EFFECTS OF FUMIGANT CHEMICALS 0N MICROBEAL ACTIVITY AND NITROGEN ‘I'RANWORMAT’ION AND ON CRO? RESPONSE IN ORGANIC SOIL Thurs Ior Hm Degree OI DI'I. D. IIIICIIISAN .3“;- IE MII’EB’SE'E‘Y James Irvin Kirkwooci 1982 IIIIII \IIIIIIIII IIIIIIIII/ 3 1293 01066 395 This is to certify that the them £ntifl¢d I . -....?" y. - «egg 1; ‘ 51.3..-. EFFECTS OF FUMIGANT CHEMICALS ON MICROBIAL- ’ ACTIVITY AND NITROGEN TRANSFORMATION AND ON CROP RF SPONSE IN ORGANIC SOIL 4:3,”: I or‘esent'ed by ’7’) ., ~...—. James Irvin Kirkwood has been accepted towards fulfillment of the requirements for $11.1)— degree in _S°_11_3_. afl/M Major professor Date June 71 1962 0-169 LIBRARY Michigan State University PLACE ll RETURN BOXtomnavombetnekouImnywncocd. 1'0 AVOID FINES Mum on or More dd. duo. DATE DUE DATE DUE DATE DUE AETRACT EFFECTS OF FUMIGANT CHEMICAIS ON MICROBIAL ACTIVITY AND NITROGEN TRAIIEFORMATION AND ON CROP IESPODBE IN ORGANIC SOIL. by James Irvin Kirkwood Field studies were conducted in 1959, 1960 and 1961 to investigate the effects of fall fumigation on seasonal accumulations of ammonium and nitrate and on yields of crops in nematode -free organic soil. 29.1.9331 (dichloropropene mixture) was used at rates of 32 to 118 gallons per acre. Pfineralization of organic nitrogen was enhanced, nitrification retarded in fumigated soil. Early applications of ammonium sulfate extended the delay in nitrification into late June. Where nitrate fertilizers were used, rapid nitrification in fumigated soil began in late May. Wide ammonium: nitrate ratios, ranging up to 6:1 in late May, were associated with marked chlorosis and retarded early growth of celery. Nitrate sidedressings corrected early injury in 1959 and 1960. Under conditions of limiting manganese and phosphorus nutrition in 1961, there was no responde to nitrate. Foliar analysis indicated that in- creased availability of iron in fumigated soil had interfered with uptake or translocation of phosphorus. Yields of celery declined as iron: phosphorus ratios increased frbm .Ol.to .02. Manganese remained below 12 ppm. ‘ In the same experiment, yields of sweet corn and lettuce were in- creased due to increased availability of manganese in fumigated soil. The manganese response of these two crops was identified by' visual James Irvin Kirkwood symptoms and foliar analysis. Laboratory incubation studies were conducted in 1961.to compare the effects of several fumigant chemicals on microbial numbers and activities in organic soil. Moisture conditions expected to prevail in the fall and spring months were simulated. Exposure and aeration periods recommended by the manufacturer were used in treating samples of organic soil with 1/2, 1, and 2 times the recommended application rates of 2329.231: Vidden-Dl, and Fumazonel. Untreated soil and a reference fumigant, chloropicrin, were included. N-Servel, a nitri- fication inhibitor, was included as a nonfumigant chemical which would specifically inhibit nitrifiers Without materially influencing hetero- trophic activities. The ventilation method of incubation was used. Multiple subsamples of soil were incubated in duplicate respiration jars for each treatment. Three different temperature regimes were used. I The rate of 002 evolution was estimated titrimetrically every third day. At periodic intervals subsamples were removed from each respira- meter jar for the determination of ammonium and nitrate and for the estimation of microbial numbers. Ammonium and nitrate were determined by microdiffusion. Enumeration of the soil microbial population was accomplished by dilution plate count. Partial sterilization effects on both heterotrophic and autotrophic components of the soil microbial population were expressed by all fum- gant chemicals. These results confirmed observations made in field studies. However, the intensity and duration of effects of all fumigants James Irvin Kirkwood were markedly different under different sequences of soil temperature imposed 3 weeks after initial exposure. Nitrification was inhibited by ohloropiorin for it to 16 weeks after treatment at 10° and 20°, but for only 9 to 10 weeks at 30° 6. Inhibition by the other fumigants at 7 weeks was observed only at 10° or 20° C. Partial sterilization effects on the heterotrophic pepulation, however, were expressed 3 to I; months after treatment at the two lower temperatures. At 30°, hetero- trophic numbers and activities were affected 7 weeks after treatment only by chloropicrin. The chloropicrin effects had largely disappeared 10 weeks after treatment. The non-fumigant chemical, NAServe, applied as a fertilizer addi- tive on (NHI)2S°h reduced levels of-nitrate and total mineral nitrogen under all three temperature regimes, without appreciable accumulation of ammonium after four weeks. This appeared to be due to chemical com- plexing of ammonium added with the chemical. 1 Products of the Dow Chemical Company, Midland, Michigan. EFFECTS OF FUMIGANT CHEMICALS ON MICROBIAL ACTIVITY AND NITROGEN TRANSFORMATION AND ON CROP RESPONSE IN ORGANIC SOIL by James Irvin Kirkwood A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Soil Science 1962 PLEASE NOTE: . Figure pages are not’ original copy. They tend to "curl". Filmed in the best possible way. University Microfilms , Inc . Dedication This work is dedicated to my wife, Jessie, and to my children,.Alledi and Paul for earnestly and patiently waiting. ACKNOWIEDGME NT The writer is indebted and grateful to Dr. A. R. Wolcott for patient assistance in completion of this study and in preparing the manuscript. He is also grateful to the National Science Foundation, the John Hay Whitney Foundation, Dow Chemical Company, and to Dr. R. L. Cook for financial support which enabled the writer to prepare for, initiate, and complete the study. Appreciation goes to Dr. L. N. Shepherd, Dr. R. E. Lucas, and Dr. C. L. SanClemente, and all the others who contributed to the com- pletion of this work. iii TABLE OF CONTENTS mTRODUCTIONOOCOOOOOOOOOO0.00000000000000.0.0000000000000000.0.0.0.... LI'I‘ERATIIRE REVIEW.OOOOOOIOOOIOOOOOOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Effects of Agricultural Chemicals on Microbial Numbers and Microbial Activities................................. Effects of Agricultural.Chemicals.on.Plant.. Nutrition and metabOIismOOOO0.0.0.0....00......OOOOOOOOOOOOOOOOO. Nitrogen Nutrition...00.000.000.000...0.0.0.000...0.0.0.0000... Relationships Among Plant Nutrients As Influenced by the Form of Nitrogen Assimilated............... SOIUble saltSOOOOIOOCO0.00.00.00.00.COCOOOOOOOOOO0.0.0.0.0.0... Methods for Studying Soil Microbial POPUlation and ACtiVitieSOOOCOOOOOOOOO0.0.0.0...OOOOOOOOOOOOOOOOO Nitrogen Transformation........................................ Respiratory Measurements....................................... Quantitative Estimation of Microbial Numbers in Soil........... iMATERIALS AND.METHODS................................................ Field ExperimntSOOOOOCOOOOCOO...00......OOOOOOOOOOOOOOOOOOOOOOOO Descfi-ptionoOOOOOOOOOOOOOOOOOO0.00000.03.000.000.00.00.00.000. Sampling of Soils and P1ants................................. Laboratory Incubation Experiment................................. Preparation of Soils for Incubation.......................... Incubation Procedure......................................... Collection of Carbon Dioxide................................. Laboratory Assays................................................ Determination of Ammonium and Nitrate........................ Determination of Chlorine in Celery Tissue................... Electrical Conductance Measurements.......................... Dilution Plate Counts of Bacteria and Fungi.................. iv PAGE 10 14 15 15 17 20 22 22 22 25 26 26 29. 29 33 33 34 35 35 TABLE OF CONTENTS (Continued) RESULTS OF FELD STUDIESOOOOOOOOOO0..IOOOOOOOOOOOOOOOOOOOOOOO0...0.0. Fumigation Effects on Soil Nitrogen Transformations................ uncropped $011.0..OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO..0... cropped SOiIOOOOOOOOOOOOOOOO...0.0.0.0.0.0...OOOOOOOOOOOOOOOOOOOO Interactions of Fumigation and Nitrogen Carriers................. Interactions of Fumigation, Nitrogen Carriers and Tillage........ smry 0f Fmigation EffeCtsOOOOOOOOOOOIOOOO0.0.0....000......O. Field SCUdieS With N‘SERVEOOOOOOOOCOOIOOO0.0.0.0...0.00000000000000 Effects of Fumigation on Celery in 1959 and 1960................... Early GrOWthooooooooooooooooooooooooooooooooooooooooooooooooooooo Fina]- Yields.0.0.0.000...00......OOOOOOIOOOOOOOOOIOOOOOOO00...... Summary of Effects on Celery in 1959 and 1960.................... FIELD STUDIES WITH CELERY, SWEET CORN AND LETTUCE IN 1961............ Sweet Corn......................................................... Lettuce............................................................ Celery...........g................................................. Summary of 1961 Field Studies with CrOps........................... INCUBATION STUDY..................................................... Nitrogen Transformation............s............................... Microbial Numbers and Evolution of Carbon Dioxide.................. Summary of Incubation Results...................................... GENERAL DISCUSSION................................................... SUMMARY.............................................................. BIBIWWHYOOOOOOOOOOOOO0.0...0.0.0.000...OOOOOOOOOOOOOOOOOCOOOOO0.0 mmDHOOOO0.0.0..O0....00.0.0000....0.0...0.00000000000000000000... PAGE 36 36 36 36 40 47 52 56 6O ‘60 66 66 68 68 70 78 85 87 87 94 102 107 111 113 120 TABLE 1. 10. 11. 12. 13. LIST OF TABLES Soil treatments imposed on field moist Houghton muck prior to inCUbation000000000000000000000000000000000000000000 Nitrate nitrogen in Houghton muck planted to celery as related to aeration, fumigation and nitrogen carriers in 1960.00......0.00.00...OC.CO.OOOOOOOOOOCCOOOOOOOOOOOO0.... Ammonium nitrogen in Houghton muck planted to celery as related to aeration, fumigation and nitrogen carriers in 1960.00.00.000000000000.0.0....OOOOOOOOOOOOOOCOOOOOOOOO... Total mineral nitrogen in Houghton muck planted to ce1ery as related to aeration, fumigation and nitrogen carriers in 1960.-.0...OOOOOOOOOOOOOOOOOIOOOOIOOOOOOOOOOOOOOOOOOOOOOO. The effects of Telone and nitrogen treatments with ammonium sulfate and ammonium sulfate + 1.6% N-Serve, on nitrate levels in uncrOpped Houghton muck. 1961.......... The effects of Telone and nitrogen treatments with ammonium.sulfate and ammonimm sulfate + 1.6% N-Serve, on ammonium levels in uncropped Houghton muck. l96l......... The effects of Telone and nitrogen treatments with ammo- nium.sulfate and ammonium sulfate + 1.6% N~Serve on levels of total mineral nitrogen in uncrOpped Houghton muck. 1961.. Dry matter yield of celery 7 weeks after transplanting in Houghton muck as related to fumigation, aeration, and nitrogen source. 1960......0...’00......OOOOOOOOOOOOOOOOOOOO Percent dry matter in celery 7 weeks after transplanting as related to fumigation, aeration and source of nitrogen. 1960.0.0...00......OOOOOOOOOOOIOOOOOOOOOOOOO0.0... Percent chloride in celery 7 weeks after transplanting as related to fumigation, aeration and source of nitrogen. 1960.00.00.000000.00....OOOOOOOOOOOOOOOOOO00...... Electrical conductance of the soil solution in Houghton muck 6 weeks after transplanting ce1ery as related to fumigation, aeration and source of nitrogen. 