v EFFECTS OF TEMPERATURE AND AMMONIUM TO NITRATE I RATIO 0N MICROBIAL RESPONSES To FUMIGATION m ORGANIC sou Thesis for rhe Degree of M. S... MECHIGAN S‘FATE UNIVERSITY V 33mg Hui Use 1953 [HESS IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 31293 006121440 LIBRARY Michigan State University ABSTRACT EFFECTS OF TEMPERATURE AND AMMONIUM TO NITRATE RATIO ON MICROBIAL RESPONSES TO FUMIGATION IN ORGANIC SOIL by Fang Hui Liao Laboratory studies were conducted to investigate the effects of rising temperature and initial ratios of ammonium to nitrate on microbial numbers and activities in organic soil fumigated with Chlor0picrin or dichloroprOpene. These chemicals were used at rates and for exPosure periods recom- mended by the manufacturer for field application. Treated and control lots of Houghton muck were subjected to eXhaustive leaching, followed by aeration, freezing and periods of low temperature (5°C.) to simulate environmental conditions to whiCh fall fumigated organic soils are subjected during the winter and Spring months. Soils were amended with ammonium and nitrate in varying prOportions before being subjected to different sequences of rising temperature. Heterotrophic microbial pepulations and activities were Characterized by measurements of CO2 evolution and by enumeration of bacteria and fungi by dilution plate counts. Changes in levels of ammonium, nitrate and total mineral nitrogen were followed by microdiffusion from soil extracts. IA! - IV! 5;. ‘l. 5“! N“. .H": I ‘0 \§‘ Fang Hui Liao In fumigated soil, bacterial numbers at low temp- eratures and respiratory losses of CO2 at higher temperatures were directly related to the level of nitrate initially supplied. At the same time, nitrate disappeared initially in fumigated soil, Whereas in unfumigated soil nitrate accumulated at rates related directly to temperature. From these observations it was concluded that hetero- trophic bacteria capable of adapting to nitrate utilization are among the first to recover in fumigated soil. Observed delays in nitrate accumulation appeared to be due to utili- zation of nitrate by these‘heterotrophes rather than to retarded recovery of nitrifiers. Longer delays in nitrate accumulation with chlorOpicrin than with dichlorOprOpene were associated with larger numbers and longer persistence of nitrate dependent bacteria. Utilization of nitrate in fumigated soil appeared to be due to assimilation by microbial cells rather than to denitrification, since no permanent differential losses of mineral nitrogen occurred. The initial assimilatory disappearance of nitrate appeared to be a function of available energy substrates and was essentially the same at temperature of 5°, 20° and 30°C. Recovery and activity of nitrifiers, on the other hand, was greatly enhanced at 20° and 30°C. Consequently, initial delays in nitrate accumulation in fumigated soil Fang Hui Liao were greatly reduced by temperature increases imposed the 16th day after fumigation. When the temperature was maintained at 5°C. for 51 days after fumigation, large numbers of bacteria with apparent nitrate dependence were observed. Increasing the temperature to 20°C. at this time resulted again in temporary disappearance of nitrate and markedly extended the delay in nitrate accumulation. Similar effects, though less striking, were observed when the temperature was in- creased from 200 to 30°C. at this time. This behavior is similar to that observed during periods of rising temperature in Spring and early summer in the field following fall fumi- gation. EFFECTS OF TEMPERATURE AND AMMONIUM TO NITRATE RATIO ON MICROBIAL RESPONSES TO FUMIGATION IN ORGANIC SOIL BY Fang Hui Liao A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Soil Science 1963 ACKN O WL EDGEI‘IEN T The writer wishes to express her sincere gratitude to Dr. A. R. Wblcott for patient assistance and interest in completion of this study and in the preparation of the manuscript. Acknowledgement goes to Dr. R. L. Cook and to the Dow Chemical Company for financial support which enabled the writer to prepare for and complete the study. TABLE OF CON TENTS Page INTRODUCTION ............................................ 1 LITERATURE REVIEW ....................................... 3 OBJECTIVES ..............................................18 EXPERIMENTAL PROCEDURES .................................l9 Fumigation and Preliminary Treatment of Soil ........20 Mineral Amendments ..................................22 Conditions of Incubation ............................26 ReSpiration Measurements ............................27 Microbial Counts ....................................29 Nitrogen Determinations .............................30 Statistical Treatment................................30 RESULTS .................................................32 ReSpiratory Activity and Microbial Numbers ..........32 Main effects of fumigants .......................32 Main effects of nitrogen treatments .............32 Relationships with temperature ..................33 Interactions of fumigants and nitrogen treatments at low to intermediate temperatures (T1) .............................39 Interactions of fumigants and nitrogen treatments at intermediate to high temperatures (T2) OOOOOOOOOOOOOOOO0.0.0.000000043 Interactions of fumigants and nitrogen treatments at constant high temperatures (T3)..45 DistSion O0.0.0....OOOOOOOOOOOOOOOOOO0.0.000... 50 ii . . .- o q o .1 ~ g a ' O - G . 1‘ < . I. r O 6 a - “ P . .. 6 f‘ "‘ O 0' L n r a a v. P o u- L o 3 r F -_ 0 a O u D U f O 0 ‘ n .- 0 n p o: f 1' n n g ‘ — b V" O 6 O Q Q ' " ' m l' 'V l‘ ' Q r 9 O 0 f0 ' (V 'i O "I I. I. r \— o o o a o c- Q C o N O O I I’ II II II II s 0 t 0" c I ' u r ' O V I. ~ 0 '5 L C 7‘ ' ' ' 0‘ n F o 4" I ‘ I: v 'l c - r s, a ‘ r» l- a. r u 1" .1 “ a Q n u v- . x. A 'l . Fl g s! n . g . C l ., r - . C I‘ a . fl 1' \ ~ 9 e II ‘ o 0 v o n 5 i '— 0 0 I. O C a O ’a 1 9 0‘ ,. . a- o u ,. . e- n H I" U Q o O a ' 0" .fi.‘ Page Nitrogen Transformations ............................54 Changes in NH4 and N03 prior to incubation ......54 Main effects of fumigants .......................56 Main effects of nitrogen treatments .............58 Relationships with temperature ..................58 Interactions of fumigants, nitrogen treatments and tel'nperature .OOOOOOOOOOOOOOO0.0.00.0000000061 Discussion C...0...OOOOOOOOOOOOCOOOOC............62 GEEEM DISCUSSION 0....COOOCOOOCCOOOOOCOOCOCO0.0.0.0....69 $11-$12?“ny 0....0.000.000.000000OOOOOOOOOOOOOOOOOO0.0.0.0...75 BIBLIOGRAPHY OCOOOOOOOOOOOOCOOOOOOOOO.000000000000000000077 iii .1 a. TABLE 1. 10. LIST OF TABLES Page Description of mineral amendments added 12 days after fumigation, 7 days after leaching and aeration ..........................................24 ReSpiratory activity and microbial numbers as related to fumigation treatment ...................34 Respiratory activity and microbial numbers as related to nitrogen treatments ....................35 Respiratory activity and microbial numbers as related to temperature ............................37 Microbial numbers as related to fumigation treatment and temperature .........................38 éicrobial numbers as related to temperature and nitrogen treatment ................................40 Early changes in ammonium and nitrate prior to controlled temperature incubation .................55 Ammonium, nitrate and total mineral nitrogen during incubation, as related to fumigation treatment .........................................57 Ammonium, nitrate and total mineral nitrogen during incubation, as related to nitrogen treatment .........................................59 Ammonium, nitrate and total mineral nitrogen during incubation, as related to temperature ......60 iv FIGURE 1. LIST OF FIGURES Page Patterns of microbial succession and respiratory activity at low to intermediate temperatures ......42 Patterns of microbial succession at inter- mediate to high temperatures ......................44 Patterns of respiratory activity at inter- mediate to high temperatures ......................47 Patterns of microbial succession at high temperatures ......................................48 Patterns of resPiratory activity at high temperatures ......................................49 Nitrogen transformations at low to intermediate temperatures ......................................63 Nitrogen transformations at intermediate to high temperatures ......................................64 Nitrogen transformations at high temperatures .....65 no... i vu .v ,o‘iu. . t, v‘, an...) I... THE Ik‘ INTRODUCTION The primary object of soil fumigation, Whether by heat or volatile chemicals is to kill pathogenic micro- organisms. The use of fumigant chemicals for the control of weeds and various pathogenic organisms in the soil has become a wide spread practice within recent years. The action of many of these Chemicals is frequently not specific and may affect organisms which have a beneficial influence on plant growth. In general, microbial numbers are initially decreased by fumigation, but certain forms quickly reinhabit or develOp in the soil, and shortly after treatment overall numbers are usually in excess of those in untreated soil. Spore forming bacteria are relatively resistant to fumigants, the organisms which oxidize ammonia to nitrite and nitrite to nitrate appear to be relatively sensitive. Organisms whiCh release ammonium from organic nitrogen complexes quick- ly return to soil following treatment, whereas the nitri- fiers are apparently slow to develoP. The apparent inhibi- tion of nitrification has resulted in an accumulation of ammonium nitrogen in fumigated soil, particularly in organic 30113. In situations Where a plant Species can utilize immnonium nitrogen as readily as nitrate nitrogen, retarded rfl1:rification will have very little effect on the availabil- itlz of nitrogen to the plants. If the concentration is too 1 2 high, ammonium sensitive plants may be injured. Under con- ditions where considerable loss of nitrogen through leaChing of nitrate nitrogen might occur, temporary inhibition of nitrification may be beneficial. Laboratory studies have shown that the intensity and duration of the suppression of nitrification vary with the chemical and with the numerous mechanical, soil and environ— mental factors which influence the effectiveness of the Chemicals as fungicides or nematocides. Field studies have shown that nitrate accumulation in organic soils is drastically delayed in the spring and early summer, even when the soil was fumigated the previous fall and no detect- able traces of the Chemical remain in the soil. In these field studies, disappearance of applied nitrate was observed during periods of wet weather or after irrigation. Nitrate disappearance was also associated with rising soil tempera- ture. These field results suggest that reduction or consump- tion of nitrate by an altered heterotrophic microflora may contribute to retarded nitrate accumulation in fall fumigated soil. The present laboratory study was undertaken to investigate the effects of varying prOportions of ammonium and nitrate nitrogen on nitrogen transformations and micro- Ibial numbers in fumigated organic soil under conditions of high.moisture and rising temperature. n , n A ova- nvv- QM ae- on.» : :0b.. 1‘ -u‘ 90:“! ‘IQD'; 0“:qu ‘ I. up... dhi‘. Q ”WA - . Wn'.