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Residual effects of soil fumigation on soil respiration and mineralization
Soil Biol Biochem. Vol. 2, pp. 99408.
Pergamon Press 1970. Printed in Great Britain
RESIDUAL EFFECTS ON SOIL RESPIRATION
OF SOIL FUMIGATION AND MINERALIZATION
D. S. JENKINSON and D. S. POWLSON Pedology Department,
Rothamsted
Experimental
Station, Harpenden,
Herts.
(Axepted 2 January 1970) Summary-Soils were taken from fields that had been fumi~ted with formalin or methyl bromide 6 months to 5&years previously. Fumigated and unfum~gated soil respired at simiIar rates when incubated in the laboratory. By contrast, after they had been exposed to chloroform vapour, the fumigated respired less rapidly and mineralized less nitrogen than the unfumigated. Irradiation (2.5 Mradf was broadly similar to chloroform vapour in its effects on soil respiration and mineralization. These results are attributed to the elimination of a section of the soil biomass during field fumigation: recovery was not complete even after several years. Field experiments sometimes show a declining crop response to repeated fumigation. Our results show that less nitrogen is mineralized after a second fumigation than after the first. Thus, when nitrogen is limiting growth, a second fumigation will be less effective than the first, quite apart from any effect on plant pathogens. INTRODUCTION
fumigants, used primarily to control plant pathogens, can increase the rate at which soils respire and mineralize nitrogen. The extra nitrogen mineralized during the flush of decomposi~on can enhance crop growth when nitrogen is limiting, quite apart from any effects on plant pathogens. Recent field experiments (Widdowson and Penny, 1970) show a declining response to repeated fumigation, possibly because less mineral nitrogen is released after the second fumigation than after the first. The aims of this work were (a), to examine respiration and mineralization in soils previously fumigated and cropped and (b), to see how the flush of decomposition that follows fumigation is affected by previous field fumigations. The soils used came from three field experiments: formaldehyde had been used in two of these and methy bromide in the other. SOIL
MATERIALS
AND METHODS
Soils Pff~t~re~~e~~. These soils were from an experiment on the effects of formalin and nitrogen on the growth of wheat (Widdowson and Penny, 1970; Salt, 1969). The site had been under pasture for 10 yr before being ploughed and sown to spring wheat in 1964. The experiment began in 1965; spring wheat was grown in 1965 and 1966; winter wheat in 1967, 1968 and 1969. A basal manuring of phosphorus and potassium was given annually; nitrogen, as calcium nitrate, was applied at 0, 63, 125 and 188 kg N/ha. Formalin, diluted with water to contain 2 ~7% formaldehyde, was applied about one month before sowing at a rate of 1140 kg formaldehyde/ha. The so& were fumigated in February of 1965 and 1966, and in September of 1966 and 1967. Soil samples were taken in March 1968, and May 1969. Butt CZosefieid. The soils came from an experiment done in 1964, 1965 and 1966 on the effects of fumigants on spring wheat ~iddowson and Penny, 3970; Slope, 1966; WiIliams, 1969). Formalin (1140 kg formaldehyde~ha) was applied twice, in December 1963 and February 1964, before the 1964 crop was sown. It was applied once, in December 1964, 99 SOIL 2/2-c
100
D. S. JENKINSON
AND D. S. POWLSON
before the 1965 crop and once, in February 1966, before the 1966 crop. At the end of the experiment the site was cropped with barley in 1967 and 1968 and fallowed in 1969. Soil sampling was in August 1969. Broadbalkfield. The soils were from a strip across the Permanent Wheat Experiment that had been fumigated with methyl bromide containing 2% chloropicrin in September 1967 (Corbett, 1969). Application (980 kg/ha) was uneven and in both 1968 and 1969 the wheat was tallest round the points where the fumigant had been released in 1967 (Brown and Jenkinson, 1969). Both soil samples were taken from plot 3 (unmanured); one was from the unfumigated control strip and the other from an area on the fumigated strip within 1 m of a methyl bromide injection point. Sampling was in July 1969. Soil sampling and preparation All samples were taken to a depth of 15 cm, using a 2.5 cm diameter corer. Eight cores were taken per sample from Butt Close and Broadbalk: two samples, of four cores each, were taken from each plot in Pastures. Discrete pieces of organic matter were removed by hand and the soils passed through a 2 mm sieve. The soils were not allowed to dry out at any stage and were stored at - 15°C. Soil was thawed at room temperature, a portion given the appropriate laboratory treatment and two portions set aside as controls. Except for the treatment itself, controls and treated soil were kept under the same conditions. When the treated and untreated incubations were started, the untreated unincubated control was returned to storage at -15°C to await analysis. Chloroform treatment. The procedure has already been described (Jenkinson, 1966): briefly, the moist soil was exposed to alcohol-free chloroform vapour for 18 hr at 25°C and the chloroform then removed by repeated evacuation. Formaldehyde treatment. The soil (150 g, containing 83 % oven-dry soil) was treated with 2.5 ml of a 5 % formaldehyde solution, made by diluting formalin (Analar grade: containing 40 % formaldehyde and 10 % methyl alcohol as stabilizer), in a closed 250 ml flask for 24 hr at 25°C. Formaldehyde was then removed by passing air through the soil for five days at 2°C: this temperature was chosen to minimize biological activity during removal of the formaldehyde. Some formaldehyde still remained; water exposed to the air stream coming from the soil after 5 days gave a positive test for formaldehyde (Feigl, 1956), although the colour intensity was much less than at the beginning. When air was passed through untreated soil for 5 days at 2°C less than 2 ppm of inorganic nitrogen was mineralized. Irradiation. Moist soil (135 g, containing 83 % oven-dry soil) in Polythene bags was given a dose of 2.5 Mrad y-radiation from a 6oCo source. The soils were kept frozen during transport to and from the source. Incubation procedure and analytical methods The soils were incubated for 10 days at 25°C in the dark. All soils, whether treated or not, were inoculated with 5 mg of finely ground air-dried soil from an unfumigated plot of the appropriate field experiment. The soils were incubated at 55% of their water holding capacity in glass vials, 4 cm in diameter, containing an amount of moist soil equivalent to 25 g oven-dry soil. Each vial was placed in a bottle connected to a differential Barcroft manometer, together with a beaker containing 20 ml N sodium hydroxide, so that oxygen uptake could be measured. The manometer bottle also contained 5 ml of water to prevent excessive soil drying by the alkali. The manometer was a modification of that used by Smith and Brown (1932). Oxygen uptake was calculated from the manometer reading by the formula of Umbreit, Burris and Stauffer (1957). The total air volume of the manometer
RESIDUAL
EFFECTS
OF SOIL FUMIGATION
101