1960........... Final yields of celery grown on.Houghton muck in 1960 as related to fumigation, aeration and source of nitrogen.... Yields of celery varieties, lettuce and sweet corn on Houghton WMCk as related to fumigations 19610000000000000000 vi PAGE 28 48 49 53 57 58 59 63 63 64 65 66 69 TABLE 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. LIST OF TABLES (Continued) Yields of celery varieties, lettuce and sweet corn on Houghton muck as related to nitrogen carriers. 1961...... Yields of sweet corn and foliar nutrient contents as related to fumigation. Houghton muck, 1961.................. Yields of lettuce and foliar nutrient contents as related to fumigation. Houghton muck, l961.................. Manganese contents and yields of lettuce as related to fumigation and nitrogen sources. Houghton muck, l961..... Yields of celery and foliar nutrient contents as related to fumigation. Houghton muck, 1961.................. Status of iron, manganese and phosphorus in ce1ery petioles in 1960 and 1961 as related to fumigation and final yields on Houghton muck............................ Geometric mean numbers of bacteria and fungi during incubation of Houghton muck as related to chemicals and incubation temperature................................... Distribution of significant F ratios among various coma parisons of the effects of tem erature and chemical treatments on total mineral nitrogen (NH4 plus N03”) in incubated Houghton muck................................................ Distribution of significant F ratios among various com- parisons of the effects of temperature and chemical treatments on levels of ammonium nitrogen in incubated Houghton muck................................................ Distribution of significant F ratios among various com- parisons of the effects of temperature and chemical treatments on levels of nitrate nitrogen in incubated Houghton WCk000000000000000000000000000000000000000000000000 Distribution of significant F ratios among various comparisons of the effects of temperature and chemical treatments on geometric mean numbers of bacteria and fungi in incubated Houghton muck............................. 121 123 125 127 PAGE 69 73 74 77 82 84 100 - 124 - 126 - 128 LIST OF FIGURES FIGURE 1. 10. 11. A single respiration unit showinng, 002- free-air inlet tube; B, respirometer jar containing soil samples in plastic cups; C, tube containing KI for removal of halogen impurities from the air flowing through the respirometer jar; D, tube containing Ag2804 to indicate when to renew KI; E, tube containing NaOH for collection Of c0200....IOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.00.... View of Operator adjusting the incubation apparatus in one Of the constant temperature roomSOOOIOOOCOOOOOOIOO00...... Effects of fall fumigation with Telone on accumulation of ammonium.and nitrate in uncrOpped Houghton muck in 1959 and 1960......I00.00.000.000.OOOOOOOOOOOOOOOOOOO....0...O Effects of fall fumigation with Telone on accumulation of ammonium and nitrate nitrogen in uncrOpped Houghton muck in 196100.00...O...0......OO...COOOOOOOOOOOOOOOOOOOOOO00...... Levels of nitrate nitrogen in fall fumigated and un- fumigated Houghton muck planted to ce1ery with and without supplemental ammonium fertilization........................... Levels of ammonium.nitrogen in fall fumigated and un- fumigated Houghton muck planted to celery with and Without supplemental ammnillm fertilizationo 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Levels of total mineral nitrogen in fall fumigated and unfumigated Houghton muck planted to celery with and without supplemental ammonium fertilization................... Effects of fall fumigation with Telone on the accumu- lation of nitrate as related to source of applied nitrogen. Houghton muck planted to celery, 1959, '60, '61............... Effects of fall fumigation with Telone on the accumu- lation of ammonium as related to source of applied nitrogen. Houghton muck planted to ce1ery, 1959, '60, '61.... Effects of fall fumigation with Telone on the level of mineral nitrogen (NH + plus N0 ') as related to source of applied nitrogen. Houghton muck, planted to ce1ery, 1959, '60, '61....0.0.00.0000...OOOOOCOOOOOOOOOOOOOCOO00...... Effects of Telone fumigation on accumulation of ammo- nium.nitrogen and nitrate nitrogen in Houghton muck as influenced by source of added nitrogen and aeration treatmentOOOOO0.000000000CODOOOOOO0.00...00.000000000000000... viii PAGE 31 32 37 38 39 41 42 43 45 46 51 LIST OF FIGURES (Continued) FIGURE 12. 13. 14. 15. 16. l7. 18. 19. 20. 21. 22. Effects of Telone fumigation on accumulation of total mineral nitrogen in Houghton muck as influenced by source of added nitrogen and aeration treatment............ Growth of celery six weeks after transplanting on fumigated (F) and unfmmigated (F0) Houghton muck with 50 pounds of nitrogen as ammonium sulfate (N1) applied at planting. 1960............................................ Growth of celery six weeks after transplanting on fumigated (F) Houghton muck with 50 pounds of nitrogen as calcium nitrate (N ) and as ammonium.sulfate (N1) applied at planting time. 1960............................... Response of sweet corn to fumigation under conditions of critically limiting manganese nutrition. Houghton Mek, 1961.0...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0...... Mbnganese deficiency symptoms in leaves of sweet corn grown on unfumigated (F1) Houghton muck in 1961. Normal leaves were from.plots treated in the fall with 48 gpa of Telone(E3)................................................ Utah 5270 celery two months after planting in unfumigated [FD and following fall fumigation with 32 gpa LP? and 48 gpa [i of Telone. Injury increased with fum gation level whe e NH4)2804 [hi] was used. Failure to respond to Ca(NO )2 NB was due to limiting Mn and P, and an interaction with increased availability of Fe in fumigated plots...0.00000COOOOOOOOOOOOOOOOOI...OOOOOOOOOOOOIOOOOOOOOOOOO Celery yields as related to the ratio of iron to phosphorus in leaf petioles six weeks before harvest on Houghton mmck with variable fumigation and nitrogen treatment. 1961........ Levels of ammonium.and nitrate nitrogen in unfumigated organic soil and in soil previously treated with N-Serve, chloropicrin, Vidden-D, Telone and Fumazone, during incubation under three temperature regimes.................... Changes in soil nitrate following chemical treatment of Houghton MCROOOOOOOOOOCOIOOOOOOOOOO.0.OOOIOOOOOOOOOOCOOOOOOOI. Changes in total mineral nitrogen following chemical treatment Of Houghton mUCkoooooooooooncoco-00.000000000000000. Levels of ammonium.and nitrate nitrogen in unfumigated muck and in muck previously treated with three levels of Fumazone, during incubation under three temperature regimes... ix PAGE 54 61 62 71 72 8O 83 88 91 92 9S LIST OF FIGURES (Continued) FIGURE PAGE 23. Effect of chlorOpicrin on the evolution of carbon dioxide and the numbers of bacteria (B) and fungi (F) in Houghton muck at three incubation temperatures............. 97 INTRODUCTION Numrous chemicals have a marked suppressive effect on microbial numbers and activities in the soil. Recognition of this fact led to the extensive use of carbon disulphide as a soil fumigant in EurOpe near tl'a close of the last century. After World War I, the merits of chlo- ropicrin as a fumigant were established, thereby increasing the interest in tin use of volatile chemicals as soil fumigants. The primary objective of soil treatment is to kill pathogenic or- ganisms. The chemicals being used, however, are frequently non-specific. As a result, non-Spore forming, nitrogen—fixing and nitrate -forming bac- teria, as well as parasitic organisms are destroyed. Nitrification is strongly inhibited. The spore forming ammonifiers increase due to this partial sterilization. Amonification, unaccompanied by nitrification, results in net accumulation of ammonium. Accumulation of ammonium may continue for weeks, capacially in soils high in organic matter. The intensity and duration of the suppression of nitrification vary with the chemical and the numerous mechanical, soil and environmental factors which influence the effectiveness of the chemicals as fungicides or nematocide s. Frequently crap injury results from soil fumigation due to the al- teration of nitrogen nutrition arising from impaired nitrification. Whether or not the ammonium nitrogen which accuImJlates in the soil after fumigation will be taken up by plants and assimilated efficiently in growth will depend on the crop, soil pH and the supply of other cations, daylongth, light intensity and the level of carbohydrates in the plant, as well as the form and placement of supplemental nitrogen fertilizer. its absorption by plants of nitrogen primarily in the ammonium form is l 2 accompanied by a tendency toward rejection or immobilization of other cations, such as potassium, calcium, magnesium, and iron, and enhanced absorption of anions, notably chloride and sulfate. This may give rise to injurious plwsiological derangements in some plants. In the alkaline range, dire ct toxicity to the roots of a large number of plants may result from the presence of ammonia (NHB) in the soil solution. In laboratory studio 3, the large quantities of ammonium which accumulate in organic soils following treatment with various fumigant chemicals are converted rapidly to nitrate as the inhibitory effect of the fumigant on nitrification wears off. These results from laboratory experiments cannot be projected directly to field conditions to explain why crap injury is sometimes associated with one chemical and not with another, or why injury from the same chemical may occur in one location or season and not in another. The field research reported here was undertaken with a view to relating seasonal patterns of ammonification and nitrification in organic soil to fumigation treatment and climatic factors on the one hand and to crop response on the other. Laboratory studies were also conducted to determine the influence of temperature, as one climatic factor, on microbial response to fumigation treatment of crganic soil. LITERATURE REVIEW Effects of Agricultural Chemicals on Microbial Numbers and Microbial Activities Some of the earlier investigators advanced many speculative theories in tin search for an explanation of the increases in plant growth fol- lowing soil treatments with heat or fumigants. These have been well reviewed by DuBuisson (16) and Kopeloff and Coleman (35). DuBuisson indicates that the first record of an "antiseptic" soil treatment seems to be that of a German, 0. Oberlin, around 1905. After using carbon disulfide as an insecticide in some of his vineyards that were attacked by Plzgy'lloxera, he noted a marked increase in the growth of vines. Hewhall (1:8) indicates, however, that we are indebted to earlier workers in the late 1800's for a number of well established facts. Among these are that treating eons with heat or "antiseptics" results in the following: (a) Non-spore -fonning, nitrogen-firdng, nitrite -foming and nitrate forming bacteria as well as parasitic organisms are destroyed, and nitrification is thereby inhibited. The spore -forming amnonifiers which sur- vive increase in number and