‘ :,y_ .1 '1 ‘5 uh“ s' ‘- 3. L5 1 N- -t E“? ‘5‘ ‘4‘. Jm LITERATURE REVIEW The main purpose of soil disinfestation is to kill detrimental microflora and to control weed seeds. In the search for an explanation of the remarkable increase in plant growth following soil treatments with heat or fumigants, many earlier investigators have pronounced a number of ingenious theories. These have been well reviewed by DuBuisson (l4) and.K0peloff and Coleman (29). DuBuisson concluded that the beneficial influences obtained by treat- ing the soil with volatile antiseptics can not be ascribed to a Change in physical condition, to a suppression of some toxic material, or to a deveIOpment of acids from the action of the antiseptics. The closely coordinated stimulation of plant and bacterial activity due to the treatment of the soil with volatile antiseptics points strongly toward a biological interpretation, with due regard for Chemical effects of altered or enhanced microbial activity. KOpeloff and Coleman ascribed the beneficial effects of "partial sterilization“ to increased amounts of plant food, eSpecially nitrogen, made available for plant use as a result of increased bacterial activity. Newhall (36) indicates that many of the earlier investigators have expressed a concern for the useful micro- organisms that must inevitably suffer the same fate as their Parasitic associates following any effective soil treatment. 3 4 It was well established by the workers in the late 1800's that treatment of soil, either by heat or by antiseptics, has the following results: 1. Non-Spore-forming, nitrogen-fixing, nitrite forming and nitrate forming bacteria, as well as parasitic organisms are destroyed, and nitrification is thereby inhibited. The spore- forming ammonifiers survive, and ammonification goes on almost uninterrupted for weeks, especially in soils high in organic matter. 2. Soluble salts are often liberated, in some cases chlorides and sulfates of ammonia, and sometimes soluble manganese. Microbiological prOperties of soil fumigated with steam or chemicals were extensively studied by Waksman and Starkey (54). They confirmed by eXperiment that "partial sterilization” results in an increase of the bacterial pOpulation and in ammonium nitrogen accumulation. They attributed these changes to a combination of factors, namely, Changes in physical and Chemical conditions of the soil, destruction of soil microorganisms thereby making their cell constituents available as a source of energy, Changing the equilibrium of the microbiological flora, and efficient conversion of organic nitrogen to ammonium nitrogen. 11) :1 r" 5 The apparent inhibition of nitrification by soil fumigants has been observed by several investigators. Stark, Smith and.Howard (45) studied the effect of Chloro- picrin fumigation on nitrification and ammonification and found that low dosages of chlorOpicrin had little effect on nitrate formation, but as the dosage was increased, nitri- fication, as measured by nitrate accumulation, was inhibited. The total amount of nitrogen made available for plant growth was not materially increased except where high dosages of ChlorOpicrin were used. Tam and Clark (48), working with pineapple plants, showed that increased growth and nitrogen composition following chlorOpicrin fumigation were related to restric- tion of the plants to predominantly ammonium nutrition as compared to the ordinary nitrate nutrition in unfumigated soil. Tam (47) also reported that D-D soil fumigant applied at the rate of 20 gallons per acre, suppressed nitrate accumulation in Hawaiin pineapple soils for a period of 8 weeks at greenhouse temperature. Kincaid and Volk (24), working with cigar wrapper tobacco in Florida, used various fumigant chemicals for con- trolling root knot. They found that, following fumigation, there was a prolonged retention of ammonia nitrogen in the soil. AldriCh and Martin (1) also found that "partial sterilization" with ChlorOpicrin and D-D mixture produced an 6 initial increase in ammonium nitrogen in soil. On incuba- tion, the ammonium was oxidized to nitrate. Recently Winfree and Cox (58, 59) reported that fumigation of an organic soil, either by ChlorOpicrin at a rate of 43 gallons per acre or methyl bromide at 21 pounds per 100 square feet, exerted a decided influence on the kinetics of nitrogen mineralization. Ammonium nitrogen accumulated at least 10 fold following the fumigation, at the eXpense of nitrate nitrogen. Soil fumigation effectively altered the activity and p0pulation of other microorganisms present in the soil. Wensley (55) investigated the action of methyl bromide, ethylene dibromide and D-D mixture on certain organisms in- volved in nitrogen transformations in soil. His data indicated that the pOpulation of nitrifiers is reduced more than that of the ammonifiers. Methyl bromide was Shown to be muCh more potent than ethylene dibromide in reducing the pOpulation and activity of the nitrifiers, while ethylene dibromide was proved to be more effective against root knot nematodes than methyl bromide. Martin (32) reported that treatment of soil with fumigant chemicals markedly altered the nature of the fungal pOpulation. After initial or near destruction of the fungal pOpulation of the soil, fungi again established them- selves, although the kinds and numbers establiShed varied 7 greatly between treatments. Klemmer (26) found that fumiga- tion of soil produced moderate inhibition of fungi and increased bacterial numbers, as determined by dilution plate counts. Overman (38), using allyl alcohol as a soil fungicide at the rate of 25 gallons per acre, showed that nitrifying bacteria were inhibited for 3 weeks after the allyl alcohol was applied. Actinomycete pOpulations were unaffected at this rate. Colony counts of Trichoderma increased greatly following the treatment with allyl alcohol. Yatazawa (62) also reported that application of allyl alcohol caused a nearly instantaneous replacement of the native fungal p0pula- tion by Trichoderma virideI POpulations of bacteria and actinomycetes were decreased at rates higher than 100 gallons per acre. The use of allyl alcohol apparently creates conditions in soil favorable for the develOpment of TriChoderma. McCants et a1. (35) have presented data which showed that certain of the soil fumigants currently used for nema- tode control can have a significant influence upon the reSponse of tobacco to applications of nitrogen in the ammonium or nitrate form. There was a greater yield response to nitrate applied with fumigant treatments which had the most suppressing effect on nitrification. However, where nitrification was inhibited and ammonium was applied, yields 8 and quality were reduced and there was a high ammonium and halogen content in the leaves. Koike (27) conducted laboratory eXperiments to deter- mine the effects of eight fumigants on nitrification of (NH4)ZSO4 and NH4 . Results indicated that under the con- ditions of the experiment the Chemicals markedly inhibited nitrification from 4 to 8 weeks. The delay in nitrification was longer with (NH4)ZSO4 than where nitrogen was supplied as NH OH. Kirkwood (25) also found that addition of (NH 4 4’2504 delayed the recovery of nitrification in fumigated soil. Wolcott et a1 (61), using Telone as a fumigant applied at recommended nematocidal rates, found nitrification to be delayed in the laboratory about 8 weeks at soil temperatures above 60°F and for longer periods at lower temperatures. In the field, following fall fumigation, they found that nitrification was delayed 6 to 8 weeks after the soil wanmed to 60°F in the Spring. Concomitant develOpment of herbicidal and inSecticidal Chemicals and control programs has aroused interest in pos- sible effects of these materials on microbial activity in the soil and resulting influences on soil fertility. A great amount of work has been done on the effect of DDT, BHC and 2,4-D on biological processes in soil. Smith et al (42) worked on the effect of certain herbicides on soil microorganisms and concluded that herbicides varied greatly in their effect on the various 9 groups of soil microorganisms. In some cases, they are definitely toxic, in others, stimulatory. They found that concentrations of 2,4-D up to 100 ppm. had no significant effect on total plate counts for bacteria, actinomyces, fungi or protozoa. The nitrite and nitrate forming organisms were definitely injured by 100 ppm. Jones (22) reported that 2,4-D at 25 pounds per acre had no detrimental influ— ence upon nitrate production in a soil to whiCh no nitrogen had been added, but 2,4-D in combination with sodium nitrate apparently inhibited the formation of nitrate during the first 2 to 3 weeks. The nitrate content of soil increased very rapidly after this initial period. This indicated that nitrate forming organisms may be temporarily inhibited by the addition of 2,4-D. Koike and Gainey (28) observed that 2,4-D at the usual field rate did not appreciably reduce total