bottle was approximately 1 litre and in no case was more than 10 % of the oxygen consumed during incubation. The carbon dioxide absorbed by the alkali was determined by titration against 0.1 N HCI using a Pye Autotitrator. Ammonium, nitrite and nitrate were determined calorimetrically in soil extracts using a Technicon AutoAnalyser. Fifty millilitres of N potassium sulphate were added to each vial; after 1 hour’s shaking the suspension was filtered. Ammonium was determined in the extract by the formation of indophenol blue with alkaline sodium phenate and sodium hypochlorite (Varley, 1966) in the presence of a sodium nitrate-sodium tartrate buffer (Dabin, 1965). Nitrite was determined by diazotization with sulphanilic acid and coupling with N-(lnaphthyl) ethylenediamine; nitrate was reduced on a zinc column and determined as nitrite (Litchfield, 1967). After irradiation, the soils contained up to 4 ppm nitrite-nitrogen: this disappeared completely on incubation (see Cawse and White, 1969). Nitrite-nitrogen was less than 1 ppm in all other treatments, both before and after incubation, and will not be considered further. The total mineral nitrogen content of the incubated soil, less that of the untreated unincubated soil, gave the nitrogen mineralized during incubation. Exchangeable manganese was determined calorimetrically (Adams, 1965). Total nitrogen in soil was determined by the Kjeldahl method, using copper sulphate and selenium as catalysts. Errors
The Pastures experiment contained no replicate plots but each formalin treatment was repeated at two different nitrogen levels. There were few significant differences in oxygen uptake, carbon dioxide evolution, or nitrogen mineralization between the same formalin treatment at different nitrogen levels. Where the differences were significant they were not consistent; an increase in the nitrogen applied to the wheat sometimes increased and sometimes decreased soil respiration and mineralization. Hence results for each formalin treatment were averaged over the two nitrogen levels which were treated as replicates in calculating the least significant differences (Table 2). RESULTS
1. Residual eflects of formalin or methyl bromide fumigation Fumigation in the field had little effect on the pH or total nitrogen content of the soils but it consistently increased their ammonium nitrogen content (Table 1). The fumigated Pastures soil contained less nitrate-nitrogen than the unfumigated soil but this effect was not TABLE 1. ANALYSIS of FUMIGATED AND UNFUMIGATED SOILP Site
Soil texture
Pastures
Silt loam
Broadbalk
Silt loam’
Butt Close
Loamy sand
Field treatment
Not fumigated Formalin in 1966 (twice) and 1967 Not fumigated Methyl bromide in 1967 Not fumigated Formalin in 1963, 1964 (twice) and 1966
PH
Total N NH4-N (%) (Ppm)
NOB-N (Ppm)
6.5 6.4 8.0 8.0 7.2
0.183 0.176 0.115 0.118 0.091
1.1 4.0 0.7 :.:
15 9 6 6 7
7.1
0.088
0.7
7
“All analyses in this and subsequent tables on oven-dry (24 hr at 105°C) basis. *Soils sampled in 1969. ‘Contains 0.3 1% carbonate C.
-
102
D. S. JENKINSON AND D. S. POWLSON
seen in the samples from Broadb~k or Butt Close. The effect of fumigation on ammonium acetate-exchangeable manganese (not shown in Table l} was still apparent in the soil from Pastures sampled 20 months after fumigation: the fumigated soil contained 8 ppm exchangeable manganese and the unfumigated soil 5 ppm. This residual effect was small compared with that in the soils studied by Smith (1963). The samples from Broadbalk and Butt Close contained too little exchangeable manganese for measurement by the method used. The effects of field fumigation on oxygen consumption, carbon dioxide evolution and nitrogen mineralization were slight by the time the soil samples used in Table 2 were taken. Respiration was slightly faster in the formalin-treated soils but the differences were mostly not significant: mineralization of nitrogen was significantly greater in soils fumigated 6 months before than in unfumigated soil or soil fumigated 3 years before. Oxygen uptake, carbon dioxide evolution and nitrogen minerali~tion all increased after exposure to chloroform vapour and inoculation with fresh soil. This is the well-known flush of decomposition of soil organic matter that follows partial sterilization. However, these increases were significantly smaller for the soils that had been fumigated in the field; even the soil given a single formalin fumigation 3 years previously gave a significantly smaller flush of decomposition than the soil that had never received formalin. This long-term effect of formalin on soil was also observed in fresh samples taken 20 months after the last fumigation (Table 4). Similar results were obtained with a sandy soil from Butt CIose field (Table 3); formahn fumigation 3Qyears before sampling decreased the flush of decomposition following laboratory chloroform treatment and even 54 years after fumigation there was still a small effect. In Broadbalk, soi fumigation with methyl bromide 22 months before sampling decreased the flush considerably (Table 3). 2. Soil respiration and mineralization after exposure irradiation
to chloroform vapour, formalin or
Two soils from Pastures field were used in a detailed comparison of treatments causing partial or complete sterilization. The soils were from the plot that had never received formalin and the plot fumigated with formalin for three consecutive years: both plots received 63 kg N/ha per year. The samples were taken 20 months after the last fumigation. Table 4 shows that oxygen uptake and carbon dioxide evolution after exposure to chloroform vapour, exposure to formaldehyde, or irradiation were broadly similar; the soil previously fumigated in the field gave a smaller flush of decomposition than the soil that had never been fumigated before. However there were differences and the treatments will be considered individually. Chloroform. There was little lag in respiration after treatmentwith chloroform vapour,and respiration was enhanced for about 7 days (Fig. 1). After this, the chloroform-treated soils consumed oxygen at almost the same rate as the untreated soils. The chloroform-treated soils mineralized organic matter with a smaller C/N ratio than the untreated soils, suggesting that chloroform treatment caused the decomposition of a highly nitrogenous section of the soil organic matter. The same effect can be seen in Table 2. irradiation. Both oxygen uptake and carbon dioxide evolution were slightly greater than after exposure to chloroform vapour (Table 4). Irradiation caused a more pronounced lag phase in respiration than chloroform vapour, almost certainly because the radiation dose of
30.9 9.8 0.11 0.85 12.7 79.1 24.3 4.30 0.82 5.7
O2 consumed, mg/lOO g soil C02-C evolved, mg/lOO g soil N mineralized, mg/lOO g soil Respiratory Quotient C/N ratio of organic matter mineralized
O2 consumed, mg/lOO g soil C02-C evolved, mg/lOO g soil N mineralized, mg/lOO g soil Respiratory Quotient C/N ratio of organic matter mineralized
60.8 17.6 2.96 0.17 6.0
Exposed to chloroform vapour 54.6 53.3 17.0 14.6 2.88 2.35 0*x3 0.73 6.2 5-9
1967’
38.3 11.6 1.34 0.81 8.7
vapour
1965, 1966 (twice) and 1967d
Not exposed to chloroform 33.1 34.5 10-4 9.7 1.16 1.14 0.81 0.77 8.4 9.1
1966 (twice) and 1967”
CHLOROFORM
64.4 20.9 3.27 0.87 6.4
34.2 10.0 0.82 0.78 12.3
1965e
VAPOUR
8.0 3.2 0.23 -
4.3 2.0 0.32 -
Least significant differences (P = 0.05)
ANRINCUBATED"
“All incubations for 10 days at 25°C. bSoils sampled in 1968 from Pastures field, 6 months after the 1967 fumigation, 3 years after the 1965 fumigation. CMeans of duplicate incubations on each of 4 soil samples, two from the plot receiving 63 kg N/ha and two from the plot receiving 125 kg N/ha. dAs c above but from the plots receiving 0 and 188 kg N/ha. eMeans of duplicate incubations on each of two soil samples, both from the plot receiving no nitrogen.
