-200~0 Hz0 in the A,-layer of site 1. But, we also found a significant positive correlation between C&H, formation and total amounts of C, N and P respectively. Litter bag experiments (Table 2B) also indicate that substrate quality influences C2H4 formation pH was not correlated with C2H4 formation at any of the sites investigated. The marked increase in &,I%, formation at temperatures above 24°C (Table 3) may be due to release of previously-formed C#, adsorbed on organic
T.
642
LINDBERG, U. GRANHALL and B. BERG
I’4 ,A I’/’amb:ent /I’
A
I
,
30
150
- 100
- 50
10 20
1 0
30
50
60
70
T,me
Ihl
Fig. 3A. Cumulative C,HI formation and CO, release in root-free humus from site 1 at 24-C. when incubated under ambient (258”:, H,O) or anaerobic gas conditions (273”/,, H,O); n = 10.
Since there was no clear evidence of the importance of bacteria in C,H, evolution in soils at the time of our investigations (1974-75) we did not look for C,H,-forming bacteria, but the marked stimulation of low 0, conditions on C,H, formation found by us indicates that anaerobic or facultative anaerobic bacteria were active in this respect. Several facultative anaerobic bacteria, such as Pseudomonas spp, have been shown to form C2H, at high rates (Primrose and Dilworth, 1976; Primrose, 1976) and are common in the soils we investigated (M. Clarholm, personal communication). The potentials for accumulation of C,H, in humus soil and various litter components reported here are well above 1 part/106, a concentration which have previously been reported as inhibiting root growth (Smith and Russel, 1969) and fungal spore germination (Smith, 1973). As our experiments were performed under laboratory conditions at 24°C and as other factors, such as COz, counteract the effect of C2H4 (Chadwick and Burg, 1967) it is difficult to draw any definite conclusions on the in situ effect of the observed CzH4 formation. The high potentials for accumulation of C2H4 commonly found in the organogenic layers of the coniferous forest soils we investigated, however, are likely to cause adverse effect on root growth, at least under wet conditions when soil temperatures exceed 10°C.
Acknowledgements-We thank Dr B. Soderstrom of the Department of Microbial Ecology, University of Lund. for providing us with fungal isolates. This investigation was Snanced by a grant from the Swedish Council for Forestry and Agricultural Research.
REFERENCES
Fig. 3B. Cumulative C,H, formation (solid line) and CO, release (dashed line) in root-free humus from site 1 (283% H,O) at 24-C when changing from ambient to anaerobic gas conditions after 17 h; n = 10.
matter pletion
(Witt and Weber, or cell lysis (Lynch
1975), increased 0, deand Harper, 1974). Such
effects are, however, not related to actual field conditions as soil temperatures seldom exceed 15°C. Regarding the origin of evolved C2H, it should be noted that living roots in untreated samples of A, soil could have contributed to C,H, formation, because Kays rr al. (1974) reported that roots when physically disturbed release C,H,. Significant C,H, formation in the mineral soil layer was in fact mainly found in the presence of roots (Table 2A). Both M. hiemnlis, which is a coloniser of decaying litter (Lynch, 1975), and the closely-related M. siluaticus are found at the sites investigated (B. Soderstrom, personal communication) and at moderate moisture contents may be responsible for shortterm (aerobic) C,H, formation in the litter and soil samples investigated. None of the Penicillium spp tested formed C,H,, although Considine and Patching (1975) found that some Penicillium spp can do so.
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Ethylene
formation
in some coniferous
Bacteria. (U. Ciranhall, Ed.) Ecological Bulletin (Stockholm) 26, 178-192. KAYS S. J., NICKLOW C. W. and SIMONS D. H. (1974) Ethylene in relation to the response of roots to physical impedance. Plant & Soil 40, 565-571. LILL R. E. and MCWHA J. A. (1976) Production of ethylene by incubated litter of Pinus radiata. Soil Biology & Biochemistry 8, 61-63. LYNCH J. M. (1972) Identification of substrates and isolation of microorganisms responsible for ethylene production in the soil. Nature 240, 45-46. LYNCH J. M. (1975) Ethylene in soil. Nature 256, 57&577. LYNCH J. M. and HARPER S. H. T. (1974) Formation of ethylene by a soil fungus. Journal of General Microbiology 80, 187-195. PRIMROSE S. B. (1976) Ethylene-forming bacteria from soil and water. Journul of‘ General Microbiology 97, 343-346. PRIMROSE S. B. and DILWORTH M. J. (1976) Ethylene production by bacteria. Journal oj’ General Microbiology 93, 177-181. RADIN J. W. and Loo~ls R. S. (1969) Ethylene and carbon dioxide in the growth and development of cultured radish roots. Plant Physiology 44, 1584-l 589. ROVIRA A. D. and VENDRELL M. (1972) Ethylene in sterilized soil: Its significance in studies of interactions between microorganisms and plants. Soil Biology & Biochemistry 4, 63-69.
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forest soils
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M. (1973) Ethylene as a cause of soil fungistasis. 246. 3 1l-3 13. M. (1976) Ethylene production by bacteria in microsites in soil and some implications to agriculture. Soil Biology & Biochemistry 8, 293-298. SMITH K. A. and RUSSEL R. S. (1969) Occurrence of ethylene, and its significance, in anaerobic soil. Narure 222, 769-771. SMITH K. A. and RESTALL S. W. F. (1971) The occurrence of ethylene in anaerobic soil. Jourtlal qf Soil Science 22, 43&443. SMITH A. M. and COOK R. J. (1974) Implications of ethylene production by bacteria for biological balance of soil. Nature 252, 703-705. SMITH K. A. and DOWDELL R. J. (1974) Field studies of the soil atmosphere. I. Relationships between ethylene. oxygen, soil moisture content, and temperature. Journal of Soil Science 25, 2 17-230. SBDERSTR~~M B. E. and BKKTH E. (1978) Soil microfungi in three Swedish coniferous forests. Holarcric Ecology 1, 62-72. STAAF H. and BERG B. (1977) Mobilization of plant nutrients in a Scats pine forest mor in Central Sweden. Sib Frnnica 11, 21G-217. WITT W. W. and WEBER J. B. (1975) Ethylene adsorption and movement in soils and adsorption by soil constituents. Weed Science 23. 302-307.