nitrate. Although there were marked temporary reductions in total nitrate accumulation with high concentrations of 2,4-D, the accumulation of nitrate nitrogen was not completely inhibited, and within 8 to 16 weeks the rate of accumulation had again reaChed that in untreated soil. Newman and Downing (37), working with 2,4-D and related phenoxyacetic acid herbicides, found that normal rates of treatment are noninjurious to the general soil microbial pOpulation, do not injure Azotobacter, and are noninjurious or only slightly injurious 10 to nitrifiers. Extremely high amounts of 2,4-D are required to inhibit ammonification. Evolution of CO2 from soils in the presence of herbicides may be used as a criterion of influence on the microbial pOpulation as a whole. Wilson and Choudhri (57), in laboratory studies, showed that DDT and BBC in amounts considerably exceeding practical field applications had no significant effect on develOpment of bacteria and molds, nor on certain of their physiological activities important in soil fertility. Smith and Wenzel (43) found that 5 to 200 ppm. DDT were not definitely injurious and in some cases were stim- ulative to heterotrophic bacteria. BHC proved definitely fungicidal and also toxic to nitrifiers. Chlordane was found less toxic than BHC, toxaphene was stimulating to bacteria and molds as shown by plate counts and apparently was utilized as a carbon source. Jones (23) studied the stability of DDT in soil and observed that no injury to nitrifiers, ammonifiers, and sulfur-oxidizing microorganisms was noted from concentrations of DDT ordinarily added to soils. No injury to the nitrogen- fixing bacteria was observed in soils containing concentra- tions of DDT as high as 1%L DDT added to the soil was remarkably stable during the first year of storage, but by the end of the second and third years appreciable breakdown had occurred. ll Bollen et al (7,8) studied various insecticides in the field and concluded that no immediate harmful effects on soil microorganisms were caused by field applications of any of the insecticides studied. None of the treatments resulted in either an approaCh to sterility or a manifold increase in molds, bacteria or streptomyces, nor were marked changes in pr0portions of these microbes produced. In solution media inoculated with soils, Gray (17,18, 19) observed that BHC and its gamma isomer were toxic to bacteria that oxidize ammonia to nitrite and those that oxidize nitrite to nitrate. They were not toxic to nitrify- ing bacteria in soil, nor to bacterial inocula from a vege— table compost in solution cultures. They were toxic to bacteria that oxidize thiosulfate in solution cultures con- taining inocula from mineral soils. BHC, but not the gamma isomer, inhibited the growth of phenol-decomposing bacteria and prevented starch hydrolysis by amylolytic bacteria. It also depressed the action of urea‘hydrolyzing bacteria in soil extracts. New Chemicals are being continuously released for use as fumigants, fungicides, insecticides and.herbicides. There are numerous contradictions among published reports dealing with the microbiological effects of these chemicals. This is to be expected, considering the varied circumstances under which the work was undertaken. 12 Studies on nitrogen transformations are further complicated by the fact that several processes are involved, each re3ponding independently to Changes in environmental conditions of aeration, temperature, moisture, pH and nutrients. The biochemical heterogeneity of the microflora bringing about nitrogen mineralization is a critical factor in determining the influence of environmental factors upon the transformation. Robinson (39) indicates that the ammonifying pOpulation includes aerobes and anaerobes. Con- sequently, organic nitrogen is readily mineralized, at moderate or at excessively high moisture levels. Ammonium is slowly formed at water levels slightly below the permanent wilting percentage, but improving the moisture status stimulates mineralization. The Optimum for ammonifi- cation generally falls between 50 and 75 percent of the wateréholding capacity of the soil (52). Alexander (3) points out that mineralization is influenced by the pH of the environment. All other factors being equal, the production of inorganic nitrogen, - ammonium plus nitrate,- is greater in neutral than in acid soils. Acidification tends to depress but does not eliminate mineralization. Temperature likewise affects the mineralization sequence, since each biochemical step is catalyzed by a 13 temperature-sensitive enzyme produced by microorganisms whose growth is in turn conditioned by temperature. Thus at 2°C, the microflora slowly mineralize. soil organic complexes, but there is no increase in ammonium or nitrate when soil is frozen. Elevation of the temperature enhances the mobil- ization of nitrogen in prcportion to the greater warmth (3). Nitrogen immobilization is a consequence of the in- corporation of ammonium and nitrate into proteins, nucleic acids and other organic Complexes contained within microbial cells. The rate of immobilization is related to the avail- ability of the organic molecule, very rapid with readily oxidized carbohydrates, moderate with less suitable materials and particularly slow with resistant tissue components such as lignin or well-rotted manure (3). Immobilization is also correlated with pH and soluble soil phosphate, results that are not unexpected because of the qualitative and quanti- tative effects of pH and phosphorus on the size and bio— chemical capacity of the microflora (60). The termination of the reactions involved in organic nitrogen mineralization occurs at the point where ammonium is formed. This, the most reduced form of inorganic nitro- gen, serves as the starting point for a process known as nitrification, the biological formation of nitrate or nitrite from compounds containing reduced nitrogen. Physical and Chemical factors affect the rate of ammonium oxidation. 14 Chief among the environmental influences is acidity. In acid environments, nitrification proceeds slowly even in the presence of an adequate supply of substrate, and the responsible Species are rare or totally absent at great acidities. An exact limiting pH cannot be ascertained since a variety of physico-Chemical factors in soil will alter any Specific boundaries (3). Wilson (56) notes that the acidity affects not only the transformation itself but also the microbial numbers, neutral to alkaline soils having the largest pOpulation. The effect of temperature on nitrification in soil has been studied by many investigators (6,16,40). Recently, Sabey et a1. (41) initiated a study to determine the influ- ence of temperature and initial p0pulation of nitrifying organisms on the maximum rate and delay period. In soils incubated at field capacity, the maximum nitrification rates increased from immeasurably low values to as great as 900 ppm. per week in some soils and the delay periods decreased from about 32 weeks to less than 1 day, as temperatures increased from 0 to 25°C. Increase in initial pOpulation of nitrifying organisms caused decreases in delay periods but did not appreciably affect the maximum rate above 10°C. In solution culture, temperature and the size of nitrifying pOpulations do influence delay periods of nitrifi- cation (2). Aeration is another factor that affects the 15 nitrification rate. Nitrifying bacteria are autotrophes and obligate aerobes. Nevertheless, nitrifying activity is Sharply curtailed only at very low partial pressures of oxygen (5). In soil systems, nitrification may proceed un- hindered by increasing moisture content to levels near saturation (30). Aleem and Alexander (2) recently Observed an increase in delay period of nitrification at excessive levels of aeration in solution culture. Certain transformations of nitrogen lead to a net loss of the element from the soil through volatilization. The sequence of steps that results in gaseous loss is known as denitrification, the microbial reduction of nitrate and nitrite with the liberation of molecular nitrogen and, in some instances, nitrous oxide. Denitrification is not the Sole means by which microorganisms reduce nitrate and nitrite. In the utilization of the two anions as nitrogen sources for growth, microorganisms reduce them to the ammonium level. Alexander (3) defines denitrification as essentially a respiratory meChanism in which nitrate replaces molecular oxygen, is. a nitrate reSpiration. The utilization of nitrate as a nutrient source may be termed nitrate assimila- tion. The rate of denitrification of the nitrate added to flooded fields is far more slow in soils low in carbon than in land riCh in organic matter. The effectiveness of organic 16 nutrients in promoting denitrification in waterlogged soils is prOportional to their availability (3). The addition of organic substances to well drained soils diminishes the nitrogen losses, the conserving action resulting from an immobilization of inorganic nitrogen (11,31). Oxygen availability is another of the critical environmental determinants. Aeration affects the transform- ation in two apparently contrasting ways: on the one hand, denitrification proceeds only when the oxygen supply is in- sufficient to satisfy the microbiological demand: at the same time, oxygen is necessary for the formation of nitrite and nitrate, whiCh are essential for denitrification. Decreas- ing the partial pressure of oxygen enhances the denitrifica- tion of added nitrate. In well-drained soils, nitrogen volatilization is related to the moisture content. Denitri- fication of added nitrate is appreciable at high water levels and in localities having imprOper drainage. The effect of water is attributed to its role in governing the diffusion of oxygen to sites of microbiological activity. (3) Alexander (3) points out that the bacteria whiCh bring about denitrification are sensitive to high hydrogen ion concentration. The pOpulation becomes large only above a pH of approximately 5.5. Denitrification is markedly affected by temperature. The transformation proceeds slowly at 2°C, but increasing the temperature enhances the rate of biologi- cal loss. The Optimum for the reaction is at 25°C and is 17 still rapid at elevated temperatures and will proceed to about 60 to 65°, but not at 70°C. OBJECTIVES Laboratory studies were conducted to investigate the effects of two fumigant chemicals, dichlorOprOpene and chlor0picrin, on microbial numbers and activities in organic soil amended with ammonium and nitrate in varying prOportions and incubated under different controlled temperature regimens. Specific objectives of the experiments were to: 1. Observe effects of rising temperature and NH4 to N03l ratio on post-fumigation adjustments in respiratory activity and numbers of heterotrophes. 