Not fumigated0
Laboratory measurement
Field treatment with formalin”
TABLE ~.SOILRESPIRATIONANJ.IMINERALIZATION AFTERFIELD-FUMIGATED SOILSWEREEXPOSEDTO
2;
2 “n
“0 F:
%
2
$I
& tn
?
Butt CIose
AFTER FIELD-FUMIGATED
23.6 l-23
O2 consumed, mg/lOO g soil N mineralized, mg/lOO g soil
54 years after the 1963 fumigation.
20.4 1.10
Exposed to chloroform vapour 19.7 18.3 o-97 0.96
“Ali ~ncu~tions for 10 days at 25°C. %Samples taken in 1969, 3Q years after the 1966 fumigation, cSamples taken in 1969, 22 months after fumigation. dMeans of duplicate incubations.
11.6 o-35
Not exposed to chloroform vapour 12-o 12.4 0.48 o-35
1963
1966
12.2 0.54
1963, 1964 (twice) and 1966
AND INCUBATED’
Broadbalk
VAPOUR
0.11 Exposed to chloroform 54.9 2.18
0.17 vapour 38.9 1.63
Not exposed to chloroform vapour 24.6 21.9
Field treatment with methyl bromideC Not fumigated 1967
SOlLS WERE EXPOSED TO CHLOROFORM
Field treatment with formalin*
MINERALIZED
Not fumigated
AND NITROGEN
OZ consumed, mg/lOO g soil N mineralized, mg/100 g soil
Laboratory m~surementd
TABLE 3. OXYGEN CONSU~D
sr-r
tr m
6
2
@ E 2:
m
P
RESIDUAL
EFFECTS OF SOIL FUMIGATION
I i: !1
’
I
:
I
:
‘
I
(l!OS~001 /fiW)
C73Wf?SNO3 N3DAXO
IO5
106
D. S. JENKINSON
AND D. S. POWLSON
TABLE 4. SOIL RESPIRATION AND MINERALIZATION AFTER FIELD-FUMIGATED SOIL WAS EXPOSED TO EITHER CHLOROFORM VAPOUR,FORMALlN,ORlRRADXATIONA~~ENINCUBATED*
Laboratory Laboratory measurement'
O2 consumed, mg/lOO g soil C02-C evolved,mg/lOO g soil N mineralized,mg/lOO g soil
Respiratory Quotient C/N ratio of organic matter mineralized
O2 consumed,mg/lOO gsoil C02-Cevolved, mg/lOO g soil N mineralized,mg/100 g soil
Respiratory Quotient C/N ratio of organic matter mineralized
None
Chloroform vapour
treatment Formalin
Irradiation
Least significant differences (P = 0.05)
Not fumigated in the field* 83.1 90.1 87.1 24.4 30.0 26.8 4.40 -0.27 3.32 0.78 O-89 0.82 5.5 8.1
3.4 1.8 0.64 0.09 2.9
Fumigated with formalin in the field in 1966 (twice) and 1967b 39.6 62.9 66.2 69.9 12-o 18.0 24.8 21.2 0.97 3‘50 0.67 2.15 0.81 0.76 1.00 O-81 12.4 5.2 9.9
3.4 1.8 0.64 0.09 2.9
42.5 12.0 1.16 0.75 IO.3
“AI1incubations for 10 days at 25°C. “Samples taken in 1969,20 months after the last field fumigation, from plots receiving 63 kg N/ha. =Means of duplicate incubations.
2.5 Mrad killed more soil organisms (see Cawse 1969) than chloroform. In contrast to the chloroform-treated soils, the irradiated soils consumed oxygen faster than the untreated soils during the whole incubation period (Fig. 1). Table 4 also shows that significantly less nitrogen is mineralized after irradiation than after treatment with chloroform. If irradiation gives a more complete kill than chloroform, it should render more and not less nitrogen mineraIizable. A compIete explanation of this anomaly cannot be given, although part of the difference is due to the method used in calculating the mineralization of nitrogen. Nitrogen mineralized during incubation is given in Table 4 by (NH4-N _1- N03-N after incubation) - (NH*-N + NO,-N in untreated unincubated soil). Irradiation decreased soil nitrate: for example the unfumigated soil in Table 4 contained I5 ppm nitrate-nitrogen before and 12 ppm immediately after irradiation. The figures in Tabie 4 for nitrogen mineralized after irradiation are therefore low by the amount of nitrate destroyed during irradiation. Formaldehyde. Here the situation was more complicated than with chloroform because formaldehyde was not completely removed from the soils before incubation. This residual formaldehyde almost certainly caused the marked lag phase in respiration (Fig. 1). The respiratory quotients of the formaldehyde-treated soils were larger than those following any other treatment, suggesting that residues from the formaldehyde treatment were mineralized during incubation: the complete mineralization of formaldehyde would give an R.Q. of I ~0. Mineral nitrogen decreased in one soil during incubation and only increased slightly in the other. Presumably mineral nitrogen was immobilized by soil organisms as residues from the formaldehyde treatment were mineralized. It is no coincidence that the soil with the longest lag phase immobilized the most nitrogen.