armnonification goes on almost uninterrupted for weeks, especially in soils high in organic matter. (b) Soluble salts are often liberated, in some cases chlorides and sulfates of ammonia, and sometimes soluble manganese. Effects of partial sterilization of the soil by heat or fumigants remained an enigma for several years. The first major breakthrough cam h with the publication of a series of three papers by Waksman and Starkey (78). Conclusions reached by them were: (a) Partial sterilization of soil by steaming brings about 03) (c) (d) (e) (f) a chemical change in the organic matter of the soil, making it more available as a scurce of energy for microorganisms. A large prOportion of the soil fungi are killed as a result of partial sterilization. The rapid increase in numbers of bacteria in the soil is at the expense of the organic matter made available. The actual amount of ammonia formed in partially sterilized soil is determined, not by the numbers of bacteria and fungi developing in the soil, but by the abundance of organic matter. The protozoa are suppressedin partially sterilized soil, but become active again long before bacterial numbers decline markedly. The more rapid the rise in bacterial numbers and the greater the maximum number reached, the sooner will the decline set in. This is true, also, of fungi. These fluctuations in numbers are related to the supply of readily available organic substrates released by the steam treatment and their subsequent exhaustion. In their work using low and high dosages of chloropicrin, Stark, Smith, and Howard (65) concluded that since the total amount of nitro- gen made available for plant growth was not increased except at high dosages then the increased plant growth obtained from low dosages could 5 not be accounted for solely by the hypothesis that more nitrogen was made available for plant growth. Because of the high cost of chloropicrin and its lachrymatory, phytocidal and corrosive properties, other safer and cheaper soil fumi- gants were needed.. The practice of soil fumigation was stimulated with the discovery of the nematocidal properties of D-D mixture (SO-SO mixture of 1,3 dichloropropene and 1,2 dichlorotpropane) by Carter (10). This led to research with this and other fumigants using chloropicrin as the standard. Tam and Clark (69), working with pineapple plants, showed that increased growth and nitrogen composition were associated with soil fumi- gated with chloropicrin. This was related to restriction of the plants to predominantly ammonium nutrition as compared to predominantly nitrate nutrition in unfumigated soil. Tam (68), Thiegs (71), and Winfree and Cox (82) observed the same pattern of ammonium accumulation, followed by delayed but rapid con- version to nitrates about two months after treatment of soils with cl'emical fumigants. Concomitant development of the insecticidal and herbicidal program created increased interest in the effect of chemicals such as D.D.T. (dichlorodiphenyl-trichloroethane) and BHC (benzene hexachloride) on specialized activities of microorganisms in soil. Wilson and Choudri(8l), in laboratory studies, showed that the mo in amounts considerably exceeding practical field applications had no significant effect on development of bacteria and molds, nor on physiological activities important to soil fertility. Bollen et a1. (6) in other studies using various insecticides in 6 the field concluded that the observed stimulations and inhibitions of microbial activities were not intensive enough to materially influence fertility of the soil. Kidson and Stanton (32) and Kidson (31) observed a long but not very pronounced "partial sterlization effect" when soil was treated with D.D. or chloropicrin. Smith et al (61;) checked the effect of 2,h-D (2,h-Dichloro-phenoxyacetic acid) on the soil. Low concentrations had no visible effect but concentrations of 100 ppm or more displayed a weak sterilization effect without an appreciable increase in ammonia production. The nitrifying organisms proved to be very sensitive to 2944-13. The results obtained by Koike and Gainey (33) are slightly dif- ferent;2,h—£-D was found to exhibit a marked bactericidal action even in concentrations applied for weed control, and to cause afterward a tem- porary increase in bacterial numbers and in ammonia content in the soil. The nitrification process was suppressed for a period of two to four months. Smith and Wenzel (63),however, found a marked suppression of the microflora and especially of the nitrifying bacteria with BHC. Chlordane and DDT were less harmful for microbes when applied in practical dosage. Smith and Bell (62) reported some observations on light sandy soils in Florida. D.D., chloropicrin, D.D.T., and 2, h-D all exhibited for a short period a partial sterilization effect. D.D.T. and 2,)4-D had the strongest effect, lasting for 70 days. Koike (3h) conducted laboratory emeriments to determine the effects of eight fumigants on the nitrification of (NHh)2 801: and NHhOH. Results indicated that under the conditions of the experiment the chemicals markedly inhibited nitrification from four to eight weeks. 7 Wolcott et al (83), using Telone as a fumigant applied at rec- onmrended nematocidal rates, found nitrification to be delayed in the laboratory about eight 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 warmed to 60°F. in the spring. Sabey et al (58) initiated a study to determine the influence of temperature and initial population of nitrifying organisms on the max- imum rate and delay period. In soils incubated at field capacity, the maximum nitrification rates increased from immeasurably low values at 0°C to as great as 900 ppm per week in some soils at 25° . Delay periods decreased from about 32 weeks to less than one day, as temperatures in- creased from 0 to 25° C. Increase in initial population caused decreases in delay but did not appreciably affect the maximum rate above 10° C. There are numerous contradictions among published reports dealing with the microbiological effects of these fungicides, insecticides, and herbicides. This is to be expected, considering the varied circum- stances under which the work was carried out. Effects of Agricultural Chemicals on Plant Nutrition and Metabolism Nitrogen Nutrition Tam (68) found that pineapple plants restricted to an ammonium nutrition due to D-D soil treatment were high in nitrogen, dark green, broad-leaved, soft, and succulent, as compared to slower growing, yellowish plants on nitrate nutrition. Wolcott et a1 (83) in work with Telone (a mixture of dichlorOpropenes) reported that celery seedlings appeared to be unable to utilize amonium 8 effectively at pH 6.3 and required nitrogen in the nitrate form during their early growth. Excessive nitrate during subsequent growth and particularly during the period immediately preceding harvest was detri- mental to yield. In his work With tomato plants Thiegs (Ti) found that the presence of nitrates seemed to decrease armnonium toxicity. As a result, he pro- posed that nitrate applied as fertilizer might counteract the detri- mental effect of too much ammonium on ammonia sensitive plants. How- ever, Winfree and Cox (82) felt that although it may be true that nitrate may counteract detrimental effects of too much ammonium, their data supported the conclusion that the benefit from applied nitrate came from substituting the more available nitrate for ammonium nitrogen. Considerable research has been done with tobacco and potatoes and the interrelations of their nutrition and metabolism to soil fumigatidn treatment. The growth and development of tobacco is known to be ad- versely effected when the major portion of the available nitrogen remains in the ammonium form. The works of Evans and Weeks (17), Gilmore (21), and 'MHav'oy‘ (146) have indicated that the yield and percentage of dry matter in tobacco is lower when grown in media in which all the nitrogen is in the ammonium form than when grown in media containing nitrate nitrogen. Plants from ammonium cultures had a high percentage of total nitrogen, amide nitrogen, nicotine and pigments and a lower percentage of sugars and organic acids than did plants from the all nitrate cultures. McCants et al (15) have presented data which showed that certain of the soil fumigants currently used for nematode control can have a sig- nificant influence upon the response of tobacco to the applications of nitrogen in the ammonium or the nitrate form. There was a greater yield response to nitrate from the fumigant treatments which had the most suppressing effect on nitrification. However, where nitrification was 9 inhibited and ammonium applied, there was a high ammonium and halogen content of leaves. This was associated with leaf abnormalities and stunting of the plants. Grogan and Zink (22) have shown that free ammonium hydroxide or free ammonia (NHB) may cause severe injury to roots and tops of lettuce plants in California fields. Application of organic nitrogenous fertilizers to cold waterlogged soil, or the use of aqua or anhydrous ammonia, produced the injury. Ammonium sulfate, amonium nitrate, or calcium nitrate caused relatively little damage. Hoff and Newhall (26) reported that a severe root rot of head lettuce on the acid mucklands of New York could be reproduced in sand culture and steamed muck by an excess of ammonium. VLorenz et al (140) reported root injury and reduction of yields in spinach, radish, peas, cabbage, lettuce and onions following the application of aqua ammonia or of anhydrous ammonia. Ammonium sulfate, in contrast, increased yields in comparison with the unfertilized check plots. Nightingale (1:9) (50) has given an excellent review of papers on nitrogen metabolism. Most of them indicate that neither nitrite nor ammonium nitrogen can accumulate in plants without causing damage . Nitrate nitrogen can be stored in considerable quantities by most plants without injury. Through the use of leHB’ Vickery et al (77) showed that nitrogen of NlSH3 is incorporated by tobacco plants into amide s, amino acids, and proteins. The results of MacVickar and Burris (142) showed that glutamic and aspartic acids become highly labeled with NIS from 15£13. Their results suggests that glutamate and aspartate are tie primary products of the assimilation of ammonium by plants. Rautenan (56) con- firmed this when he showed that glutamate, aspartate, their amides, and lO alanine are the major initial products of ammonium uptake in plants. Grogan and Zink (22) have expostulated that if assimilation or detoxification of ammonium or nitrite keeps pace with absorption so that no accumulation occurs in the plant, injury is prevented. How- ever, if ammonium accumulates because of slower assimilation at low pH values or because of too rapid absorption at high pH levels, injury to the plant may result. Lack of carbolrwdrate s, which are necessary for the detoxification of ammonium by conversion to amines, may also result in damage from excessive ammonium uptake. Response of plants to nitrate and ammonium varies with a number of environmental factors. Webster (80) has indicated, for example, that pH of the nutrient medium exerts a considerable influence on the relative utilization of nitrate and ammonium. Arrington and Shiva (2) found that a low pH favors nitrate uptake, while a high pH favors the uptake of ammonium. Tiedjens and Robbins (73) reported that both absorption and assim- ilation of nitrate nitrogen was most rapid from acid media, whereas ammonium nitrogen is assimilated most rapidly from alkaline media. Other conditions of the soil such as aeration, temperature, and soil moisture have their effect upon nitrification also. Relationships Among Other Plant Nutrients as Influenced by the Form of Nitrogen Assimilated As has been stated, the ability of plants to take up or assimilate either NHh-r or HOB“ depends upon other nutritional factors which are strongly controlled by pH. The availability of many other nutrients is controlled by pH, and when the cation-anion balance is altered the metabolic patterns of the plant are likely to be altered. 