2. Observe effects of rising temperature and NH4 to NO3 ratio on post fumigation patterns of accumulation or disappearance of NH4, NO3 and total mineral nitrogen. 3. Test the hypothesis that adjustments in heterotrophic metabolism during periods of rising temperature contri- bute to delayed accumulation of nitrate in fumigated soil. 1NH4=NHZ, N03=N03-. This convention is followed throughout the thesis. 18 EXP ERIl‘dENTAL P ROCEDURES Because of previous field and laboratory eXperienceS associated with its use, diChloroprOpene was the chemical of primary concern. ChlorOpicrin was used as a reference chemical with general, non-selective sterilizing action. Lots of soil material treated with these two chemicals and a control lot from the same homogeneous organic soil source were amended with ammonium and nitrate in varying prOportions. Amended soil systems were incubated for 94 days under three controlled temperature regimens. All samples were frozen for three days and equilibrated at 5°C. for eight days prior to incubation. Two temperature regimens involved abrupt temperature increases at the beginning of incubation from the 5°C. imposed prior to incubation. One of these involved a second abrupt temperature increase midway through the incubation. Under a third regimen, pre-incubation low temperature was maintained until the middle of the incubation period and then increased abruptly. Effects of the three treatment variables on the general microbial population were characterized periodically by collecting CO evolved and by enumeration of bacteria and 2 fungi. Changes in level of ammonium, nitrate and total mineral nitrogen were followed. Details of treatment and analytical procedures are described in the succeeding sections. 19 20 Fumigation and Preliminary Treatment of Soil A bulk lot of Houghton muck (75 to 80 percent organic matter, pH 6.3) was collected from a recently irthgated area in the field in late June, 1962. Soil from the surface eight inches was taken, forced through a 4-mesh screen and thoroughly mixed. The moisture content of the soil was found to be 232 percent, dry soil basis. Previous eXperience in the same muck area indicates that this is approximately field capacity for this soil. The bulk lot was divided into three parts. Two lots were fumigated with dichlorOpr0pene2 or ChlorOpicrin3 at rates and for exposure periods recommended by the manufacturer for field application. A third lot was untreated. DichlorOprOpene was used at the rate of 32 gallons per acre (0.53 ml. per Kg. dry soil). ChlorOpicrin was used at the rate of 70 gallons per acre (1.16 ml. per Kg. dry soil). The fumigants were dribbled on the surface of the soil in a drum mixer which was sealed while the soil was thoroughly mixed. The soils were then transferred to plastic bags and sealed for the apprOpriate eXposure period (2 weeks for dichlorOprOpene, 2 days for chlor0picrin). 2 prOpene). 3 Telone, Dow Chemical Co. (90 to 95% l, 3 dichloro- Picfume, Dow Chemical Co. (99% ChlorOpicrin). 21 The addition of chlorOpicrin was delayed so that eXposure periods for both chemicals terminated on the same date. This terminal exposure date is used as zero on the time scale for all subsequent observations. All three lots of soil (control and fumigated with dichlorOprOpene or chloropicrin) were kept in sealed plastic bags at 20°C. during the two-week period required for expo- sure to dichlorOprOpene. After SXposure to the fumigants, the soils were placed in tall metal cylinders and leached with a constant 6-inch head of distilled water until no nitrate could be detected with diphenylamine in the leachates. This required four days. After drainage by gravity had ceased, the soils were forced through a 4-mesh screen and Spread out on large canvas sheets to aerate and partially dry for 24 hours. The soils were stirred periodically with a rake during the day- light hours. Moisture content after aeration ranged from 150 to 165 percent in the three soil lots. After aeration there were no detectable fumes of either fumigant in treated soil. After thorough mixing, the soils were placed in sealed plastic bags. These were placed in a deep freeze (-10°C.) for three days, then equilibrated at 5°C. for eight days prior to incubation. This was done, in part, to simulate temperature SXperienceS to which fall fumigated 22 soils may be subjected during the winter months. It was also necessary to minimize microbial activity during the time necessary to carry out chemical determinations and calculations on which mineral amendments were based, to add the amendments and to dispense soil samples for incubation. Mineral Amendments A major objective of the experiment was to determine to what extent disappearance of nitrate in fumigated soil might contribute to delayed accumulation of nitrate suCh as is commonly observed in the field. It was recognized that both dissimilatory and assimilatory reduction of nitrate might be involved. It was anticipated that the presence of other reducible ions, suCh as sulfate, might compete with nitrate as terminal electron acceptors in facultative metabolism leading to nitrate reduction. For this reason, ammonium was added as (NH4)2HPO4, rather than as (NH4)ZSO4. Nitrate was added as KNOB. Ammonium and nitrate found in the soil after fumiga- tion, leaching and aeration was augmented by salt additions to give 400 ppm. total mineral nitrogen and ratios of NH44N to NO3-N equal to 9, 3, l, and l/3. The resulting variations in P and K were compensated by adding KHZPO4 oerHPO4 in quantities calculated to give minimal variations in P, with both P and K in excess. The schedule of mineral amendments is presented in table 1. 23 Mineral amendments were Sprayed on thin soil layers in water solutions of concentration and volume calculated to bring moisture content up to 230 percent, dry soil basis. Previous experience in the field had indicated that this was close to field capacity for this soil. It would represent the maximum moisture that could be taken up by the soil without serious loss of aggregate integrity. Beyond this point, it would be difficult to establish uniform aeration characteristics for the different treatments without increas- ing water to saturation levels. To promote nitrate reduction under conditions approaching those observed in the field, maximum moisture consistent with stable aggregation and uni- form aeration prOperties appeared to be a desirable Characteristic common to all experimental soil systems during subsequent incubation. As noted earlier, the soil at the time of collection in the field had contained 232 percent moisture. However, it was found that the soil, -after fumigation, leaching and aeration, - could no longer absorb this amount of water without serious loss of aggregation. It was, therefore, necessary to force the amended soils again through a 4-mesh screen to reestablish aggregation. Additional drying in a stream of warm air was necessary before the aggregated soils could be homogenized satisfactorily by mixing. The amount of drying was gauged by the visual appearance and mixing 24 .eHemo HHoe sot ce>o .ooeocoo moooeHos HmcHe x .eoezmx no eoemmx mm a com a meooxo .mozx no voezmxvmzo em smote ceeonon ** .AcofipmmHESM Haven m>mo mv cowpmnmm tam ocHrommH seven venom * meH eeeH mew mew a on we m\H e2 meH oemH new oeH m mmH we H m2 meH omHH new we a one as m Nz Home someone oeH mmHH new mm m mHm me e Hz .oHoHooHo moH oHeH oee oom o an we m\H vz oeH ommH oee oom o mmH me H mz meH eHmH oee ooH o use me o Nz Ammo cfisowe oeH mHeH oee ov o «Hm me e Hz .oHoHto omH ommH mom eom mm em HH m\H v2 eeH onoH new eoH mm eeH HH H m2 eeH omoH one eo mm ewe HH m «2 H AH”: mmH omoH one e no eem HH e z ecoz R Eon Edd Son and Eon Eon \pcopcou **owoo< **omoo¢ **omoo< *pcsow **omoo< *ocsom m02\vrz acmEHmme bcmepmmnp magpmHoz x d Zumoz nllllllflflUfiufllll owpmm z coHHmoHssm .coHpmmnm pew mcHrommH sebum m>mo h .coHummH83m Hmumm m>oo NH omoom mucmeocmEm Hmnmcfls mo cofipownummosa.a mHomh 25 prOperties of the aggregates. The moisture contents of amended soils after thorough mixing are given in the last column of table 1. It is not known to what extent water holding capacity of the soils was reduced by losses of soluble dry matter during leaching or by irreversible dehydration during aera- tion. The final moisture content of amended soils shown in table 1 is certainly high on the scale of available moisture, but it is probably less than field capacity. The variations between treatments are relatively minor. However, at these high moisture levels, such relatively small variations may have influenced oxygen diffusion to sites of microbial activity within aggregates or in thicker moisture films associated with meniscuses around points of contact between aggregates (9). No direct relationships between moisture content and observed indices of microbial activity could be observed. Nevertheless, variations in moisture, as well as in salt concentrations apparent in table 1, may have contributed to variation in data taken during subsequent incubation. No consideration was given to pH buffer effects of the varying = to H PO - used. Phosphate was added in 4 2 4 total amounts equivalent to 0.01 molar in the Soil solution. prOportionS of HPO The prOportions of HPO4 = to H2P04- used with the two higher NH4/NO3 ratios (treatments N and N2) would have tended to raise 1 pH above the initial 6.3. With the two lower NH4/NO3 ratios (treatments N3 and N4), the compensating salt was principally 26 KH2P04, which would have tended to buffer at a pH lower than the initial 6.3 (50). There was evidence that initial buffer effects may have influenced early transformations of ammonium and nitrate. The extent to which pH was altered by mineral amendments was not determined. Conditions of Incubation After moisture adjustment and mixing, the twelve lots of soil amended as Shown in table 1 were returned to the 5°C. storage temperature for the balance of the 8-day equilibration period (3 to 4 days). During this time, weighed aliquots con- taining 10 g. oven dry Soil were diSpensed in 60 ml. plastic cups. Fifteen cups of soil were placed in each of six gallon jars for each treatment. Thus, there were 72 jars in all, each containing the equivalent of 150g. oven dry soil. The jars were sealed and maintained at 5°C. for the remainder of the equilibration period. On the sixteenth day after the end of the fumigation exposure period, duplicate jars of each treatment were placed in each of three constant temperature rooms maintained at 5°, 0 and 30°C., reSpectively. The jars were connected to the 20 aspiration apparatus described by Kirkwood (25). Except for periods during whiCh CO2 was collected (see next section), the jars were aerated for 30 minutes every fourth day using water- saturated air purified by passing through NaOH and H2804 to remove CO2 and NH3. 