REtSIDUAL EFFECTS OF SOIL FUMIGATION
LO7
DISCUSSION
Our results show that formaldehyde fumigation in the field eliminates a fraction of the soil organic matter, so that a second exposure to formaldehyde, chloroform vapour, or irradiation causes a smaller flush of decomposition. Furthermore, the elimination of this fraction does not appreciably alter the respiration rate of the soil. Restoration of this part of the soil organic matter is not complete even after several unfumigated crops have been grown. Jenkinson (1966) attributed the flush of decomposition following fumigation with a reagent such as chloroform, which does not leave residues in the soil, to the killing of organisms and their subsequent mineralization by surviving or recolonizing organisms. On this hypothesis, field fumigation eliminates a section of the biomass that had not been completely replaced by the time the samples were taken for the laboratory investigations. This is most likely to be the zymogenic spore population: its elimination would not appreciably influence soil respiration in the absence of fresh substrate. The slowness with which the eliminated population recovers suggests that the section of the biomass involved has a half-life in soil measured in years rather than days or months, although the results in Tables 2 and 3 are not precise enough for quantitative calculations on the half-life of the eliminated population. These observations of a long-term effect of fumigation on the soil biomass are consistent with earlier work. Warcup (1951) found that soil exposed to formalin or steam 18 months before contained fewer species of fungi than untreated soil: colony numbers were also less. Similar effects from steam or formalin were found by Mollison (1953) ; even after 25 months fungal numbers had not completely recovered. Martin, Baines and Ervin (1957) reported that the fungal populations of many soils were still depressed 2 or 3 years after fumigation with a mixture of 1, 3-dichloropropane and 1, 3-dichloropropene (D-D). Reber (1967) showed that fungal numbers had not recovered appreciably 13 weeks after field fumigation with methyl bromide: bacterial and actinomycete numbers increased briefly but by 13 weeks were little different from the unfumigated control. Field fumigation with formahn decreased the biomass considerably. Assume, as a first appro~mation, that the amount of biomass carbon in a soil is proportional to the ~dditj~nui carbon mineralized by the soil in 10 days after exposure to chloroform vapour{i.e. the carbon dioxide-carbon evolved after chloroform treatment less the carbon dioxide-carbon evolved by untreated soil: Jenkinson 1966). On this basis, a single fumigation 6 months before sampling decreased the biomass carbon by roughly half (Table 2); the biomass nitrogen decreased similarly. The effects offumigation on crop yield Widdowson and Penny’s (1970) experiments on cereals (from which most of the soil samples used in our work were taken) were particularly concerned with the effects of formalin on crop yield. Formalin usually increased the crop immediately following fumigation: the increase was most marked in straw yields but was often seen in the grain yields as well. The second crop after fumigation was little better and often worse than that on soil that had never been fumigated. Fumigation in two consecutive years did not increase yields as much as a single fumigation in the harvest year only. Changes in the incidence of nematodes in these experiments have been followed by Williams (1969); in the incidence of fungal pathogens by Slope (1966) and Salt (1969). The declining response to repeated fumigation in these field experiments may be partly due to less effective control of pathogens : this is particularly likely in the Butt Close experiment
108
D. S. JENKINSON
AND D. S. POWLSON
where cereal cyst eelworm (~eter~dera avenue) numbers were greater a year after fumigating than in the unfumigated control plots (Williams, 1969). Our work shows that the flush of mineral nitrogen accompanying a second fumigation is less than that from the first. Thus, when nitrogen is limiting growth, a second fumigation will be less effective than the first in increasing yield, quite apart from any effects on plant pathogens. Even if the second fumigation takes place after an interval of as much as 5 years, the effect of the first treatment still persists and less nitrogen will be mineralized than if the soil had never been fumigated. Acknowledgements-We thank Mr P. A. CAWSE of the United Kingdom Atomic Energy Authority for arranging the soil irradiations; Miss B. MESSERfor analyses using the AutoAnalyser; Mr J. H. A. DUNWC~~DY for the statistical analyses and Professor E. W. RUSSELLfor helpful discussion.
ADAMSF. (1965) Manganese. In methods of Soil AnaZysis (C. A. Black, Ed.) pp. 1013-1014, American Society
of Agronomy,
Madison.
BROU?~I G. and JENKIN~OND. S. (1969) Fumigation
with methyl bromide. Rep. Rothamstedexp. S&for 1968, pp. 71-72. CAWSE P. A. (1969) The Use of Gamma Radiation in Soil Research, United Kingdom Atomic Energy Authority Reoort R 6061. HMSO. London. CAWSE P. A. and~Wmrn T. (1969) Rapid changes in nitrite after gamma-irradiation of fresh soil. J. agric. Sci. Camb. 73, 113-I 18. CORBETTD. C. M. (1969) Root lesion nematodes. Rep. Rathamsted exp. Stn for 1968, p. 164. DABIN B. (1965) Application des dosages automatiques a I’analyse des sois. Cab. O.R.S.T.O.M. Pedal. 3, 335-348. FEIGL F. (1956) Spot Tests in Organic Analysis, 5th edn, pp. 331-332, Elsevier, Amsterdam. JENKINSOND. S. (1966) Studies on the decomposition of plant material in soil. ILPartial sterilization of soil and the soil biomass. J. Soil Sei. 17, 280-302. LITCHFLELD M. H. (1967) The automated analysis of nitrite and nitrate in blood. Ann&s& Load. 92,132-136, MARTIN J. P.. BAIN~~ R. C. and ERVINJ. 0. (1957) Influence of soil fumigation for citrus replants on the fungus populations of the soil. Proc. Soil Sci.‘Soc.‘Am. 21, 163-166. MOLLISON J. E. (1953) Effect of partial sterilization and acidification of soil on the fungal population. Trans. Br. mycol. Sot. 36,215-228. REBERH. (1967) Untersuchungen uber die Wiederbesiedlung eines chemisch entseuchten Bodens. Z. Pfl Krankh. PfPath. PjSchutz 74,427438. SALT G. A. (1969) The effects of repeated fumigation. Rep. Rothamsted exp. Stn for 1968, pp. 139-140. SLOPED. B. (1966) Effect of formalin on take-all. Rep. Rothamsted exp. Stn for 1965, pp. 127-128. SMITHD. H. (1963) Effect of fumigants on the soil status and plant uptake of certain elements. Proc. Soil Sci. Sot. Am. 27, 538-541. SMITHF. B. and BROWN P. E. (1932) Methods for determining carbon dioxide production in soils Res. BuiI. Iowa agric. Exp. Stn No. 147. UMBREITW. W., BURRISR. H. and STAUFFERJ. F. (1957) Muaometric Techniques, 3rd edn, p. 83, Burgess Publishing Co., Minneapolis. VARLEY J. A. (1966) Automatic methods for the dete~ination of nitrogen, phosphorous and potassium in plant materials. Analyst, Land. 91, 119-l 26. WARCUP J. H. (1951) Effect of partial sterilization by steam or formatin on the fungus flora of an old forest nursery soil. Trans. Br. ~nycaf. Sot. 34, 519-532. WIDDOW~~N F. V. and PENNYA. (1970) The effects of partially sterilizing agricultural soils with formalin, and of applying nitrogen fertilizers, on the yields and nitrogen contents of spring and winter wheat, of barley and of grass. Rep. Rothamsted exp. Stn for 1969, Pt. 2, pp. 113-134. WILLIAMST. D. (1969) The effects of formalin, nabam, irrigation and nitrogen on Heterodera auenae Woll., Ophiobolus gram&is Sacc. and the growth of spring wheat. Ann. appI. Biol. 64, 325-334.