11 The absorption by plants of nitrogen primarily in the ammonium form.is accompanied by a tendency toward rejection or immobilization of the cations (K, Ca, Mg, and Fe) and enhanced adsorption of anions (01",soh'). Bear (h) observed that the cation-anion equivalent ratio in plants can be represented as Ca:g§vK:ga a a constant. From the equation it follows that increased absorption of any one cation results in reduced absorption of some other cation or cations or increased uptake of one or more anions. Increased absorption of an anion.results in reverse effects. These rela- tionships may give rise to injurious physiological derangements in some plants. ‘Wallace et a1 (79) have shown evidence that increased chloride absorption results in decreased nitrogen in plants. The exact function of chloride in.plant nutrition is undefined, although evidence has been forwarded that it interferes with carbohydrate metabolism, modifies chlorOphyll content and affects relations between cation and anion ab- sorption. Baslavskaja (3) has shown that large doses of chloride lower the content of chlorophyll in plants. The most apparent effect observed by Slatz et al (60) was the large increase in chloride content of plants associated with increasing chloride level in the nutrient medium. This increase did not cause a corresponding decrease in the content of phos- phorus or sulfur. Buchner (8) showed, in.water culture experiments on various crops, that a shift in carbohydrate fractions associated with chloride ions was dependent on the kind of nitrogen supplied. Content of reducing sugars and sucrose declined much.more as a result of chloride supply'with ammonium than with nitrate nutrition. Absolute proportions of starch and total carbohydrate content were less influenced. . 12 Harvard et a1 (25) made a study of the role of chloride and sulfate anions in the nutrition of Irish potatoes when different forms of nitrogen were used. Increasing Cl" and decreasing 50h” concentrations in the presence of NH]: resulted in marked reduction in the yield of fibrous roots. Addition of Cl“ generally resulted in decreases in the percentage of dry matter in the tape. This effect was more pronounced in the presence of NEH: The chloride content of the leaves was markedly increased in the presence of NHh+. Vladmirov (76) reported that chloride and nitrate in culture solu- tions stimulated production of organic acids in tobacco, while sulfates and ammonium nitrogen retarded their accumulation. Corbett and Gausman (l2) concluded that chloride may affect potato tuber quality by affecting the uptake of phosphorus. Kretschmer et a1 (36), in their work on chloride versus sulfate ions in nutrient-ion absorption, found that the most consistent response phenomenon involved an inverse relationship .between nitrogen and chloride contents in plants. Tester (70) using ammonium chloride as nitrogen fertilizer studied the effects of the chloride ion on yields and uptake of nutrients by crops. In general summary, he found that there appeared to be no great problem associated with the use of ammonium chloride for grain craps. Even though chloride accumulated in plants, there was little evidence that its interference with absorption of other essential anions was of any importance. Another element that is affected by fumigation is manganese. Over three decades ago, Gilbert et al (20) found conclusive evidence that, through heavy liming, soil conditions are created in which many plants 13 develop chlorosis. This chlorosis was prevented by application of manganese sulfate. Alexander (1) points out that manganese occurs in the soil in the divalent and tetravalent forms. The exchangeable divalent cation is water soluble, while the tetravalent cation is essentially insoluble, occurring as 141102. The ion that predominates is a function of pH. However, since there are manganese oxidizing microorganisms in the soil, they can also produce the insoluble form of manganese. Lingle and Wright (38) studied the growth and manganese content of onions as influenced by source and rate of applied nitrogen, lime, and soil fumigation. The growth of onions on very acid coastal California soils was inversely related to the manganese content of the tissue. The manganese content was directly related to the soil pH 'as influenced by the rate and source of applied nitrogen, lime, and soil fumigants. It was also found that heavy nitrogen applications increased the man- ganese content independently of the effect of nitrogen on soil pH. Soil affected manganese uptake differently depending on lime application. Sherman and Harmer (61) were the first to recognize the need for manganese on organic soils in Michigan. McCall and Davis (M4) used manganese carriers effectively to increase the yield of onions on or- ganic soils by foliar spraying and dusting, and by soil application. Lucas and Davis (bl) have indicated that the availability of man- ganese is influenced more by soil reaction than any, other plant nutrient. Total manganese content in organic soils is of little value in predicting the need for manganese fertilizers. Availability decreases above pH 5.5. Shickluna and Davis (50) showed that the manganese content of onion tops dropped from 1125 ppm to 1414 ppm when the pH of an organic soil was it; raised by liming from 14.1 to 5.6. Another peat showed a drop of man- ganese in onion tops from 875 ppm to 25 ppm when the soil pH was raised from 14.9 to 7.0. Manganese toxicity is often credited with causing poor growth in very acid soils, emecially if fumigated or sulfured. Soluble Salts Steam treatment of soil may release enough adsorbed salts to produce plant injury. The soluble salts may be, in some case 3, chlorides and sulfates of ammonium, and sometimes soluble manganese . (’48) A study was made by Markle and Dunkle (143) on the use of the soluble salt content of greenhouse soils as a diagnostic aid. Some observations and conclusions were: (a) There is a close relationship between the total soluble matter and the electrical conducoance of aqueous soil extracts. (b) There is a close relationship between the inorganic soluble matter and the electrical conductivity of aqueous soil extracts. (c) The electrical conductivity of the extract is a reliable measure of the soluble matter content . Specific conductance has been used as a criterion for determining the range in soluble salt content over which plants will grow satis- factorily. In correlating specific conductance with growth, a range between 61 and 106 x 10.5 mhos would appear favorable for celery plants, the critical electrical conductivity for a 1:5 (V/V) soil: water extract being about 110 x 10'; mhos (15). Methods for'Studying Soil Microbial Population and Activities Nitrogen Transformations Harmsen and Van Schreven (2h) have given a short review of the dif- ferent methods used for determining the rate of nitrogen transformation in the soil. They divide the methods into three groups, field trials, pot experiments, and different procedures for incubation of soil samples under laboratory conditions. Field trials, being the most empirical, provide reliable results, but they are laborious, time- and space—con- suming and subject to many external influences such as the effects of weather, season, crop grown, etc. Pot experiments are similar to field trials but the external conditions can be controlled and standardized better. Incubation experiments have numerous limitations, also. By using both field and incubation data, however, the experimenter can develop a practically'useful prediction of the pattern of nitrogen mineralization and transformation to be expected under a given set of field conditions. It is generally accepted that organic nitrogen must be mineralized before it is available for plant uptake. In normal soil, nitrate is the end product of mineralization. The transformation of organic nitro- gen to nitrate takes place in three steps by two general types of processes: Nitrifipatipn . r . \ rganic N-—-—>Ammon1a ——9Nitrite——+Nitrate Ammonification Soil microorganisms involved in the decomposition of organic matter set free more nitrogen than they are able to assimilate into their own protOplasm. Under aerobic conditions the excess nitrogen appears in the 15 16 soil as ammonium. In the nitrification process nitrate is formed from the ammonium. Harmsen and Van Schreven observed that the majority'of earlier investigators determined the N03, - N or the total mineral nitrogen (NO " — N + NH}: - N + NO " - N) only in the field. They deduced from 3 2 these periodically collected data their conclusions about the changes in the mineral nitrogen content as influenced by season, crops, climate, moisture, temperature, structure of the soil, and its total nitrogen and humus content. With the introduction of incubation methods, the release of mineral nitrogen during incubation under standardized conditions could be fol- lowed: Alexander (I) noted that early microbiologists chose to limit their analyses to ammonium, the first inorganic product. The usually rapid conversion of ammonium to nitrate in soil invalidated the deter- mination of ammonium alone as a measure of mineralization. Nitrate production has also been used as a mineralization criterion. This usually'provides a valid measure of mineralization since ammonium and nitrite accumulate only under abnormal conditions. In some situations, however, ammonium and nitrite may accumulate, or denitrification losses may occur, so that the rate of accumulation of nitrate does not always reflect the rate of mineralization. Furthermore Jansson (29) has shown by'NlS tracer studies that extensive cyclical reautilization (immobili- zation) of mineralized nitrogen occurs. Usually it is not feasible to account for nitrogen recycled or that lost by denitrification. The most acceptable measure of mineralization is the total of all mineral products, - ammonium, nitrite and nitrate. The rate of mineral nitrogen accumulation is not an absolute measure of the rate of release of nitro- 17 gen from humus, proteins, nucleic acids or related materials. It is a measure only of net mineralization. Respiratory'Measurements The rate of nitrogen mineralization in the soil can be estimated by measurements of CO2 evolved by a sample during incubation. However, this is only an indirect measurement and may be entirely misleading if organic materials low in nitrogen are being decomposed, with extensive concomitant immobilization of nitrogen. Its more usual application is as a measure of microbial activity. Starkey (66) in evaluating the usefulness of the C02 measurement stated that under aerobic conditions, with a mixed microbial population, it is the principal carbonaceous end product of decomposition. Deter- minations of 002 liberated from soils may be interpreted as indicating fairly accurately the speeds of decomposition of organic materials in soils. He felt this method to be much more accurate in estimating some of the influences of plant development on soil organisms than are plate counts. Russell (57) stated that the number of microorganisms in a soil gives no direct measure of the activity of the microbial population. The activity of the total papulation is not a concept that can be given a quantitative definition. For many purposes,however, it is a useful concept. Total activity can be estimated by the amounts of either 00 2 or heat evolved by the population. In general, 002 evolution does” increase as the number of microorganisms increases. Gainey (18) studied the parallel formation of 002, NHh", and N03 in soil, air being drawn through the soil. There was a similarity 4. between the curves for 002 and NHL release from soils incubated under 18 similar conditions. Insufficient moisture retarded both 002 and ammonium formation. Insufficient aeration also caused a depression or marked delay in 002 and NHh+ production. Where water content was varied, rate of nitrate accumulation was directly proportional to moisture con- tent, the maximum rate not being reached until the maximum moisture that would be retained by the soil was reached. Where aeration was varied, insufficient aeration retarded the initial accumulation of nitrate, but after nitrification became active in all samples the rate of accumulation was inversely proportional to aeration. Corbet (13) derived the following formula to describe, mathe— matically, the evolution of 002 from a soil sample, under laboratory conditions and at constant temperature: PW“ where: F=yield of CO in unit time at the begigning of the experiment y=tota1 002 evolved in time t. m=constant less than 1, representing the progressive retardation in rate of gas evolution due to the restrictive experimental conditions of incubation. This expression was shown to fit the pattern of CO2 evolution from pure cultures of actinomycete species during the decline phase of the generalized bacterial growth curve. Since the same expression could be applied to the data from incubated soil samples, it was deduced that F represents the activity of the climax population present in the soil at the time of sampling in the field. Vandecaveye (7h) (75) in studies of microbial activities in soils concluded that, since increased rates of 002 production did not coincide 19 with increased microbial numbers, - the maximum CO production preceding 2 maximum number of microbes by 2-3 weeks, - then 002 production is by no means an accurate index of microbial numbers. Less and Porteous (37) stated that, in practice, the direct measurement of carbon retention in a medium naturally so highly organic as soil is difficult if not impossible. If assimilation is to be measured in a soil, it must be measured indirectly; The method they used involved the titrimetric estimation of 002 released from a known weight of an organic compound percolated through a soil. The decom- position of the perfused compound was measured by the surplus of 002 released over that released by the soil alone where no carbonaceous substrate was added to the perfusate. The difference between carbon added and carbon recovered as 002 represented carbon retained, or im- mobilized, in the soil. Norman and Newman (52) collected 002 by diffusion into alkali in test tubes inserted with the soil samples in sealed jars. Replicate soil samples were sacrificed periodically to follow chemical and physical changes in the soil itself and changes in microbial.numbers. The authors concluded that no single measurement could be used to adequately' char- acterize microbial activities in soils. However, the pattern of CO2 evolution over a period of time does provide a composite picture of the behavior of the organisms present acting on the substrates available under the environmental conditions that prevail. Delay periods and the cumulative total 602 evolved over a period of time can.be used to make inferences about the size of the initial population and the biochemical capabilities of the population which develops during incubation. Damirgi (1h) used the "simultaneous absorption" method described by the previous authors for studying microbial pOpulations and activity 20 in soils of a Prairie—Forest biosequence. Only small differences in 002 evolution were encountered between soils when incubated alone. This indicated a basic similarity in microbial activity in soils. No correlation appeared to exist between initial microbial numbers and total 002 evolved over 2h days from unamended soils. Stotzky and Norman (67) studied the influence of several environ- mental factors on glucose decomposition in soil. Carbon dioxide evolution, substrate disappearance, formation of intermediates, and changes in soil pH were followed by periodic assay over intervals up to h8 days. Although good correlation was obtained between these parameters, microbial activity was not directly correlated with the numbers of bacteria and fungi estimated by dilution plating. High res- piratory quotients were obtained during the early part of the incubation. Maximum numbers of bacteria and fungi deve10ped after the rate of 002 evolution had decreased. A sequential devolpment of microbial popu— lations differing qualitatively was indicated, with the early popu- lations being characterized by a dominance of anaerobic types. Quantitative Estimation of Microbial Numbers in Soil According to Thornton (72), no fully'satisfactory counting methods have been devised for the quantitative determination of microorganisms in soil. Colony counts from platings of diluted soil suspension have been the standard method for bacteria, actinomycetes, and fungi. Thornton indicates that one of the defects of this method is that no one medium is suitable for all the nutrionally varied organisms present. Wbrk by Lochhead and Chase (39) showed that, just for bacteria, a quite complex medium was required that included unknown growth substances from yeast 21 extract and soil extract. On the other hand if the medium used for plating was too rich, competition between organisms on the plate reduced the colony count. In the case of fungi, there is additional uncertainty with regard to plate counts, because there is no way of knowing whether a colony is derived from a Spore or from a fragment or from a clump of mycelium. Actinomycete colonies are usually'counted along with bacterial colonies because a positive and complete separation of bacteria and actinomycetes is impossible on the basis only of colony characteristics. Russell (57) offers as a major defect in the method that it gives only limited information about the reactions the various bacteria actually'carry out in the soil, since bacteria can grow on organic sub- stances in pure culture that they would be unable to use in the com- potitive environment of the soil. In spite of the fact that plate counts inherently create uncertainty as to the absolute numbers of organisms in soil, they do have definite value (72). They are quite useful in comparing estimates made by the same technique from a number of soil samples. Hence, it is possible to demonstrate differences in number of organisms in soils variously treated. Johnson et a1 (30) have given details of this method and various modifications that can be used. MATERIALS AND METHODS Field Experiments Description Field studies in 1959 and 1960 were directed towards evaluating the effects of fumigation with Telone (l, 3 dichloropropene) on seasonal patterns of nitrogen transformation in crOpped and uncropped muck, and on celery yields. To eliminate nematodes as a factor in celery response, areas were selected on Houghton muck at the Michigan State University Muck Experimental Farm, Bath, Michigan, where previous crop performance had shown no evidence of nematode infestation. Houghton muck is an organic soil containing 85 percent organic matter. It was developed from organic deposits more than b2 inches deep. The pH averages 6.0 to 6.3 with a range of 5.0 - 7.0: Depending upon the crop, major and minor nutrients must be added. The water table at the muck experimental farm is controlled by a combination drainage and water level contrdl pumping system. In 1959, a split-plot design in four replications was used involving two levels of fumigation (none and 32 gpa Telone) as main plots, and no nitrogen or nitrogen applied as ammonium sulfate, ammonium nitrate or calcium nitrate as subeplots. The fumigation treatment was made by injection, using a tractor'mounted applicator, October 2h, 1958. The entire experimental area was then aerated two weeks later by deep cul- tivation and again by'plowing prior to planting celery on May 7, 1959. A basic fertilizer application of 1500 pounds per acre of 0—10-30 was plowed down. Nitrogen treatments were made in a split application of 50 pounds nitrogen at planting time and 50 pounds sidedressed on July 7. A sub-plot not planted to celery and receiving no nitrogen was included. 22 23 A split-split—plot design in h replications was used in 1960. Main plots were unfumigated or fumigated with 32 gpa of Telone on October 26, 1959. The entire experimental area was immediately'cultipacked. One- half of each main plot was aerated by plowing just before transplanting celery, Utah 5270, May 5, 1960. All plots were subjected to three very shallow sweep cultivations during the growing season. weeds which escaped cultivation were pulled by hand. All.plots in 1960 received 1500 pounds per acre of 0-10-30 before planting. Sub-subeplots received 50-pound sidedressings of nitrogen at planting and on June 2h. Nitrogen was applied as calcium nitrate, ammonium nitrate, or ammonium sulfate. A fourth sub-SUb—plot received no nitrogen and a fifth sub-subeplot was uncropped with no applied nitro— gen. Both in 1959 and 1960, ammonium and nitrate nitrogen were determined on field fresh soil samples taken periodically through the growing season, by methods to be described later. In 1960, soluble salt determinations were made on June 20. Estimates of dry matter and chloride content in celery also were made at this time. Final yields of celery were taken both years . Field studies in 1961 were directed towards evaluating the effects of recommended and excessive rates of Telone fumigation on seasonal patterns of nitrogen transformation in cropped and uncropped muck and on yields of corn, celery, and lettuce. Soil population studies by J. A. Knierim, Nematologist, Entomology Department, Michigan State University, showed no evidence of parasitic nematodes in the selected plot area, which was located again on Houghton muck at the Michigan State University Muck Experimental Farm. 2b A split-split-plot design of four replications was used in 1961. Main.plots were unfumigated or fumigated on October h, 1960, with 32 gallons per acre and h8 gallons per acre of Telone. The entire ex- perimental area was cultipacked as in 1959. The entire area was aerated by plowing on April 1h, 1961. subeplots were either uncropped or planted to lettuce, ce1ery or sweet corn. Lettuce was planted with 800 pounds 0-10-20 plus l/h% boron + l/h% capper + 2% manganese banded 2 inches below the seed and 1/2 inch to the side. The variety used was Great Lakes 659. Two celery varieties, Utah 5270 and Spartan 162, were planted on two rows of each celery subeplot on May 11 and May 17 respectively. Basic fer- tilizer used was 500 pounds of 0-10-20 before planting. CrOpped and uncrOpped plots were further subdivided for broadcast nitrogen treatments. Subeplots of lettuce and uncropped sub—plots received 50 pounds of nitrogen as a broadcast application on April 27, 1961. The nitrogen was applied to lettuce plots as calcium nitrate, ammonium sulfate, and ammonium sulfate treated with 1.6% N-Serve)‘ The uncropped plots received nitrogen as ammonium sulfate and treated ammonium sulfate. Nitrogen was sidedressed on celery and sweet corn sub-sub-plots on May 26 at the rate of 50 pounds per acre as ammonium sulfate and calcium nitrate. A treatment involving no supplemental nitrogen was included on all three crOps. Soil samples were taken.periodica11y from all replicates of all treatment combinations beginning April 5 and continuing through September w 1 N-Serve is the trade name for 2-chloro-6 (trichloromethyl) pyridine, a nitrification inhibitor manufactured by the Dow Chemical Company, Midland, Michigan. 