27 On the Slst. day after fumigation, the temperature in the 50 room (T1) was raised to 20°C., that in the 200 room (T2), to 30°C. The temperature in the 300 room (T3) was not changed and was held constant from the 16th. through the 110th. day after the end of the fumigation SXposure period. No attempt was made to determine losses of dry matter or moisture during the incubation period. Estimates based on carbon loss suggest that up to 20% of the initial dry weight may have disappeared due to organic matter decomposi- tion where the temperature was maintained at 30°C. over the 94-day period of incubation. Water loss was minimized by discontinuous aeration (4 hours each 4 days, at most) and by saturating the incoming air with water in two stages. This was done by bubbling air through a 12-inch column of water at ambient temperature before entering the controlled temp- erature room, and again through a lZ—inch column of water at incubation temperature inside the room. Moisture losses may have tended to parallel dry matter losses. Nevertheless, at the two higher temperatures, soils were sensibly drier at the end of incubation than at the beginning. ReSpiration Measurements During major portions of the incubation, the general level of microbial activity was Characterized by measurements of CO evolution. Carbon dioxide formed during a 4-day 2 period was displaced by passing water—saturated, COZ-free 28 and NH3-free air through the jars for four hours. DiSplaced CO2 was collected in NaOH, after passing first through KI and AgZSO4 to remove volatile chlorine or Chlorides (4). HBix-inch (100 ml) plastic centrifuge tubes containing 2% inches of fine glass beadsvere used for collecting C02. ApprOpriate quantities of N/2 NaOH were measured into the tubes and CO -free distilled water added to cover the glass 2 beads. Effluent air from the incubation jars was introduced into the bottom of the tube through a pin hole in a flexible plastic tip which could be inserted through the column of glass beads. UnSpent NaOH was titrated in the same tubes, using a Similar arrangement for bubbling a vigorous stream of CO -free air through the column of beads for agitation and 2 mixing. Excess BaCl was added to remove carbonate for 2 titration with standard acid to the phenolphthalein end point. Final standardization of NaOH was based on titration of a blank tube of NaOH aSpirated simultaneously from a common manifold with each group of six incubation jars. Carbon dioxide production was calculated from the difference in acid required by blank and sample tubes. Respiration rates were recorded as mg. C per 100 g. oven dry soil per day for the median day of each 4-day collection period. Ten-gram soil samples sacrificedfrom time to time for plate counts and mineral N determination were accounted for in these calculations. No allowance was made for losses 29 in soil weight which occurred by decomposition of organic matter during incubation. Microbial Counts Periodic plate counts for bacteria and fungi were facilitated by the fact that known quantities of soil with known moisture content had been weighed prior to incubation and diSpensed in plastic cups. Initial 1:10 dilutions were made in wide-mouth, pint freezer jars containing a volume of sterile water calculated to allow for the water contained in the soil sample as determined prior to incubation. The use of wide mouth jars made it possible to transfer a plastic cup and its contained soil aliquot directly from the incuba— tion jar into the initial water blank contained in the freezer jar. Subsequent dilutions were made in standard milk dilution bottles. Thornton's standardized medium (49) was used for bacterial counts, no distinction being made in counting be- tween bacterial and streptomycete colonies. Martin's rose bengal medium with streptomycin (33) was used for fungi. Duplicate plates were poured for each of the following dilutions: l to 104, 105 and 106 for bacteria, and l to 103, 4 and 105 for fungi. Plates were incubated in the dark 10 for 5 days at room temperature (220 to 25°C.). Colony counts were recorded for plates containing 20 to 300 bacterial colonies or 10 to 100 fungal colonies. 3O Stastical analyses were performed on the log trans- formations, and geometric, rather than arithmetic, means are reported (13). Results are reported as numbers per gram of oven dry soil initially present, no allowance being made for losses of dry matter or moisture during incubation. Nitrogen Determinations Periodic extractions for ammonium and nitrate were made in a manner similar to the initial 1:10 dilutions for microbial counts. Plastic cups and their contained soil ali- quots were transferred directly to pint freezer jars contain- ing 100 ml. of N.K in N/lO H2504. These were placed on 2304 a rotary Shaker for 30 minutes. Ammonium and nitrate were determined on suitable aliquots of the filtered extract by distillation at room temperature in Conway microdiffusion cells, as described by Bremner and Shaw (10). The formula- tion of titanous sulfate distributed by the British Drug Houses, Ltd., and recommended by the above authors was used for reduction of nitrate. Ammonium, nitrate and total mineral nitrogen (NH4+N03) were calculated to ppm. N, oven dry soil basis, allowing for dilution by the water initially contained. No allowance was made for losses of dry matter or moisture during incubation. Statistical Treatment Only three contr011ed temperature rooms were used. Consequently, there was no replication of temperature 31 treatments. The three rooms were side by side, and it was suSpected that systematic temperature variations might arise within a given room by conductance from adjacent rooms at different temperatures. For this reason, duplicate jars were assigned to random positions, one on each side of each room. Data for each room was analyzed separately in accord- ance with a randomized block design. Data for the three temperature rooms were then combined as locations, as des- cribed by Snedecor (44). Multiple range and multiple F tests as described by Duncan (15) were used to test for the Signi- ficance of differences between observed means for various treatment combinations. Significant variations in microbial numbers and in the different forms Of nitrogen were associated with blocks. The precaution taken to associate within-room temperature differentials with replications was, therefore, justified. Within-room relationships to inferred temperature gradients were consistent with those observed between rooms during the first half of the incubation period. After temperatures were raised in two rooms at mid-incubation, significant variations associated with replications were less frequent, and trends with temperature were occasionally in a direction Opposed to those observed between rooms. RESULTS ReSpiratory Activity and Microbial Numbers Main effects of fumigants Main effects of fumigation treatments on CO2 evolu- tion and microbial numbers are shown in table 2. When interactions with temperature and nitrogen treatment were ignored, neither ChlorOpicrin nor diChlorOprOpene influenced the respiration rate. Data for 0 days Show that, during the exposure period, diChlorOprOpene stimulated both bacterial and fungal numbers, whereas, chlorOpicrin depressed them both. During subsequent incubation, numbers Of bacteria increased sharply in both fumigated soils but declined dur- ing the latter half of the incubation period. The pOpulation of bacteria in fumigated soil was higher than that in Check soil throughout the incubation. There was some recovery of fungal numbers, during incubation in soil treated with ChlorOpicrin, but numbers remained lower than in the con- trols arin soil treated with diChlorOprOpene. Main effects of nitrogen treatments CO evolution was not affected by any of the nitrogen 2 treatments, when interactions with temperature and fumigation were ignored (table 3). There were no consistent main effects on microbial numbers over the major portion of the 32 33 incubation period. However there was a tendency for bacterial numbers to be increased, fungal numbers to be decreased, by increasing nitrate level, at the beginning of the incubation. These relationships with initial nitrate level were reversed at the end of the incubation period. Both at the beginning and at the end of incubation, fungal numbers tended to be inversely related to bacterial numbers. This is a Character- istic competitive relationship between these two groups (3). Relationships with temperature Respiration rates and microbial numbers as related to temperature are shown in table 4. Significant differences in respiration rates for the three different temperatures were eXpressed. The highest rate was noted in the 30°C room at the beginning of the incubation, but the rate declined during subsequent incubation at this temperature. Low res- piration rates were Observed at 50 and 20°C. prior to the temperature changes on the Slst. day. Immediately after the temperature in the T1 room was raised to 20°C. and that in the T room was raised to 30°C., rates were quantitatively 2 similar to those observed earlier at the same temperatures in the T2 and T3 rooms. During subsequent incubation, rates declined from these new levels. Bacterial numbers increased dramatically during incubation at low temperatures, whereas numbers of fungi were depressed at the lowest temperature and increased at the two higher temperatures. In both cases, 34 .Ocmoumo n so econommwp >HpcmonHcmHa no: out HoppmH mean any >n ochmoEooom momma .mmnnp mo ozone HmoHpum> come awry“: .oocoHa>Hoom mo mmucam o .n .m .wcmoonoonoHcoHo on muowooxe .oxmmz N .cHnoHoonoHnu on anomooxm .a>mo 039 * a m.HH a H.HH a m.mH a «.mH Ammo ecoeooeonoHoOHo o e.m o e.e o H.o o o.o Ammo :HaoHeoooHto m o.oH om m.e o m.m o o.v HHmV mooz x m eoH H can m v.e m e.vH m H.mm a e.e Ammo ecoeoueotoHcoHo m e.e o o.HH m e.om o o.o Ammo oHuoHeouoHto o o.o o H.m o e.w o o.o AHev mooz OOH x manmvowm m H.O a o.o a o.