Pergamon Press 1970. Printed in Great Britain
RESIDUAL EFFECTS ON SOIL RESPIRATION
OF SOIL FUMIGATION AND MINERALIZATION
D. S. JENKINSON and D. S. POWLSON Pedology Department,
Rothamsted
Experimental
Station, Harpenden,
Herts.
(Axepted 2 January 1970) Summary-Soils were taken from fields that had been fumi~ted with formalin or methyl bromide 6 months to 5&years previously. Fumigated and unfum~gated soil respired at simiIar rates when incubated in the laboratory. By contrast, after they had been exposed to chloroform vapour, the fumigated respired less rapidly and mineralized less nitrogen than the unfumigated. Irradiation (2.5 Mradf was broadly similar to chloroform vapour in its effects on soil respiration and mineralization. These results are attributed to the elimination of a section of the soil biomass during field fumigation: recovery was not complete even after several years. Field experiments sometimes show a declining crop response to repeated fumigation. Our results show that less nitrogen is mineralized after a second fumigation than after the first. Thus, when nitrogen is limiting growth, a second fumigation will be less effective than the first, quite apart from any effect on plant pathogens. INTRODUCTION
fumigants, used primarily to control plant pathogens, can increase the rate at which soils respire and mineralize nitrogen. The extra nitrogen mineralized during the flush of decomposi~on can enhance crop growth when nitrogen is limiting, quite apart from any effects on plant pathogens. Recent field experiments (Widdowson and Penny, 1970) show a declining response to repeated fumigation, possibly because less mineral nitrogen is released after the second fumigation than after the first. The aims of this work were (a), to examine respiration and mineralization in soils previously fumigated and cropped and (b), to see how the flush of decomposition that follows fumigation is affected by previous field fumigations. The soils used came from three field experiments: formaldehyde had been used in two of these and methy bromide in the other. SOIL
MATERIALS
AND METHODS
Soils Pff~t~re~~e~~. These soils were from an experiment on the effects of formalin and nitrogen on the growth of wheat (Widdowson and Penny, 1970; Salt, 1969). The site had been under pasture for 10 yr before being ploughed and sown to spring wheat in 1964. The experiment began in 1965; spring wheat was grown in 1965 and 1966; winter wheat in 1967, 1968 and 1969. A basal manuring of phosphorus and potassium was given annually; nitrogen, as calcium nitrate, was applied at 0, 63, 125 and 188 kg N/ha. Formalin, diluted with water to contain 2 ~7% formaldehyde, was applied about one month before sowing at a rate of 1140 kg formaldehyde/ha. The so& were fumigated in February of 1965 and 1966, and in September of 1966 and 1967. Soil samples were taken in March 1968, and May 1969. Butt CZosefieid. The soils came from an experiment done in 1964, 1965 and 1966 on the effects of fumigants on spring wheat ~iddowson and Penny, 3970; Slope, 1966; WiIliams, 1969). Formalin (1140 kg formaldehyde~ha) was applied twice, in December 1963 and February 1964, before the 1964 crop was sown. It was applied once, in December 1964, 99 SOIL 2/2-c
100
D. S. JENKINSON
AND D. S. POWLSON
before the 1965 crop and once, in February 1966, before the 1966 crop. At the end of the experiment the site was cropped with barley in 1967 and 1968 and fallowed in 1969. Soil sampling was in August 1969. Broadbalkfield. The soils were from a strip across the Permanent Wheat Experiment that had been fumigated with methyl bromide containing 2% chloropicrin in September 1967 (Corbett, 1969). Application (980 kg/ha) was uneven and in both 1968 and 1969 the wheat was tallest round the points where the fumigant had been released in 1967 (Brown and Jenkinson, 1969). Both soil samples were taken from plot 3 (unmanured); one was from the unfumigated control strip and the other from an area on the fumigated strip within 1 m of a methyl bromide injection point. Sampling was in July 1969. Soil sampling and preparation All samples were taken to a depth of 15 cm, using a 2.5 cm diameter corer. Eight cores were taken per sample from Butt Close and Broadbalk: two samples, of four cores each, were taken from each plot in Pastures. Discrete pieces of organic matter were removed by hand and the soils passed through a 2 mm sieve. The soils were not allowed to dry out at any stage and were stored at - 15°C. Soil was thawed at room temperature, a portion given the appropriate laboratory treatment and two portions set aside as controls. Except for the treatment itself, controls and treated soil were kept under the same conditions. When the treated and untreated incubations were started, the untreated unincubated control was returned to storage at -15°C to await analysis. Chloroform treatment. The procedure has already been described (Jenkinson, 1966): briefly, the moist soil was exposed to alcohol-free chloroform vapour for 18 hr at 25°C and the chloroform then removed by repeated evacuation. Formaldehyde treatment. The soil (150 g, containing 83 % oven-dry soil) was treated with 2.5 ml of a 5 % formaldehyde solution, made by diluting formalin (Analar grade: containing 40 % formaldehyde and 10 % methyl alcohol as stabilizer), in a closed 250 ml flask for 24 hr at 25°C. Formaldehyde was then removed by passing air through the soil for five days at 2°C: this temperature was chosen to minimize biological activity during removal of the formaldehyde. Some formaldehyde still remained; water exposed to the air stream coming from the soil after 5 days gave a positive test for formaldehyde (Feigl, 1956), although the colour intensity was much less than at the beginning. When air was passed through untreated soil for 5 days at 2°C less than 2 ppm of inorganic nitrogen was mineralized. Irradiation. Moist soil (135 g, containing 83 % oven-dry soil) in Polythene bags was given a dose of 2.5 Mrad y-radiation from a 6oCo source. The soils were kept frozen during transport to and from the source. Incubation procedure and analytical methods The soils were incubated for 10 days at 25°C in the dark. All soils, whether treated or not, were inoculated with 5 mg of finely ground air-dried soil from an unfumigated plot of the appropriate field experiment. The soils were incubated at 55% of their water holding capacity in glass vials, 4 cm in diameter, containing an amount of moist soil equivalent to 25 g oven-dry soil. Each vial was placed in a bottle connected to a differential Barcroft manometer, together with a beaker containing 20 ml N sodium hydroxide, so that oxygen uptake could be measured. The manometer bottle also contained 5 ml of water to prevent excessive soil drying by the alkali. The manometer was a modification of that used by Smith and Brown (1932). Oxygen uptake was calculated from the manometer reading by the formula of Umbreit, Burris and Stauffer (1957). The total air volume of the manometer
RESIDUAL
EFFECTS
OF SOIL FUMIGATION
101