25 13. Tissue samples were taken periodically through the growing season for all crops. Harvest and final yield measurements were made for lettuce on July 13, for celery on August lb and for sweet corn on August 25. Sampling of Soils and Plants As indicated in the previous section soil samples were taken peri- odically during the field experiments. Twenty soil cores to an eight inch depth were composited per plot. These were passed through a four- mesh screen, thoroughly mixed and sub-samples taken. Soil samples in 1959 and the April and May samplings in 1961 were frozen immediateLy and stored in a deep freezer for later analysis. Aliquots of all later samplings in 1961 and all samples taken in 1960 were extracted immedi- ately for determination of ammonium and nitrate nitrogen as described in a later section. The tissue samples taken of celery in June, 1960 consisted of five whole plants per plot; in 1961, ten petioles per plot were taken peri- odically through the growing season. Prior to thinning, ten whole lettuce plants were taken per'plot for tissue analysis in 1961; in later samplings ten to twenty'leaves were taken. In the case of corn, tissue samples in 1961 consisted of ten basal midribs per plot. These tissue samples were dried at a temperature of 7o-80° c. and stored in paper sacks. For the present study, samples from selected sampling dates were analyzed in the De- partment of Horticulture for N, P, K, Ca, Mg, Mn, Fe, Cu, Zn, Mo, B, Na and Al.2 Nitrogen was determined by'Kjeldahl procedure and potas- 2 Under the supervision of A. L. Kenwortby and S. J. Gamble. 26 sium by flame photometer. The remaining elements were determined spectro- graphically, using a photoelectric spectrometer. Laboratory Incubation Experiment .Most of the fumigant chemicals used extensively in commercial agri- culture have been compared in incubation studies with regard to their immediate and residual effects on microbial numbers and activities. None of the reported studies, however, have taken into account the sequence of soil and climatic conditions which intervenes between fall fumigation and the planting of early spring crops. The objective of the laboratory incubation studies reported here was to compare the effects of several fumigant chemicals on microbial numbers and activities in organic soil under moisture and temperature conditions which might be expected to prevail for significant periods of time in the field during the fall and spring months. Preparation of Soils for Incubation Virgin, uncropped, unfertilized Houghton.muck was taken June 21, 1961, one day after two days of rain totalling 0.85 inches. At this time, the soil contained 263 percent moisture. Although wetter than normal for the growing season, it was not saturated. Prior to incubation, the field moist soil was fumigated in S-pound ' lots (oven dry basis) with 1/2, 1 and 2 times the recommended appli- cation rates of Telone, ViddenéD and FUmazone. In addition to these treatments, a check, and a reference chemical, chloropicrin, were used. The nitrification inhibitor, N-Serve, was included as a twelfth treatment. After addition of the volatile chemicals, the 5-pound lots of soil were sealedfixl plastic bags for an.exposure period recommended by the manu- 27 facturer. Following exposure, water saturated air was forced through the samples for the minimum aeration period recommended by the manu- facturer as necessary before shallowrooted crops may be planted. Chemical identification of the materials used and their rates of application, eXposure times, and aeration periods are shown in Table 1. Soils were treated at such times as to allow for completion of the indicated exposures and aeration at the beginning of incubation, on July 12, 1961. All of the lots of soil were amended with 100 ppm nitrogen (50 pounds N per acre) as ammonium sulfate just before diSpensing in subsamples for incubation. 28 Table l. - Soil treatments imposed on field moist Houghton muck prior to incubation. Rates Emposure Aeration Material Per Acre Per Splbs. Time at 65°F. Period at 65°F. 1 Check - - 2 weeks 1 week 2 Picfume 70 gal. 2.65 ml. 2 days 2 weeks 3 Telone 16 gal. 0.60 ml. 2 weeks 1 week Telone 32 gal. 1.21 ml. 2 weeks 1 week Telone 6h gal. 2.h2 ml. 2 weeks 1 week h ViddenéD 20 gal. 0.76 ml. 2 weeks 1 week ViddenéD ho gal. 1.51 ml. 2 weeks 1 week ViddenéD 80 gal. 3.02 ml. 2 weeks 1 week 5 Fumazone M-777 300 lbs. 1.37 g. 2 weeks 1 week Fumazone M5777 600 lbs. 2.7h g. 2 weeks 1 week Fumazone M-777 1200 lbs. 5.h8 g. 2 weeks 1 week 6 N-Serve h lbs. 18.16 mg. (added at beginning of incubation) # 1 - All soils, including the check, were held at field moisture content in sealed plastic bags through the exposure periods shown, ter which they were aerated by forced passage of water-saturated air. 2 - Picfume contains 99 per cent active chloropicrin. 3 - Telone contains 90% active dichlorOpropenes plus 10% related hydro- carbons. h -'ViddenéD is a mixture of dichloropropenes and 1,2 dichloropropane. Rates used supply the active dichloropropenes in the same quantities as the correSponding rates of Telone. 5 - M-777 is a granular formulation containing 10 percent active 1,2- dibromo-B-chloroprOpane on 30-60 mesh attaclayu Rates used correspond to 5, 10 and 20 gpa. of Fumazone 70E, the liquid formulation containing 8.6 lbs. active in redient per g on. 6 - Active 2-chloro-6- trichloromet yl pyridine supplied at a rate equal to 8% of the N added as (NHh)2 80h to all soils (50 lbs. N per acre). 29 Incubation Procedure In this experiment, the "ventilation" method of incubation was used. Figure 1 shows a single respiration unit and Figure 2 shows the arrange- ment of the apparatus in one of the constant temperature chambers. Ten- gram samples (oven dry basis) of moist soil, after exposure to chemicals and after aeration, were dispensed in 60 ml. plastic cups. Groups of 15 were placed in large reapirometer jars. Duplicate jars were set up for each treatment. Incubation was carried out at 10°, 20°, and 30‘0 C., beginning July 12, 1961. Due to mechanical failure, the temperature in the 100 room was raised to 23°C. an October 3, and in the 200 room, to 2hoC. on October 1h. The rate of CO2 evolution was estimated every third day. At periodic intervals, subsamples of soil were removed from each respirometer jar for the determination of ammonium and nitrate and for the estimation of microbial numbers. Collection of Carbon Dioxide Carbon dioxide evolved from the incubating soil was swept out of the respirometer jars by a current of moist, carbnn dioxide-free air. The incoming air was cleaned by passing through a series of absorbents con- sisting of a carboy of LN sodium hydroxide to remove CO followed by'a 2: carbqy of concentrated sulfuric acid to remove ammonia. An absorption tower of granular zinc removed sulfuric acid from the air stream. From this point the air moved into each constant temperature room. The air was remoistened by passing through a column of water which served also as a manostat. It was distributed through manifolds into the appro- priate jars and subsequently bubbled through tubes containing sodium 30 Figure 1. - A single respiration unit showingaA, COZ - free-air inlet tube; B, respirometer jar containing soil samples in plastic cups; C, tube containing KI fer removal of halogen impurities from.the air flowing through the respirometer jar; E, tube containing Ag2804 to indicate when to renew KI; D, tube containing NaOH for collection of C02. 31 Figure 1. 32 Figure 2. - View of operator adjusting the incubation apparatus in one of the constant temperature rooms. 33 hydroxide to collect the evolved carbon dioxide. Prior to passing into the sodium hydroxide, halogens were removed by passing through a tube of potassium iodide solution. A tube of silver sulfate served as an in- dicator to show when the potassium iodide had been spent. There were 21; respirometer jars in each room, each treatment being replicated twice at each temperature. Carbon dioxide evolution was determined for a 2h-hour period every third day, using one replication only. The other replication in each room was aerated for two hours prior to switching the air current to the replication used for col- lecting carbon dioxide . Carbon dioxide in the incoming air stream was monitored by passing through a blank tube of sodium hydroxide. The titration value for this blank was used directly to calculate, the initial normality of the sodium hydroxide in the tubes used for collecting carbon dioxide from the corresponding respirometer jars. Unused sodium hydroxide was titrated. against standard sulfuric acid in the presence of phenolpthalein and excess barium chloride. Carbon dioxide absorbed was calculated by dif- ference and is reported as mgm. carbon per 100 gm. soil (oven dry basis) per day. Laboratory Assays Determination of Ammonium and Nitrate The microdiffusion method described by Brenner and Shaw (7) was used for determination of ammonium and nitrate in both field and lab- oratory studies; The method is a modification of the procedure developed by Conway (11). A 1:10 extracting ratio (moist soil basis) was used, with a 30-minute extracting period (on a shaker). The extracting solu- tion was 1 N potassium sulfate in 0.1 N sulfuric acid, having a pH between 1 and 2. 