> o o.o Ammv ocooonoouoHnoHo a H.o a a.» a m.e a e.m Huey oHuoHeonoHto a o.o a o.e a o.o e o.o HHmv ocoz >mo\.mooH\U.me .muan coHpmuHoamm ooH we.om oe-oo me oe-om ev ev.om o *coHvamHSSm stoma o>oo pcamHSSE .ucesvoonu SOHO-massm Op omuoHon an «Hones: Hngouows ecu >9H>wpoc >Hopanuooomau.m oHnaH 35 .pcoonoo m an ucwnmmme >HucoonHcmHm no: who nmuamH mama orb >n ooHcmoeoooa memos .nsom mo ozone Hmompum> some awry“; .mocmHm>Hoom mo neocom .u .n .m .csoru acoHpnooono Se 2 and cos on HHou em moz mzHo vrz mcHun on coHvamH83m umumo m>mo NH ommaooo cmmonufiz * a o.o on o.e o H.o a e.o m\H e.z o e.o o o.e oo v.e m e.o H oz o o.o on m.m a o.oH m e.o m «z o o.o . m.aH on e.o a e.o e Hz voH x Hesse o o.o a v.m a o.oH a o.o m\H «z a u.m m o.o m o.mH a o.o H mz om H.e a o.HH . «.mH a o.o m N2 m o.o a e.e a e.HH m o.o e Hz 00H x manopomm a v.o a u.e a e.e m o.o m\H «z e v.o a e.e a e.m m o.o H mz a H.o a m.e a H.m a e.m m «z a e.o . m.e a o.o o o.o e Hz >mo\.mooH\o.os .opmn coHumHHQaOm * ooH me.oo me.oo me oe.om ea ev-om o cofipmmHSSm seven o>mo m02\g:z ucoauamnu comonvfiz .npcoSvoonu cemOHOH: Op popmHou an newness HuwnonoHs new >OH>Huoa >novanmooomau.m pooH 36 maximum, or near maximum, numbers were attained by the 33rd. day of incubation (49 days after fumigation). Prior to the temperature increases on the 5lst. day, large numbers of bacteria at the two lower temperatures were associated with low rates of CO2 loss. Data in table 5 Show that large initial increases in numbers of bacteria occurred only in fumigated soil and that these increases were inverse- ly related to temperature. ReSpiration rates (not shown) were not affected by fumigation (ignoring nitrogen treatment) at any temperature. Thus it appears that decreasing temper- ature promoted increasingly efficient assimilation of carbon into microbial tissues and that this effect was strikingly expressed on the bacterial components of the recovery pOpula- tion in fumigated soil. As regards their effects on bacteria, both fumigants behaved similarly under all three temperature regimens throughout the incubation period. There were Significant differences, however, in their effects on fungi. ChlorOpicrin appeared to be more injurious to fungi than diChlorOprOpene. This difference was apparent initially at the two lower temperatures, and, at the end of incubation, under all three temperature regimens. No clear-cut first-order interactions between temperature and nitrogen treatments were observed (table 6). The tendency noted earlier (table 3) for initial increases meow may >O omHCmoeooom memos .moncp mo ozone Haowpnm> some cHanz .pcmonoo m we Oceammmwo >HvaumecmHu was who nonbmH .ommcmnoc: auspmanSOH .mocoHo>Hsom mo «mesmm o .n .m m. * .uoom on confine enabmnooaeh mm .ooom Op Common waspauooamh e .oom pm memo m use ooH- on ease m neoo< * 37 a «.mH H o o.mH a o.HH as a o.oH o ¢.v * o m.m voH x “meow o H.e “ o o.o a o.oH as o m.nH a o.HH * m m.om ooH x ownopoum o H.m o m.e M m e.oH m o.e e ~.HH *4 o H.@ o o.v o o.o e o m.H >wm\.mooH\o.mS .opmn COwOmHHOmmm mhuoo me oenmm Hm av hvumm coHpomHeom nevus u>oo Hmeo.ooom Hmeo.ooom HHHo.ooe Hmeo.ooom Aeao.ooou HHeo.ooo HmHV.Ooom Hmsv.o°om HHHV.oom an eat tooH co onsuanmoaoh .onopaumoeop o» ooOaHmu on oneness HmHnOHoHS new >OH>Hpoa >nouwnaonomuu.v mHnuH 38 Table 5.--Microbia1 numbers as related to fumigation treatment and temperature. Temperature Days after fumigation regimen Fumigant 49 75 100 49 75 100 * ** Bacteria x 106 Fungi x 104 T1 1:1 7.29 4.11 6.88 3.86 3.84 9.04 e d cd c d ab 8 a a d d c ab a ab c cd b e c f b ab ab F2 21.1 9.93 10.7 11.8 8.81 7.26 cd b a b bc b F3 23.8 21.6 9.61 29.7 15.9 12.1 bc a abc a ab ab T3 F 2.06 6.83 4.81 11.6 18.8 12.2 1 f bc of a ab e b bcd ab b F3 12.2 6.89 6.28 12.8 15.8 15.7 do be de b ab 3 * cf. Table 4. ** cf. Table 2. a, b, ......f Ranges of equivalence. different at 5 percent. Within a given column, means accompanied by the same letter are not significantly 39 in bacterial numbers to be prOportional to nitrate level was most clearly eXpressed at the lowest temperature. At the two higher temperatures, fungi tended to reach maximum numbers at intermediate levels of nitrate, with significant reductions at the highest level (N4). A similar tendency for microbial numbers or activity at intermediate levels of nitrate to differ from those at higher or lower levels appears frequently when second order interactions between fumigation, nitrogen treatment and temperature are considered in the following sections. Interactions of fumigants and nitrogen treatments at low to intermediate temperatures (Tl) Numerous investigators have observed that peaks in respiratory activity in soils tend to occur 2 to 3 weeks prior to corresponding peaks in microbial numbers (20, 46, 51). For this reason, CO2 was collected for 2 to 3-week periods preceding each sampling for plate counts during the course of incubation. Under the T1 temperature regimen, major differences associated with nitrogen treatment were Observed between the two lower initial nitrate levels (N1 and N2), on the one hand, and the two higher levels (N3 and N4), on the other. The correSponding means are presented graphically in figure 1. Nothing can be said about changes which may have occurred during the 2-week period after nitrogen was added. Table 6.--Microbial numbers as related to tenperature and nitrogen treatment. 40 Tenpera ture Nitrogen Days after fumigation regimen treatment 49 75 109 49 75 i ** Bacteria x IOVR Fungi x 10 abc ab abc de cde N2 23.7 11.7 8.55 3.61 3.84 abc abc abcd d e a abcd ab e de N4 30.3 11.5 8.64 2.38 3.99 ab abc abcd de de abc abcd a ab ab N2 13.0 16.1 7.15 19.7 13.1 ed a abcd 8 abc bcd abcd bcde a bcd N4 17.8 6.21 6.69 11.4 12.2 abc d cde bc abc e cd de ab 8 N? 5.85 7.10 5.91 17.0 11.0 e cd de ab abc e d abcd bc abc N4 7.85 8.21 4.82 8.44 18.5 de bcd e c a 100 4.96 e 5.55 do 6.55 cde 8.81 bcde 7.75 cde 17.2 ab 6.49 cde 10.5 abcd 9.53 bcde 12.1 abc 6.96 cde 20.4 a * of. Table 4. ** cf. Table 3. a, b,....e Ranges of equivalence. different at 5 percent. Within a given column, means accompanied by the same letter are not significantly 41 and the first collection of CO2 30 days after fumigation. Nonetheless, there is strong evidence that patterns of micro- bial succession were influenced by both fumigation and the prOportions of NH4 and N03 added 16 days after fumigation. Between the 30th. and 45th. days, marked adjustments in reSpiration rate occurred. These adjustments were dis- tinctly different at higher nitrate levels (N3 and N4) in both fumigated soils than those in unfumigated soil or at lower nitrate levels in fumigated soil. After the temperature change, maximal increases in 002 evolution were associated with the N3 andN4 treatments, and these occurred earlier in fumigated than in unfumigated soil. Large increases in numbers of bacteria on the 49th. day occurred only in fumigated soils. These increases were greater with the N and N4 treatments and reflect the 3 earlier adjustments in CO evolution observed in fumigated 2 soils. The association of larger numbers of bacteria in the initial recovery pOpulation, and of higher rates of loss of C02, with higher nitrate additions in fumigated soils suggests that fumigation promoted physiological types capable of utilizing nitrate for growth and for electron exchange. Fungi in the recovery pOpulation appeared to be suppressed competitively by these bacterial types on the 49th. day. At the end of incubation, however, maximum recovery of fungi 42 Figure l.--Patterns of microbial succession and reSpiratory activity at low to intermediate temperatures. Houghton muck was eXposed 2 days to chlorOpicrin or 2 weeks to diChlorOprOpene, after Which control and treated soils were eXhaustively leached, aerated, frozen for 3 days and then maintained at 5°C. through the Slst. day after fumigation. At this time the temperature was raised to 20°C. On the 12th. day after fumigation, all soils were amended to 400 ppm. N with NH4 and N03 in ratios of 9:1 (N1), 3:1 (N2), 131 (N3) and 133 (N4). 42a 00. 05 ac O ._.. mmeHm Om wk on q H _ 23: cz+nz 0 23:: «2+ .2 0 >7 0\ o o o /.,,H\...fim. 138'; 2% l 9 ,on x lawns O N 23.2 5.12 I 23: N2.1,. D AIIIIIIUOD 00. n5 m¢ 0 £— .n... .t mzmaomaoconzus ¢ AVG/'9 oouo 'ow-Zoo O O V’ N 9on x Vlaalove O 0 00. oh ac o om Oh no ._.zw2._.L-30°C—>x _zo°c——>~ao°C-—> a / 32°“ ' /’ h II. I o I l l f CHLOROPICRIN (TZFZ) Key __ V i‘ N. o 8 940— "2 ' ' o x ‘ :3 3 V\/ a V \ 220*- / ~ — Lu. P" O \ I l l 0,._/' 4 _ fiuem DICHLOROPROPENE (T2F3) 8 T- ‘ 1" V 240~ O O Q t " ./v \\ 32° N P” 'L 9 \ v—v J l I O l i l I o 49 75 I00 0 49 75 I00 45 nutrition (N1) would likely have been qualitatively as well as quantitatively different from those in the presence of increasing prOportions of nitrate. ReSpiration rates under the T2 regimen are shown in figure 3. In unfumigated soil after the temperature change, maximum rates were obtained with the highest additions of NH4 (N1) and the lowest rates with the lowest NH additions 4 (N4). By contrast, reSpiration rates associated with the N1 treatment were depressed, - relative to unfumigated soil, - in soil treated with chlorOpicrin or diChlorOprOpene. Maximum rates with chlorOpicrin were associated with the N treatment. 