bottle was approximately 1 litre and in no case was more than 10 % of the oxygen consumed during incubation. The carbon dioxide absorbed by the alkali was determined by titration against 0.1 N HCI using a Pye Autotitrator. Ammonium, nitrite and nitrate were determined calorimetrically in soil extracts using a Technicon AutoAnalyser. Fifty millilitres of N potassium sulphate were added to each vial; after 1 hour’s shaking the suspension was filtered. Ammonium was determined in the extract by the formation of indophenol blue with alkaline sodium phenate and sodium hypochlorite (Varley, 1966) in the presence of a sodium nitrate-sodium tartrate buffer (Dabin, 1965). Nitrite was determined by diazotization with sulphanilic acid and coupling with N-(lnaphthyl) ethylenediamine; nitrate was reduced on a zinc column and determined as nitrite (Litchfield, 1967). After irradiation, the soils contained up to 4 ppm nitrite-nitrogen: this disappeared completely on incubation (see Cawse and White, 1969). Nitrite-nitrogen was less than 1 ppm in all other treatments, both before and after incubation, and will not be considered further. The total mineral nitrogen content of the incubated soil, less that of the untreated unincubated soil, gave the nitrogen mineralized during incubation. Exchangeable manganese was determined calorimetrically (Adams, 1965). Total nitrogen in soil was determined by the Kjeldahl method, using copper sulphate and selenium as catalysts. Errors
The Pastures experiment contained no replicate plots but each formalin treatment was repeated at two different nitrogen levels. There were few significant differences in oxygen uptake, carbon dioxide evolution, or nitrogen mineralization between the same formalin treatment at different nitrogen levels. Where the differences were significant they were not consistent; an increase in the nitrogen applied to the wheat sometimes increased and sometimes decreased soil respiration and mineralization. Hence results for each formalin treatment were averaged over the two nitrogen levels which were treated as replicates in calculating the least significant differences (Table 2). RESULTS
1. Residual eflects of formalin or methyl bromide fumigation Fumigation in the field had little effect on the pH or total nitrogen content of the soils but it consistently increased their ammonium nitrogen content (Table 1). The fumigated Pastures soil contained less nitrate-nitrogen than the unfumigated soil but this effect was not TABLE 1. ANALYSIS of FUMIGATED AND UNFUMIGATED SOILP Site
Soil texture
Pastures
Silt loam
Broadbalk
Silt loam’
Butt Close
Loamy sand
Field treatment
Not fumigated Formalin in 1966 (twice) and 1967 Not fumigated Methyl bromide in 1967 Not fumigated Formalin in 1963, 1964 (twice) and 1966
PH
Total N NH4-N (%) (Ppm)
NOB-N (Ppm)
6.5 6.4 8.0 8.0 7.2
0.183 0.176 0.115 0.118 0.091
1.1 4.0 0.7 :.:
15 9 6 6 7
7.1
0.088
0.7
7
“All analyses in this and subsequent tables on oven-dry (24 hr at 105°C) basis. *Soils sampled in 1969. ‘Contains 0.3 1% carbonate C.
-
102
D. S. JENKINSON AND D. S. POWLSON
seen in the samples from Broadb~k or Butt Close. The effect of fumigation on ammonium acetate-exchangeable manganese (not shown in Table l} was still apparent in the soil from Pastures sampled 20 months after fumigation: the fumigated soil contained 8 ppm exchangeable manganese and the unfumigated soil 5 ppm. This residual effect was small compared with that in the soils studied by Smith (1963). The samples from Broadbalk and Butt Close contained too little exchangeable manganese for measurement by the method used. The effects of field fumigation on oxygen consumption, carbon dioxide evolution and nitrogen mineralization were slight by the time the soil samples used in Table 2 were taken. Respiration was slightly faster in the formalin-treated soils but the differences were mostly not significant: mineralization of nitrogen was significantly greater in soils fumigated 6 months before than in unfumigated soil or soil fumigated 3 years before. Oxygen uptake, carbon dioxide evolution and nitrogen minerali~tion all increased after exposure to chloroform vapour and inoculation with fresh soil. This is the well-known flush of decomposition of soil organic matter that follows partial sterilization. However, these increases were significantly smaller for the soils that had been fumigated in the field; even the soil given a single formalin fumigation 3 years previously gave a significantly smaller flush of decomposition than the soil that had never received formalin. This long-term effect of formalin on soil was also observed in fresh samples taken 20 months after the last fumigation (Table 4). Similar results were obtained with a sandy soil from Butt CIose field (Table 3); formahn fumigation 3Qyears before sampling decreased the flush of decomposition following laboratory chloroform treatment and even 54 years after fumigation there was still a small effect. In Broadbalk, soi fumigation with methyl bromide 22 months before sampling decreased the flush considerably (Table 3). 2. Soil respiration and mineralization after exposure irradiation
to chloroform vapour, formalin or
Two soils from Pastures field were used in a detailed comparison of treatments causing partial or complete sterilization. The soils were from the plot that had never received formalin and the plot fumigated with formalin for three consecutive years: both plots received 63 kg N/ha per year. The samples were taken 20 months after the last fumigation. Table 4 shows that oxygen uptake and carbon dioxide evolution after exposure to chloroform vapour, exposure to formaldehyde, or irradiation were broadly similar; the soil previously fumigated in the field gave a smaller flush of decomposition than the soil that had never been fumigated before. However there were differences and the treatments will be considered individually. Chloroform. There was little lag in respiration after treatmentwith chloroform vapour,and respiration was enhanced for about 7 days (Fig. 1). After this, the chloroform-treated soils consumed oxygen at almost the same rate as the untreated soils. The chloroform-treated soils mineralized organic matter with a smaller C/N ratio than the untreated soils, suggesting that chloroform treatment caused the decomposition of a highly nitrogenous section of the soil organic matter. The same effect can be seen in Table 2. irradiation. Both oxygen uptake and carbon dioxide evolution were slightly greater than after exposure to chloroform vapour (Table 4). Irradiation caused a more pronounced lag phase in respiration than chloroform vapour, almost certainly because the radiation dose of
30.9 9.8 0.11 0.85 12.7 79.1 24.3 4.30 0.82 5.7
O2 consumed, mg/lOO g soil C02-C evolved, mg/lOO g soil N mineralized, mg/lOO g soil Respiratory Quotient C/N ratio of organic matter mineralized
O2 consumed, mg/lOO g soil C02-C evolved, mg/lOO g soil N mineralized, mg/lOO g soil Respiratory Quotient C/N ratio of organic matter mineralized
60.8 17.6 2.96 0.17 6.0
Exposed to chloroform vapour 54.6 53.3 17.0 14.6 2.88 2.35 0*x3 0.73 6.2 5-9
1967’
38.3 11.6 1.34 0.81 8.7
vapour
1965, 1966 (twice) and 1967d
Not exposed to chloroform 33.1 34.5 10-4 9.7 1.16 1.14 0.81 0.77 8.4 9.1
1966 (twice) and 1967”
CHLOROFORM
64.4 20.9 3.27 0.87 6.4
34.2 10.0 0.82 0.78 12.3
1965e
VAPOUR
8.0 3.2 0.23 -
4.3 2.0 0.32 -
Least significant differences (P = 0.05)
ANRINCUBATED"
“All incubations for 10 days at 25°C. bSoils sampled in 1968 from Pastures field, 6 months after the 1967 fumigation, 3 years after the 1965 fumigation. CMeans of duplicate incubations on each of 4 soil samples, two from the plot receiving 63 kg N/ha and two from the plot receiving 125 kg N/ha. dAs c above but from the plots receiving 0 and 188 kg N/ha. eMeans of duplicate incubations on each of two soil samples, both from the plot receiving no nitrogen.