3h Aliquots of the extract were placed in plastic microdiffusion units. Ammonia was distilled off at room temperature in the presence of MgO and collected in boric acid in the center well of the microdiffusion'unit. In separate units, nitrate was reduced to ammonia by titanous sulfate in the presence of magnesium oxide. In both units ammonia collected in the boric acid was determined by direct titration with .005 N sulfuric acid. Ammonium in the extracts was calculated directly, nitrate by difference. Ammonium and nitrate in the soil were calculated to ppm., oven dry basis. Determination of Chlorine in Celery Tissue The method described by Piper (Sh) was used for dry-ashing of the plant material for the determination of chlorine in celery tissue in 1960. Chlorine is readily'lost during the ordinary ashing of many plant materials. For retaining it in the ash the sample must be ignited in the presence of an alkali. Chlorine-free lime was used in this case. . After ashing, the residue was dissolved, filtered, and brought to volume as described by Husband and Godden (27). A microdiffusion.procedure described by Conway (11) was used to determine the chlorine in the final extracting solution. The Cbrink Madified Conway Unit as developed by thunk (53) was employed in the determination. The essentials of the method are as follows: Chloride in the extracting solution is oxidized to chlorine by an oxidizing agent such as potassium.permanganate. The chlorine volatilizes from the acid extract and is absorbed by'a solution of potassium iodide in the center well of the microdiffusion.unit. The iodide is oxidized and free iodine produced. The iodine is then titrated using sodium 35 thiosulfate with starch as the indicator. Electrical Conductance measurements Electrical conductance of the soil solution in samples taken June 20, 1960 was measured in 1:2 (weight/volume) suspensions, using a Wheatstone bridge (Solu-Bridge). The general procedure followed is outlined by Jackson (28). Measured conductances were calculated to field moisture basis as recommended by Geraldson (19). Dilution flats Count: of Bacteria anC Fungi The soil dilution and plate count methods outlined by Johnson et al (30) were used for enumerating the soil microbial population. Thornton’s standardized medium was used for estimating numbers of bacteria plus Streptomyces spp. and Martin's peptone dextrose medium was used for estimating fungal numbers. Dilutions of 1/10, l/T, l/lOT, l/lOOT, and l/M were made. For bacteria one milliliter of the l/lOT, l/lOOT, and l/M dilution was placed aseptically in each of two disposable petri dishes. For fungi, one milliliter of the 1/T, l/lOT, and l/lOOT was used. Twelve to 15 milliliters of the appropriate agar medium, cooled to just above solid- ifying, were added to each dish. The dishes were rotated by hand in a broad swirling motion. The plates were incubated in the dark at room temperature (23°C.) for h-6 days for fungi and 5-7 days for bacteria. A magnifying colony viewer was used for counting. Data was recorded only for those dilution plates containing 20 to 200 colonies per'plate. In the case of bacteria, usually these were the l/lOOT and l/M dilution plates. For fungi, the usual dilution plates used for counting were those of the l/T and l/lOT dilutions. M$WHSOFFHEDSNDD§ Fumigation Effects on Soil Nitrogen Transformations UncrOpped Soil Fall fumigation with Telone markedly interfered with nitrié fication in the field through the middle of June. The seasonal pattern of interference can best be seen in data for uncropped plots where crOp uptake was not a factor (Figs. 3 and h). The difference in levels of ammonium and nitrate in fumigated and unfumigated soil was less in a cool season, 1960, than in the other two years. Soil temperatures during 1960 remained in the 50's and low 60’s through May. Recovery of nitrifying capacity after about June 20 was rapid fol- lowing the strongly retarding effect df fumigant in 1959 and 1961. This recovery may have commenced about one week earlier at the lower rate of fumigant used in 1961. CrOpped Soil Although nitrification was retarded in fall fumigated soil, it was not completely suppressed. A slow accumulation of nitrate was apparent by early May. This may be observed in data for cropped plats in Fig. 5. The greatly accelerated nitrification rates observed in uncropped plots after June 20 were not so apparent here since the celery was removing nitrate rapidly. However, the rapid rate of conversion to nitrate may be inferred from the fact that nitrate levels in fumigated soil were equal to or greater than in unfumigated soil during late July, a period of maximum growth and nitrogen requirement by celery. During the period of retarded nitrification, ammonium accumulated in fumigated soil, reaching seasonally maximum levels in late may or early June. Where 50 pounds per acre of ammonium nitrogen (100 ppm) 36 37 I959 +Ioo~ ' .\./ . / T . ’5 + son 0 g \ LL / a ’ l g, a) 2: CW )1. I .T ..... z 10.40 1 J j 1 o 2 \ / -50- o 0 a. a. \O / ' \Q _loo 41 54 5] 61 L l ’23 ’7 ’22 ’5 649 7’7 7’22 DATE - TELONE, 329m. TELONE,40gPo. 'VFiqfl' ‘.---‘l ‘.""".l wo3- o--—o o—o A + IOO - __|960 o u. i + 60- ___. Z SOT .‘\.___. a. l ’ 0' O I”O_—-O/ '-(),. _ 60 1 l l I L l l 4 4 5 ' 6 6 7 ’2: ’30 ’2: ’e ’20 ’5 749 DATE Figure 3. - Effects of fall fumigation with Telone on accumulation of ammonium and nitrate nitrogen in uncrOpped Houghton muck in 1959 and 1960. (F - F = fumigated minus unfumigated.) o 37 I959 + I00~ 0 ' \./ / T ' ’5 + 50r o g \ LL / o . s. ' T m T v 0LT" ...... T ..... z lea—lo 1 l l 1 o 2 \ / - 50 - 0 0 a. (L \O / . \Q _l00 4L 51 5] 61 L 1 ’23 ’7 ’22 ’5 649 7’7 7’22 D A T E , TELONE, 329m. TELONE,40gPo. NH4+ O———O O—O N03” O--—O O——O ..... + :00 — ——'96° C) u. + 60 I .— ..--CD a“: .‘-O——-—""’." 3‘ \ 0‘ Z a) "---o O‘O’I’ ' __ 60 l l I l l 1 1 4 4 5 ' 6 6 7 ’2: ’30 ’2: ’e ’20 ’5 7’19 DATE Figure 3. - Effects of fall fumigation with Telone on accumulation of ammonium and nitrate nitrogen in uncr0pped Houghton muck in 1959 and 1960. (F - F = fumigated minus unfumigated.) o 38 o A.ewummfi55mc= m5¢wfi woumwwEsw u m 1 mV .HomH CH #058 ecunmsom emaaouoc: CH cowouufis oumuufia mam SufisoEEm mo cofluma35200m no mcoHoH £uw3 defiumwflafim Hamm mo muoommm I .s shaman meqo m_\m $8 9m owe. :9 mud. Sm a? TA _ _ xxtfir _\\\\\\\MW AunvN.I O\\ o\\\oa oo. . d .d 0/ W mkbUrJMm mT/IoL LNA o N // fl 0,, 00. + Jr nu . . ( / oom + 00» + o o Olllo umoz hwme o o cull. «:2 e N e .93 we 23 mm + |. 0m. A Izv mo ~23”: mzonm» 5m. 3:93 2 :00 50“ N op lied on: 50“ N a lied on: 400 p I959 _ pp I960 5/28 7/7 —- 5/7 5/24 'm300' / " O 2 .// . :E 21)C)”' ‘..,r”" l” — \\\\\ .’ a. O ’ “r 0' éo‘x/I .7L.\ '00 “- O ,O/ \O - . //.---. O . ’ .__ \ / I ...... \ 31/0" 0 ‘~8 2%8—0/0 \‘0 l l l J Q l J L 3 6/5 5/19 7/7 7/22 5/2l 6/5 6/20 7/5 7/:9 DATE No N (NH ) so CHECK 0—--0 o—i-Z-o 4 TELONE 0---0 c---o 50“ N applied on: \ \ C) 1 1 l 1 4 J 4/5 5/4 5/5 6/2I 7/12 7/253/9 DATE Figure 5. - Levels of nitrate nitrogen in fall fumigated and unfumigated Houghton muck planted to celery with and without supplemental ammonium fertilization. ho had been side dressed previously, 250 to 300 ppm of ammonium nitrogen was found in the soil at this time. In 1959 and 1960, the ratio of ammonium to nitrate was 6 to l in fumigated soil which had received ammonium sulfate, as compared with ratios less than 1 in unfumigated soil without ammonium fertilizer. The maximum ratio found in 1961 was 2 to 1. (Fig. 6) In addition to the retarding effect on nitrification, there was evidence that the fumigant exerted a partial sterilization effect which gave rise to heightened microbial activity and a greater re- lease of inorganic nitrogen from soil organic matter (Fig. 7). The sum of ammonium and nitrate recovered was frequently significantly higher in fumigated than unfumigated soil. This was particularly true during the early part of the season, although significant increases for fumigation were also observed during later periods of high temper- atures. Interactions of Fumigation and Nitrogen Carriers The effect of fall fumigation with Telone on nitrogen trans- formations in the soil was markedly influenced by the fertilizer form of supplemental nitrogen used. The effects of fumigation on soil nitrate levels, expressed as the difference between fumigated and any fumigated soil (F-Fb), is plotted in Fig. 8 for plots which received no supplemental nitrogen and those sidedressed with ammonium sulfate or calcium nitrate. The suppression of nitrate accumulation by Telone was strikingly and significantly enhanced by ammonium sulfate in the first two samplings in 1960. A similar tendency towards a synergistic suppression of nitrate levels by fumigation and ammonium fertilizer is expressed in 1959 and 1961, although statistical significance was PPM NH4+-N N PPM NH4— 41 SO‘N applied on: SOnNapplied on: |960 400F569 F 5/7 6/24 — 300* " .\ \ 200- V o \ \ O\\ \k |()C)' ~:‘~‘.,,”".t:;::::L ?\ _‘ .__“ -e-_._--§3 6/5 6/I9 7/7 7/22 5/2: 5/6 6/20 7/5 7/19 DATE NON (NH4)2504 CHECK 0—0 0—0 400 _ 50" N applied on: '96'TELONE o-—-o .___. 5/26 —— 300~ 0“ . w\\ / \ \ // O\ \ / \ \ IOO-O ————— -O’ \\O\ \\ x .\ \ 0‘ ~0/ .\o————-——-Q-———"‘ 339 4/5 5/4 6I5 6/2: 7/:2 7/263/9 DATE Figure 6. - Levels of ammonium nitrogen in fall fumigated and unfumigated Houghton muck planted to ce1ery with and without supplemental ammonium fertilization. (1959, '60 and '61). 42 SOTN applied on: ’(625) SOnN applied on: V 5/28 7/7 I I959 5/7 6/24 |960 400 ’i — — — A I I m T.____ .\\ I ‘9.(523) ;— T O . \\ I V Z 300 .. \. C l. g \: ‘- \ Z 200— '- \ ,/ \ Z 0” \‘~0\ ‘ ' 2 __ O \\ O—-'"O“~ ;—--O a. [00 ‘0 i- ~o —— ‘x l I l 1 1 1 1 1 in 6 ’5 6’19 7’7 7’22 5’2: 6/6 6’20 7’5 7’I9 DATE N0. N (NH4)2$O4 CHECK 0—0 0—0 TE LONE O— —-o ....... 500 L 5’ 2‘5 l96| I 3:; f‘ ‘\ 400 *- 4. \ I \ z \ V \ z 200 \\ 2 \ 0L \ ‘1 IOO O 1 l l l l 1 4 5 6 6 ’5 ’4 ’5 ’2: 7’12 7’25 8’9 D A T E Figure 7. - Levels of total mineral nitrogen in fall fumigated and unfumigated Houghton muck planted to ce1ery with and without supplemental ammonium fertilization. 43 + I00 50" N applied on: '959 50"!“ applied on: l960 5/28 7/7 — 5/7 ‘ 6/24 '— 5 T T K:— T . I + 50*- t / 5 ' ———4\A ? 0“/ _../Q’<\C.D ..... .\...O 'm 0/0 [3/ A/ c2) 0/ ' . 2 — 50— O/A/A _ 0 O’— 3, 0,-8/ \6/6 -lOO ' ' ' ' ' \. 4 L 1 6I5 6/I9 7/7 7/22 5/2I 6/20 7/5 7/:9 DATE - NoN (NH ) SO F-Fo O—o 0—4-3 4A—A CO(NO3)2 SOtN applied on: |96| + IOO 5,26 __ A E i xvi/3 LL O .0/0 .................. 07A. .. z -Ioo~ // II .\ IQ. N5 0 z —2oo~ \. 2 A a -300 L L l l l l i 4/5 5/4 5/5 5/2: 7/:2 7/25 3/9 ‘DATE ‘ Figure 8. - Effects of fall fumigation with Telone on the accumulation of nitrate as related to source of applied nitrogen. Houghton muck, planted to ce1ery, 1959, '60, '61. (F - F = fumigated minus unfumigated.) ' o 43 +IOO 50"Napplied on: '959 50":N applied on: |960 5/28 7/7 — 5/7 ‘ 6/24 — :5. T T [7— T I + 50*- '- 3: o ___4: I: . . ... )//( .. .é’,z”’<>/ \<3’///.\ [3 "’ o 3 o/ 0 — 50- 0%”...13 - . 2 3 I00 2:2/. 1 n\6’6 . L — \ o 6/5 6us 7/7 7/22 5x2: 5x20 7/5 "/19 DATE - NoN (NH ) so F-Eo o—o 0-4-3 4A—A CO(N03)2 + '00” 503“] applied on: l96| 5/26 — A " N(F-Fo) I 6 (3 Cl 6? 