3 With diChlorOprOpene, maximum rates were associated with the N3 and N4 treatments, although inversions occurred after 75 days. These relationships are consistent with the View that fumigation promoted a type of metabolism with a relatively *V' higher dependence upon N03 rather than hn4. Interactions of fumigants and nitrogen treatments at constant high temperatures (T3) Again under the T temperature regimen, effects of N 3 treatment on bacterial numbers were Observed only in fumigated soils (figure 4). With chlorOpicrin on the 49th. day, there was a strikingly direct relationship between numbers of bacteria and the level of nitrate added on the 16th. day. With diChlorOprOpene, increased numbers of bacteria appeared at this time only at the lowest nitrate level (N1). However, 46 numbers of fungi at this time were inversely related to nitrate additions, which suggests that bacterial competition prior to the 49th. day may have been greater at the higher levels of nitrate. With both fumigants, fungi increased markedly after 49 days with the N3 and/or N4 treatments. This again suggests that primary bacterial successions were consummated earlier in the presence of higher nitrate addi- tions. In unfumigated soils under both T2 (figure 2) and T3 (figure 4) regimens, terminal increases in fungi were associated with the N2 treatment only, indicating that NH4/NO3 ratios may have influenced microbial succession in normal soil pOpulations also. Interaction effects of fumigation and N treatment on patterns of respiratory activity were even more pronounced under the T regimen (figure 5) than under the T2 temperature 3 sequences (figure 3). Losses of CO2 were markedly depressed by fumigation where nitrogen was added principally as NH4 (N1). In chlorOpicrin treated soil, rates prior to the 45th. day were directly related to nitrate additions, as were bacterial numbers on the 49th. day (figure 4). With dichloro- prOpene, maximum respiratory rates were associated with intermediate nitrate additions (N2 and N3). Inversions in relative respiratory activity for N1 and N occurred after the 75th day (figure 5). Changes in 3 relative numbers of fungi for these two treatments (figure 4) indicate that correSponding major adjustments in the 47 Figure 3.--Patterns of respiratory activity at intermediate to high temperatures. Houghton muck was exposed 2 days to chlorOpicrin or 2 weeks to diChlorOprOpene, after which control and treated soils were eXhaustively leadhed, aerated, frozen for 3 days and then maintained at 5°C. through the 16th. day after fumi- gation. At this time the temperature was raised to 20°C. The temperature was raised again to 30°C. on the 51st. day. On the l2th.day after fumigation, all soils were amended to and NO 400 ppm. N with NH in ratios of 9:1 (N1), 3:1 (N2), 4 3 1:1 (NB) and 1:3 (N4). 47a om he no he on _ J H . Illo.om mum/Rb />> m > Pa 0 m? D / m 1 no we”. Team .9 D. Ju\\ > .2 on or Ilplunz /w>> o «z- /\ IoIa D xoxi inane. mzmaomaomoazo_o hzu2k:I. > nz M\O\O . ~21 \ Ilol..z 0 so} tau: ouh V IO A C) FUNGIX m C) P V 30°C I o/° /v lééééfi:"flfiv/' O ;//’Y>?‘-1 o )- DICHLOROPROPENE (1'3 F3) e I. ’ 2 O ><4O O - 5 b \ V 2 / / Dzor/O 9 / < V “- o< >> I >0! ’\ at»: I amazon—04:0 om ms mm m¢ on M . 4 . coon v vzlblnz I 0 N2 D mum b» Iolmaz Agnew/a» o a. I / o Ob \o w g l o /W\/m\ o w»? a) tent oxo/om owh<0_2:mza 0. v. we /'9 om 10 an - zoo 50 micrdbial pOpulation were occurring at this time. Discussion In evaluating microbial counts in the foregoing sections, it must be recognized that culture plates were in- cubated aerobically at 220 to 25°C. Obligate anaerobes would not have develOped on the plates. Organisms with specific low temperature requirements would also have been discouraged. The bacterial medium supplied minerals, plus nitrogen as as- paragine and as nitrate, but no other growth factors. Organisms with more fastidiOus requirements would not have been counted. It is impossible to say whether fungal numbers represent vegetative forms or viable Spores. Nor can anything be said about short term fluctuations in numbers, or about effects on reSpiratory activity during the first two weeks of incubation. Nevertheless, the observed second-order interaction effects on both numbers and losses of CO consistently sup- 2 port the inference that recovery pOpulations in fumigated soil were dominated by bacterial types with a marked prefer- ence forNO3 rather than N14. This reflects an abnormal type of metabolism for soils. Normal soil pOpulations are characterized by a high degree of preference for NH4 over N03 (3, 21). Utilization of nitrate by microorganisms, as well as by plants, involves nitrate reductase systems which have 51 been shown to develop adaptively in a number of bacterial and fungal Species When grown on media supplying nitrate (2, 53). Under strict anaerobiosis, nitrate reduction by bacteria leads to gaseous denitrification products, principally N2 and N20. With Spore-formers, reduction to N54 may also occur. Such dissimilatory or reSpiratory nitrate reduction is accompanied by assimilation of part of the nitrogen from nitrate into cellular protein. Dissimilatory processes are increasingly inhibited, assimilation of nitrate is enhanced, by increasing oxygen supply. With full aeration, nitrate re- duction leads only to protein synthesis. Fumigation effects a partial pasteurization of the soil. The degree to which the soil fauna and flora are decimated depends upon the conditions of treatment. Spore- formers are most likely to survive initially, although the recovery of certain non-Spore-forming bacteria is frequently rapid. Their numbers are greatly reduced initially, but, in the absence of normal competitors and antagonists, certain fast growing types quickly dominate the population, reaChing numbers frequently several fold greater than the total pOpula- tion before treatment. Among such fast growers, fluorescent IPseudomonas Species are frequently abundant in soils soon after incubation (34). The ability to reduce nitrate and nitrite is an adapt- ive characteristic of many bacterial Species, including pseudo- monads and other fast growing non-Spore-formers, as well as 52 numerous spore-formers (12). The adaptation is promoted by oxygen stress. The soil systems used in the present study involved large aggregates, up to 1/4 indh (6 mm.) in diameter. Moisture initially was high, although short of field capacity. Such a physical matrix would have provided an intimate associ- ation of anaerobic micro-habitats conducive to nitrate reduc- tion with fully aerobic environments inhibitory to denitrifi- cation and favorable for assimilation of nitrate (9). Under these conditions of partial anaerobiosis, the adaptation to nitrate utilization would have been promoted by increasing initial nitrate levels in both fumigated and un- fumigated soil. However, the apprOpriate bacterial Species would have encountered little competition in fumigated soil from more normal soil microflora. Thus bacterial reSponses to varying nitrate levels were observed only in fumigated soil, Whereas fungal reSponses were observed in both. In both cases, however, fungal reSponses may have reflected competitive ad- justments to changes in the bacterial flora. The fact that no effects of NH4/NO3 ratio on bacterial numbers were observed in unfumigated soil may have been due to failure of species with complex growth factor requirements to grow on the medium used. Differential reSponses to initial nitrate level in fumigated soil were most clearly eXpressed by bacterial numbers at low temperature and by respiratory losses of CO2 at high temperatures. This was undoubtedly due, in part, to more 53 efficient assimilation of carbon at low temperatures. How- ever, microbial successions would also have occurred more rapidly with increasing temperature. At the higher tempera- tures, recovery pOpulations of bacteria comparable to those observed on the 49th. day at low temperatures may have develOped and exhausted apprOpriate energy materials earlier. The earlier recovery of fungi at high temperatures supports this View. Differences in incubation behavior between the two fumigants reflect differences in initial kill. Initiating recovery pOpulations were both more numerous and more hetero- geneous immediately after treatment with diChlorOprOpene than with ChlorOpicrin. These initial differences influenced patterns of microbial succession throughout the incubation period. Notably fungi recovered more slowly in soil treated with chlorOpicrin, suggesting that this chemical may have been more effective than diChlorOprOpene in killing fungal Spores. Relatively large counts for fungi immediately after treatment with diChlorOprOpene were likely due principally to surviving Spores. 54 Nitrogen Transformations Changes in NH4 and N03 prior to incubation Early Changes in NH4 and N03 prior to controlled temperature incubation are shown by data in table 7. No determinations were made before exposure of soils to fumigants. Immediately after 2 days' eXposure to chlorOpicrin and 2 weeks' eXposure to diChlorOprOpene, ammonium levels were higher, nitrate was lower, than in the control. Little change occurred in NH4 during subsequent leaChing and aeration. Leaching was continued for 4 days, at Which time no traces of NO were 3 detected in the percolate. Nitrate, to the extent of 33 ppm., had accumulated during the subsequent 24—hour aeration period in control soil only. Immediately after aeration, soils were frozen for 3 days and then held at 5°C. for 8 days, except for an 8-hour period during Whidh nitrogen additions and moisture adjust- ments were made. Nitrogen additions were calculated on the basis of NH4 and N03 found after leaching. Calculated amended totals are shown for the 12th. day after fumigation, When nitrogen additions were made. Comparison of these totals with those found 4 days later reveals that marked differential ad- justments had occurred during freezing and low temperature storage imposed after leaching and aeration. Net mineraliza- tion of organic N had occurred in all soils, leading to accu- mulations of N03 in unfumigated soil and accumulations of both one nausea mama any >n poficmdsoooa memos .cssaoo co>fim a swap“; .pcoonod 0 pa pconommwo >chaowmficmfiu no: .oocoda>w:oo mo condom m........ .n .m .oomw op oou pa >ao H acflvmumm can manna“: anon ow u>av v mcfinowoa novm< * a ham com a N am a has ooH a me he at a eem oom n 0 he 4 and cod a me am we a «Hm com a mm ave e «as can a HA 4 HA «2 a sea com a N am we aha com a we as me can was com a 0 mm a mom com a me am me up new com a mm ass a «am com A AA a He m2 no ems ooH a N am on mam com a we as me can ova can a 0 am oh Ohm oom a me am we 5 5 can ooH cos a mm ass a esm com a as a He Nz a we as h N am a mse can a «a as me a ens ea a 0 he a hoe com a me am we a he as a an ass a mom com a as a at Hz * w wt 0. no N. to n - an o no OH to N: to m .o o wcmmwesm accepaouu on newcommH cowummwsam on ocwcouofl cowpwmwe:m z venom concos< Hmawa nouma venom ooocos< nopmu acum- zHQz .saa 2-32 .see £03335 35939.3 3:93:00 ow .3qu 33:: one 53:08.8 5 39.23 :3qu.» 033. 56 NH4 and N03 in fumigated soils. Applying standard errors associated with the determina- tions on the 16th. day, increases in NH4 over calculated amended totals in fumigated soils would have been statistical- ly Significant, whereas none of the apparent increases in N03 would have been. It is of interest, however, that Kirkwood (25) observed similar increases in N03 during freezing or sub- sequent thawing of organic soil samples taken in early Spring from field plots fumigated the previous fall and from unfumigated plots. Main effects of fumigants Soils were placed under differential temperature regimens on the 16th. day after fumigation. Main effects of fumigants, during subsequent incubation, on NH4, N03 and total mineral N may be seen in table 8. Conversion of NH4 to NO was retarded by both fumi- 3 gants, maximally by chlorOpicrin. Between the 16th. and 26th. days, there was a marked reduction in recovery of mineral N in all soils. This was due to the disappearance primarily of NH4 in control soil and of NO with chlorOpicrin. With dichlorOpropene, marked 3 reductions in both NH4 and N03 occurred during this initial lO-day period, and the mineral N total was more sharply reduced. During the balance of incubation, average values for N03 remained lower for diChlorOprOpene than in the control, 57 Table 8.--Ammonium, nitrate and total mineral nitrogen during incubation, as related to fumigation treatment. Days after fumigation* Fumigant 16 26 41 57 69 94 ppm. NH4-N None (F1) 253 b 69 c 28 c 2 c 9 b 2 b ChlorOpicrin (F2) 286 a 273 a 238 a 119 a 104 a 15 a Dichloropropene (F3) 282 a 230 b 79 b 72 b 11 b l b ppm. mg-N None (F1) 195 a 325 a 396 a 477 a 522 a 582 a ChlorOpicrin (F2) 195 a 122 b 211 c 362 b 464 b 567 a Dichloropropene (F3) 195 a 133 b 325 b 381 b 512 a 543 b ppm. mineral N (NH4+N03) None (F1) 448 a 394 a 426 b 480 ar 532 b 585 a Chloropicrin (F2) 481 a 395 a 449 a 481 a 568 a 583 a DichloroprOpene (F3) 476 a 363 b 404 c 453 b 523 b 544 b a, b, c Ranges of equivalence. Within each vertical group of three, means accompanied by the same letter are not significantly different at 5 percent. * TWO days' exposure to Chloropicrin, 2 weeks' exposure to diChlorOprOpene. 58 and mineral N means remained lower than for either control or chlorOpicrin. The behavior with diChlorOprOpene might suggest that this chemical may have retarded mineralization of organic N or may have promoted dissimilatory losses of N03. This was only apparent in the means, however, and derived from the fact that temporary periods of NO disappearance with dich- 3 lorOprOpene occurred at different times with different comb- inations of temperature and nitrogen treatment. Mineral N totals were Significantly depressed at these times relative to control and ChlorOpicrin treated soil but recovered in later samplings. It appeared likely that similar recoveries would have followed significant depressions encountered in the last sampling. Main effects of nitrogen treatments When effects of temperature and fumigation were averaged out, significant differences in NH4 and N03 were related essentially to differences in amounts added (table 9). Declines in total mineral N during the initial lO-day period tended to be greater and subsequent recoveries less rapid at the lower levels of NO3 addition. Totals at the end of incu- bation were essentially indentical for all N treatments. Relationships with temperature The rate of conversion of NH4 to N03 increased with increasing temperature (table 10). Total mineral N declined 59 Table 9.--Ammonium, nitrate and total mineral nitrogen during incubation, as related to nitrogen treatment. Nitrogen Days after fumigation treatment treatment NH4/N03 16 26 41 57 69 94 * ppm- NH4-N N1 9 395 a 271 a 166 a 84 a 56 a 8 a N2 3 335 b 234 b 137 b 82 a 47 b 13 a N3 1 245 c 162 C 95 C 57 b 36 C 2 b N4 1/3 120 d 97 d 63 c 37 c 26 d 2 b ppm. N03-N N1 9 101 c 105 d 258 d 384 b 471 b 555 a N2 3 144 c 146 c 284 c 385 b 504 a 558 a N3 1 199 b 232 b 322 b 404 b 504 a 575 a Na 1/3 335 a 290 a 380 a 453 a 519 a 570 a ppm. mineral N (NH4+N03) N1 9 496 a 376 a 424 ab 468 ab 528 a 562 a N2 3 478 a 380 a 421 ab 466 ab 550 a 570 a N3 1 444 a 394 a 417 b 461 b 540 a 576 a N4 1/3 455 a 387 a 443 a 490 a 545 a 572 a * Nitrogen applied 12 days after fumigation to bring NH4 plus N03 in soil to 400 ppm. N in proportions shown. a, b, c, d Ranges of equivalence. Within each vertical grOUp of four, means accompanied by the same letter are not significantly different at 5 percent. 60 Table lO.--Ammonium, nitrate and total mineral nitrogen during incubation, as related to temperature. Temperature Days after fumigation treatment igpfisggyon 16 26 41 51 57 69 94 * ppm. NH4-N 5°C. (T1) 274 a 227 a 183 a 4 159 a 105 a 14 a 20°C. (72) 274 a 170 b 91 b 44 30 b 11 b 2 b 30°C. (T3) 274 a 176 b 73 c I 5 c 8 b 2 b ppm. 1103-11 5°C. (71) 195 a 154 c 194 c 4 250 c 378 c 481 c 20°C. (T2) 195 a 194 b 338 b 4+ 440 b 537 b 592 b 30°C. (13) 195 a 232 a 400 a 1 592 a 583 a 619 a ppm. mineral N (NH4+ m3) 500’ (T1) 469 a 381 b 377 C + 409 C 483 C 496 C 20°C. (T2) 469 a 364 b 429 b :4 471 b 548 b 595 b 30°C. (T3) 469 a 407 a 473 a 1 534 a 529 a 621 a * After 3 days at -100 and 7 days at 5°C. 4 Temperature raised to 20°C. 44 Temperature raised to 30°C. : Temperature unchanged. a, b, c Ranges of equivalence. Within each vertical group of three, means accompanied by the same letter are not significantly different at 5 percent. 61 Sharply during the first 10 days at the two lower tempera- tures, less Sharply at 30°C. Subsequent release was rapid at the two higher temperatures, Whereas mineral N remained depressed through the 4lst. day at 5°C. The extent and duration of mineral N disappearance were directly related to numbers of bacteria Observed on the 49th. day (cf. table 4). This indicates that temporary reductions in mineral N at the beginning of incubation were due primarily to immobiliza- tion in microbial tiesues. Net release of mineral N from soil organic matter, as observed in the last sampling, increased with increasing average temperatures eXperienced during incubation. Interactions of fumigants, nitrogen treatments and tempera- ture Numerous first and second-order interactions between the three treatment variables in their effects on NO NH 3' 4 and total mineral N were expressed with statistical signifi- cance during the course of incubation. Mean N03 values for duplicate jars of each ultimate treatment are presented graphically in figures 6,7, and 8. The totals of NH4 plus N03 shown in these figures represent averages for the three fumigation treatments, except Where significant differences between them were encountered within a given combination of temperature and nitrogen treatment. 62 The most striking feature of the data in these figures is the fact that early declines in mineral N were accompanied by a disappearance of NO in fumigated soils, 3 Whereas in control soils, NH4 disappeared and N03 accumulated at rates which were increasingly rapid with increasing temperature. This confirms the earlier inferences from microbial numbers and respiratory activities (pages 50-53) that the heterotrOphic microflora in fumigated soils were characterized by an abnormal dependence upon N03. Discussion The patterns of delayed NO accumulation observed in 3 figures 6, 7, and 8 for fumigated soils are not characteris- tic for lag periods associated with Slow recovery in numbers of nitrifiers. Rather, they represent a masking of early nitrifying activity by reason of the fact that associated heterotrOphic bacterial pOpulations were concurrently consum— ing NO The larger these bacterial pOpulations were, or 3. the longer they persisted in the soil, the longer was N03 accumulation delayed (figures 1, 2, 4). There is little to indicate whether or for how long nitrifiers were Specifically inhibited by fumigation. With dichlorOprOpene, N03 accumulated at rates equal to or greater than in controls as soon as net disappearance of mineral N ceased. This indicates that full recovery of nitrifying 63 Figure 6.--Nitrogen transformations at low to inter- mediate temperatures. Houghton muck was exposed 2 days to chlorOpicrin or 2 weeks to diChlorOprOpene, after which control and treated soils were exhaustively leached, aerated, frozen for 3 days and then main- tained at 50C. through the Slst. day after fumigation. At this time the temperature was raised to 20°C. On the 12th day after fumigation, all soils were amended to 400 ppm. N with NH4 and N03 in ratios of 9:1 (N1), 3:1 (N2), 1:1 (N3) and 1:3 (N4). Insets Show effects of leaching unfumigated soil (F1) and soil treated with chlorOpicrin (F2) or diChlorOprOpene (F3). 63a hzwihdwmk ['3 mDOHm «.0 mm #0 .0 2‘ mm m. n O N d d _ . _ — - ............ m u— IIIIII N... ... o noz \\0. o noz... e:2 \me Imam ...... o../ >0 0‘ ... o” T..\.. yo x xx . o .d \\\\ ... .I. \w\\ll\l|. 0‘ ... \.\.& .IIIII /. ....... ‘ TIUCONIIJTIUomL 1 2 . q _ _ q 1 u C .. O ..O....\\vO/h. 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