Not fumigated0
Laboratory measurement
Field treatment with formalin”
TABLE ~.SOILRESPIRATIONANJ.IMINERALIZATION AFTERFIELD-FUMIGATED SOILSWEREEXPOSEDTO
2;
2 “n
“0 F:
%
2
$I
& tn
?
Butt CIose
AFTER FIELD-FUMIGATED
23.6 l-23
O2 consumed, mg/lOO g soil N mineralized, mg/lOO g soil
54 years after the 1963 fumigation.
20.4 1.10
Exposed to chloroform vapour 19.7 18.3 o-97 0.96
“Ali ~ncu~tions for 10 days at 25°C. %Samples taken in 1969, 3Q years after the 1966 fumigation, cSamples taken in 1969, 22 months after fumigation. dMeans of duplicate incubations.
11.6 o-35
Not exposed to chloroform vapour 12-o 12.4 0.48 o-35
1963
1966
12.2 0.54
1963, 1964 (twice) and 1966
AND INCUBATED’
Broadbalk
VAPOUR
0.11 Exposed to chloroform 54.9 2.18
0.17 vapour 38.9 1.63
Not exposed to chloroform vapour 24.6 21.9
Field treatment with methyl bromideC Not fumigated 1967
SOlLS WERE EXPOSED TO CHLOROFORM
Field treatment with formalin*
MINERALIZED
Not fumigated
AND NITROGEN
OZ consumed, mg/lOO g soil N mineralized, mg/100 g soil
Laboratory m~surementd
TABLE 3. OXYGEN CONSU~D
sr-r
tr m
6
2
@ E 2:
m
P
RESIDUAL
EFFECTS OF SOIL FUMIGATION
I i: !1
’
I
:
I
:
‘
I
(l!OS~001 /fiW)
C73Wf?SNO3 N3DAXO
IO5
106
D. S. JENKINSON
AND D. S. POWLSON
TABLE 4. SOIL RESPIRATION AND MINERALIZATION AFTER FIELD-FUMIGATED SOIL WAS EXPOSED TO EITHER CHLOROFORM VAPOUR,FORMALlN,ORlRRADXATIONA~~ENINCUBATED*
Laboratory Laboratory measurement'
O2 consumed, mg/lOO g soil C02-C evolved,mg/lOO g soil N mineralized,mg/lOO g soil
Respiratory Quotient C/N ratio of organic matter mineralized
O2 consumed,mg/lOO gsoil C02-Cevolved, mg/lOO g soil N mineralized,mg/100 g soil
Respiratory Quotient C/N ratio of organic matter mineralized
None
Chloroform vapour
treatment Formalin
Irradiation
Least significant differences (P = 0.05)
Not fumigated in the field* 83.1 90.1 87.1 24.4 30.0 26.8 4.40 -0.27 3.32 0.78 O-89 0.82 5.5 8.1
3.4 1.8 0.64 0.09 2.9
Fumigated with formalin in the field in 1966 (twice) and 1967b 39.6 62.9 66.2 69.9 12-o 18.0 24.8 21.2 0.97 3‘50 0.67 2.15 0.81 0.76 1.00 O-81 12.4 5.2 9.9
3.4 1.8 0.64 0.09 2.9
42.5 12.0 1.16 0.75 IO.3
“AI1incubations for 10 days at 25°C. “Samples taken in 1969,20 months after the last field fumigation, from plots receiving 63 kg N/ha. =Means of duplicate incubations.
2.5 Mrad killed more soil organisms (see Cawse 1969) than chloroform. In contrast to the chloroform-treated soils, the irradiated soils consumed oxygen faster than the untreated soils during the whole incubation period (Fig. 1). Table 4 also shows that significantly less nitrogen is mineralized after irradiation than after treatment with chloroform. If irradiation gives a more complete kill than chloroform, it should render more and not less nitrogen mineraIizable. A compIete explanation of this anomaly cannot be given, although part of the difference is due to the method used in calculating the mineralization of nitrogen. Nitrogen mineralized during incubation is given in Table 4 by (NH4-N _1- N03-N after incubation) - (NH*-N + NO,-N in untreated unincubated soil). Irradiation decreased soil nitrate: for example the unfumigated soil in Table 4 contained I5 ppm nitrate-nitrogen before and 12 ppm immediately after irradiation. The figures in Tabie 4 for nitrogen mineralized after irradiation are therefore low by the amount of nitrate destroyed during irradiation. Formaldehyde. Here the situation was more complicated than with chloroform because formaldehyde was not completely removed from the soils before incubation. This residual formaldehyde almost certainly caused the marked lag phase in respiration (Fig. 1). The respiratory quotients of the formaldehyde-treated soils were larger than those following any other treatment, suggesting that residues from the formaldehyde treatment were mineralized during incubation: the complete mineralization of formaldehyde would give an R.Q. of I ~0. Mineral nitrogen decreased in one soil during incubation and only increased slightly in the other. Presumably mineral nitrogen was immobilized by soil organisms as residues from the formaldehyde treatment were mineralized. It is no coincidence that the soil with the longest lag phase immobilized the most nitrogen.
REtSIDUAL EFFECTS OF SOIL FUMIGATION
LO7
DISCUSSION
Our results show that formaldehyde fumigation in the field eliminates a fraction of the soil organic matter, so that a second exposure to formaldehyde, chloroform vapour, or irradiation causes a smaller flush of decomposition. Furthermore, the elimination of this fraction does not appreciably alter the respiration rate of the soil. Restoration of this part of the soil organic matter is not complete even after several unfumigated crops have been grown. Jenkinson (1966) attributed the flush of decomposition following fumigation with a reagent such as chloroform, which does not leave residues in the soil, to the killing of organisms and their subsequent mineralization by surviving or recolonizing organisms. On this hypothesis, field fumigation eliminates a section of the biomass that had not been completely replaced by the time the samples were taken for the laboratory investigations. This is most likely to be the zymogenic spore population: its elimination would not appreciably influence soil respiration in the absence of fresh substrate. The slowness with which the eliminated population recovers suggests that the section of the biomass involved has a half-life in soil measured in years rather than days or months, although the results in Tables 2 and 3 are not precise enough for quantitative calculations on the half-life of the eliminated population. These observations of a long-term effect of fumigation on the soil biomass are consistent with earlier work. Warcup (1951) found that soil exposed to formalin or steam 18 months before contained fewer species of fungi than untreated soil: colony numbers were also less. Similar effects from steam or formalin were found by Mollison (1953) ; even after 25 months fungal numbers had not completely recovered. Martin, Baines and Ervin (1957) reported that the fungal populations of many soils were still depressed 2 or 3 years after fumigation with a mixture of 1, 3-dichloropropane and 1, 3-dichloropropene (D-D). Reber (1967) showed that fungal numbers had not recovered appreciably 13 weeks after field fumigation with methyl bromide: bacterial and actinomycete numbers increased briefly but by 13 weeks were little different from the unfumigated control. Field fumigation with formahn decreased the biomass considerably. Assume, as a first appro~mation, that the amount of biomass carbon in a soil is proportional to the ~dditj~nui carbon mineralized by the soil in 10 days after exposure to chloroform vapour{i.e. the carbon dioxide-carbon evolved after chloroform treatment less the carbon dioxide-carbon evolved by untreated soil: Jenkinson 1966). On this basis, a single fumigation 6 months before sampling decreased the biomass carbon by roughly half (Table 2); the biomass nitrogen decreased similarly. The effects offumigation on crop yield Widdowson and Penny’s (1970) experiments on cereals (from which most of the soil samples used in our work were taken) were particularly concerned with the effects of formalin on crop yield. Formalin usually increased the crop immediately following fumigation: the increase was most marked in straw yields but was often seen in the grain yields as well. The second crop after fumigation was little better and often worse than that on soil that had never been fumigated. Fumigation in two consecutive years did not increase yields as much as a single fumigation in the harvest year only. Changes in the incidence of nematodes in these experiments have been followed by Williams (1969); in the incidence of fungal pathogens by Slope (1966) and Salt (1969). The declining response to repeated fumigation in these field experiments may be partly due to less effective control of pathogens : this is particularly likely in the Butt Close experiment
108
D. S. JENKINSON
AND D. S. POWLSON
where cereal cyst eelworm (~eter~dera avenue) numbers were greater a year after fumigating than in the unfumigated control plots (Williams, 1969). Our work shows that the flush of mineral nitrogen accompanying a second fumigation is less than that from the first. Thus, when nitrogen is limiting growth, a second fumigation will be less effective than the first in increasing yield, quite apart from any effects on plant pathogens. Even if the second fumigation takes place after an interval of as much as 5 years, the effect of the first treatment still persists and less nitrogen will be mineralized than if the soil had never been fumigated. Acknowledgements-We thank Mr P. A. CAWSE of the United Kingdom Atomic Energy Authority for arranging the soil irradiations; Miss B. MESSERfor analyses using the AutoAnalyser; Mr J. H. A. DUNWC~~DY for the statistical analyses and Professor E. W. RUSSELLfor helpful discussion.
ADAMSF. (1965) Manganese. In methods of Soil AnaZysis (C. A. Black, Ed.) pp. 1013-1014, American Society
of Agronomy,
Madison.
BROU?~I G. and JENKIN~OND. S. (1969) Fumigation
with methyl bromide. Rep. Rothamstedexp. S&for 1968, pp. 71-72. CAWSE P. A. (1969) The Use of Gamma Radiation in Soil Research, United Kingdom Atomic Energy Authority Reoort R 6061. HMSO. London. CAWSE P. A. and~Wmrn T. (1969) Rapid changes in nitrite after gamma-irradiation of fresh soil. J. agric. Sci. Camb. 73, 113-I 18. CORBETTD. C. M. (1969) Root lesion nematodes. Rep. Rathamsted exp. Stn for 1968, p. 164. DABIN B. (1965) Application des dosages automatiques a I’analyse des sois. Cab. O.R.S.T.O.M. Pedal. 3, 335-348. FEIGL F. (1956) Spot Tests in Organic Analysis, 5th edn, pp. 331-332, Elsevier, Amsterdam. JENKINSOND. S. (1966) Studies on the decomposition of plant material in soil. ILPartial sterilization of soil and the soil biomass. J. Soil Sei. 17, 280-302. LITCHFLELD M. H. (1967) The automated analysis of nitrite and nitrate in blood. Ann&s& Load. 92,132-136, MARTIN J. P.. BAIN~~ R. C. and ERVINJ. 0. (1957) Influence of soil fumigation for citrus replants on the fungus populations of the soil. Proc. Soil Sci.‘Soc.‘Am. 21, 163-166. MOLLISON J. E. (1953) Effect of partial sterilization and acidification of soil on the fungal population. Trans. Br. mycol. Sot. 36,215-228. REBERH. (1967) Untersuchungen uber die Wiederbesiedlung eines chemisch entseuchten Bodens. Z. Pfl Krankh. PfPath. PjSchutz 74,427438. SALT G. A. (1969) The effects of repeated fumigation. Rep. Rothamsted exp. Stn for 1968, pp. 139-140. SLOPED. B. (1966) Effect of formalin on take-all. Rep. Rothamsted exp. Stn for 1965, pp. 127-128. SMITHD. H. (1963) Effect of fumigants on the soil status and plant uptake of certain elements. Proc. Soil Sci. Sot. Am. 27, 538-541. SMITHF. B. and BROWN P. E. (1932) Methods for determining carbon dioxide production in soils Res. BuiI. Iowa agric. Exp. Stn No. 147. UMBREITW. W., BURRISR. H. and STAUFFERJ. F. (1957) Muaometric Techniques, 3rd edn, p. 83, Burgess Publishing Co., Minneapolis. VARLEY J. A. (1966) Automatic methods for the dete~ination of nitrogen, phosphorous and potassium in plant materials. Analyst, Land. 91, 119-l 26. WARCUP J. H. (1951) Effect of partial sterilization by steam or formatin on the fungus flora of an old forest nursery soil. Trans. Br. ~nycaf. Sot. 34, 519-532. WIDDOW~~N F. V. and PENNYA. (1970) The effects of partially sterilizing agricultural soils with formalin, and of applying nitrogen fertilizers, on the yields and nitrogen contents of spring and winter wheat, of barley and of grass. Rep. Rothamsted exp. Stn for 1969, Pt. 2, pp. 113-134. WILLIAMST. D. (1969) The effects of formalin, nabam, irrigation and nitrogen on Heterodera auenae Woll., Ophiobolus gram&is Sacc. and the growth of spring wheat. Ann. appI. Biol. 64, 325-334.