3 PPM N03 '- n'» C) C) OI ‘ /\ / i. A -300 l l l l l L 4/5 5/4 6/5 6/2: 7/I2 7/25 8/9 DATE ' Figure 8. - Effects of fall fumigation with Telone on the accumulation of nitrate as related to source of applied nitrogen. Houghton muck, planted to ce1ery, 1959, '60, '61. (F - F = fumigated minus unfumigated.) ' o 14h not attained. After dissipation of the retarding effect of the fumigant, nitrate accumulation in 1959 and 1960 rose'to'significantly higher levels in fumigated ammonium sulfate plots than in those which received calcium nitrate or no nitrogen. This happened during a period when nitrate was being removed rapidly by the crop.. The fumigation effect on nitrate levels in plots which received calcium nitrate was erratic. This appeared to be due to differences in rate of removal of nitrate by the celery in the different seasons and at different times during each growing season. However, there was evidence in 1960 and 1961 that periods of rapid nitrate accumulation in fumigated soil receiving calcium nitrate preceded full recovery of nitrifying capacity in those plate which received no nitrogen or received nitrogen as ammonium sulfate. Since nitrate levels were depressed by fumigation to a greater extent when combined with an ammonium fertilizer source, it would be expected that fumigation would also give rise to greater accumulations of ammonium where this form of nitrogen was used. Such was actually the case, as may be seen in Fig. 9. Differences in fumigation effect between ammonium sulfate, on the one hand, and no nitrogen or calcium nitrate, on the other, were statistically significant during June of 1959 and 1960. The combination of fumigation and ammonium sulfate significantly enhanced net mineralization of soil organic nitrogen through most of the season 1959 and in June of 1960 (Fig. 10). This was not true in 1961. It is important to note in Fig. 9 that, during 1959 and 1960, the increase in ammonium levels due to fumigation was never as great with calcium 'ttit‘rate' as it was where ammonium sulfate was used. ‘- In 1960, the earlier decline in stimulus to ammonification in calcium '45 50a N applied an: 5013 N applied on: T . T 27‘ v '+I50- ./ ~— O\O\.\. 3/.\ + 50* - o\ . \O\% \gflézll: O 1 l J 1 1’ L 5/5 6/l9 7/7 7/22 5/2l 6/6 5/20 7/5 7/I9 D'ATE NON (NH4)2$O4 * F-Fo 0—0 0—0 A—A 50 N applied an: Ca(NO3)2 5/26 T 135. PPM NH4+-N (F-Fo) + + - “D C) C) C) C) l I (D 3 lob I \\\ - \ _|Oo l l l 1 l l 4/5 5/4 6/5 6/2: 7/12 7/26 3/9 DATE Figure 9. - Effects of fall fumigation with Telone on the accumulation of ammonium as related to source of applied nitrogen. Houghton muck planted to ce1ery, 1959, '60, '61. (F - F = fumigated minus unfumigated.) o 46 50" N applied on: '959 50” N applied on: |960 ’3 + I50 "5/28 7/7 ' 5/7 6/24 u. i T T [‘7‘ T :: + Ioo— " — '0 g ///t\\\~/// //0 4:1; +’ 55C)”' l. ‘. *' :::::k<2!_____.L_._::é%“\\\vs g o o/O\O o \%// \o v or. ......... AMAW... _m\.... z —"""“' 2 \A o/ a "" 50 L ‘ * 3 14 l l I L 1 6/5 5/I9 7/7 7/22 5/2I 6/5 5/20 7/ 7/I9 DATE No N (NH4)2$O F-Fo 0—0 0—0 4A—A CO(N03)2 ’3 % . E: 50" N applied on: l96| 173+ 200- 5/26 '— 3 3 ++ + IOOP o A f, o’/’///’ ‘\ <%’”Q><% Z /0/ /A \0 V 0.. .........._..<. \/ z / A E _IOO l 1 .A l J l 4 °- 4/5 574 6/5 55/2: 7/12 7/25 8/9 DATE Figure 10. - Effects of fa 1 fumigation with Telone on the level of mineral nitrogen (NH plus N0 ') as related to source of applied nitrogen. Houghton muck, planged to ce1ery, 1959, '60, '61. (F - Fo= fumigated minus unfumigated.) ‘ h? nitrate plots is distinctly paralleled by an earlier recovery of nitrifying capacity in the fumigated plots (of. Fig. 8 and 9). Effects of the nitrate fertilizer on the fumigation stimulus to mineralization were erratic and generally nonsignificant (Fig. 10). However, a statistically significant synergistic relationship was ex- pressed in the May 21 and June 6 samplings in 1960. Interactions of Fumigation, Nitrogen Carriers and Tillage In 1960, one half of the plots in the field fumigation experiment were not disturbed after they were heavily compacted immediately after injection of the Telone in the fall of 1959. The other half of the plots were plowed just before planting celery in the spring. Plowed and unplowed plots, therefore, represented a differential compaction and . aeration status which was maintained through the growing season. Numerous significant first and second order interactions between fumigation, aeration treatment and nitrogen carriers in their effects on the accumulation of ammonium and nitrate were encountered (Tables 2 and 3). The LSD noted for the FxN within A comparison is appro- priate for comparing two differences (F-Fo) in the data plotted in Fig. 11. Where no supplemental nitrogen was applied, there was no effect of the plowing on the extent to which ammonium accumulated after fumigation (Fig. ll-A). The accumulation of nitrate was retarded to a greater extent by the fumigant in unplowed soil (Fig. ll-B). By contrast, in the may 21 sampling, the accumulation of ammo- nium was dramatically enhanced in unplowed soil by calcium nitrate (Fig. ll-A) and by ammonium nitrate (Fig. ll—C). Concurrently, a marked disappearance of the nitrate added on May 7 was noted in the fumigated, unplowed plots. This appears in Figs. ll-B and 114D as a D8 Table 2. - Nitrate nitrogen in Houghton muck planted to celery as re- lated to aeration, fumigation and nitrogen carriers in 1960 Isration Fumigation Nitrogen PPHT_N5§ - N 63’ treatment1 treatmentz carriers3 may 21 dune 6 JunE'QO July 5 July 19 A F (NHh)ZSO 5& 123 10& 182 17& & NHhNo3 159 16& 118 236 15& Ca(N03)2 228 172 108 195 160 NO N 38 6& 7h 96 52 A F0 (NHh)ZSOh 98 220 125 138 90 NHhNOB 138 223 11& 208 1&0 Ca(N03)2 13& 232 131 1&8 187 NO N &8 55 38 82 && A0 F (NH&)2SO& 27 109 121 190 1&6 NHhNO3 82 208 153 30& 160 Ca(N03)2 120 230 1&6 2h7 218 NO N 29 S9 58 66 &2 A0 F6 (NHh)ZSOh 92 2&0 151 170 120 NHhNo3 1&0 225 8& 216 8& Ca(NO3)2 230 15h 1&6 232 15& N0 N 5h 59 56 hl ‘ ho LSD .05 (A within FN) 90 NS NS NS NS LSD .05 (F within AN) 90 89 63 NS 56 LSD .05 (N within AF) 9& 79 53 103 56 LSD .05 (FVN within A) 93 NS 60 NS 56 I - A11.plbts compacted after fumigation on OEtober 26, 1959. A0 = Soil undisturbed except for planting operations in 1960. A = Plowed May 2, 1960, prior to planting celery on May S. 2 - Fo - no fumigant. F== 32 gpa Telone. 3 - SO poufids per acre N (100 ppm) applied May 7, 1960, and again on June 2 . h9 Table 3. - Ammonium nitrogen in Houghton muck planted to celery as related to aeration, fumigation and nitrogen carriers in 1960. Aeratibn' Fenigation,'Nitrogen FFM7“NHZ:Z'N' on F“‘—_““_ treatment treatment carriers3 May 21 June 6 Hune 2O July:§_guly'12 A F (NHh)ZSOh 290 131 56 80 100 NHbNOB 188 83 3& 61 &0 Ca(N03)2 93 &9 38 33 20 N0 N 76 52 38 26 19 A F NH so 1 26 O ( &)2 & 93 5 &3 &7 NH&NO3 95 33 9 39 28 Ca(N03)2 112 22 7 12 1& NO N &3 12 1& 13 15 A0 F (NHh)280h 221 82 5& 120 59 NHbNOB 227 86 &6 9& &8 Ca(N03)2 158 &8 h2 && 23 N0 N 76 &2 2& 25 26 A F NH o o ( h)250& 187 17 5 136 83 NHhNO3 53 10 22 85 22 Ca(N03)2 1& 6 15 ' 21 10 N0 N 16 8 7 11 13 LSD .05 (A within FN) Ns NS NS &5 38 LSD .05 (F within AN) 9& 37 22 Ns 31 LSD .05 (N within AF) 95 37 22 &5 29 LSD .05 (FXN within A) 96 38 22 38 31 1 - All plots compacted atter»ifinigation on.0ctOBer‘26, 1959. A a Soil undisturbed except for planting operations in 1960. A = Plowed May 2, 1960, prior to planting celery on May S. 2 - Fo - no fumigant. F a 32 gpa Telone. 3 - 50 pounds per acre N (100 ppm) applied May 7, 1960, and again on June 2h. 50 markedly enhanced suppression of nitrate on May 21 associated with the two nitrate carriers in unplowed soil. Apparently the nature of the recovery population in fumigated soil was such as to effect direct reduction of nitrate to ammonium in the poorly aerated environment represented by compacted, unplowed soil at high moisture content. Nitrate had completely disappeared on May 21 in all fumigated, unplowed plots in one replication which happened to fall on a slight depressional area which had not drained as quickly as the rest of the plots. In the 17 days immediately following this marked disappearance of nitrate, it appeared that essentially complete recovery of nitrifying capacity had been achieved in unplowed plots which had received cal- cium nitrate or ammonium nitrate. Nitrate accumulated at rates of hS to 50 ppm.per week. This compared with rates of 50 to 60 ppm per week in unfumigated plots which had received ammonium sulfate and in which maximum rates were expressed during this period. These rates exceeded those in unplowed, unfumigated plots and appear in Figs. ll-B and ll—D as marked differential accumulations of nitrate on June 6. This dramatic recovery of nitrifying capacity was reflected also in the very rapid differential disappearance of ammonium in unplowed, fumi- gated plots when nitrate was added (of. Figs. ll-A and 114D). During this same period, differentially enhanced crop removal or microbial immobilization in fumigated, aerated plots resulted in a marked differential disappearance of nitrate, similar to that expressed in all plots which received ammonium sulfate. This differential nitrate disappearance, as plotted in Figs. ll-B and 114D, leads to an erroneous inference regarding the onset of rapid nitrification in plowed soil following application of the two nitrate carriers. Large 51 l960 50"“Napplied on: _ SO‘Nopplied on: + 20°F 25/? 6/24 5/7 6/24 E, (7- .\ . + Iso- . - \ LL \\ \ V \ ll-A \\ ll-C 2+ IOO — \ t 8—————-{0 +' \ \ \ <1- \\ \\ :\ I . ‘\ 2+ 50» o\\;€ r /0.\ 9\ o 2 I RQ 3‘: ~ ‘0: —-Q‘ I. O. I ‘8- \~ ‘./"0 a_ 0.. ........ I (‘ ... ............ I O C) “‘~.~‘. _ 50 1 L l l ’L l J l l 4 5x2: 6/6 6/20 7/5 7/I9 5/2l 5/6 6/20 7/5 7/19 KEY DATE KEY NOT No N C0(N03)2 NOT (NH4)ZSO4 NH4NO3 PLOWEDO—O O——O PLOWEDO—O O--O SPRING O—O O-—O SPRING O—o O-—O “00f 0‘ PLOWED F‘ PLOWED J. O a’ “- A \ . a /. lf?4’ SCJF' \ I \\x l’ _ LL \\ \ 0 ’O>/ \I o/\ /8$\e [840 Z Op...6%.m.( ............ \. . t." .......... ,I .X o/ \ '0 I \ / g - 50- ’ \ ’ - I o’ \ 2 I a I II-B O ll-D - a I _ IOO . . a -|50 l l l l l l J l I 4 5/2l 6/6 6I20 7/5 7/l9 5/2: 6/6 5/20 7/5 7/I9 DATE Figure 11. - Effects of Telone fumigation on accumulation of ammonium nitrogen and nitrate nitrogen in Houghton muck as influenced by source of added nitrogen and aeration treatment. (F - F = fumigated minus unfumigated). o 52 accumulations of nitrate were observed already on may 21 in fumi- gated, aerated plots which had received these two materials (Table 2). Thus, it may be inferred that plowing and the use of nitrate con- taining fertilizer maximally reduced the period of retarded nitri- fication resulting from fumigation. Rapid nitrification apparently commenced in plowed and fumigated plots receiving fertilizer nitrate even earlier than when these materials were applied on plowed plots that had not been'fumigated‘ (Table 2). Totals of mineral nitrogen (NH f plus N037) are shown in Table h. The effects of nitrogen carriers and plowing on the basic min- eralization response to fumigation are depicted in Fig. 12. In unplowed soil, fumigation tended to depress the level of mineral nitro- gen early, leaving it unchanged at the end of the season, in plots which received ammonium sulfate (Fig. l2-B);in.plots fertilized with ammonium nitrate,fumigation greatly enhanced net mineralization all through the season. Plowing resulted in a marked reduction of the fumigation - enhanced mineralization in plots which received ammonium sulfate. Results with calcium nitrate (Fig. l2-A) were erratic, but the relative effect of plowing on June 6 and July 19 was the same as that observed where ammonium nitrate was used. There was a tendency during June for behavior in plots not sup- plemented with nitrogen (Fig. 12qA) to parallel that in ammonium sulfate plots (Fig. l2-B). Summary of Fumigation Effects The interactions described are complex. They suggest that the nature of the recovery population following partial sterilization of soil is strongly'influenced by environmental conditions imposed during 53 Table h. - Total mineral nitrogen in Houghton muck planted to celery as related to aeration fumigation and nitrogen carriers in 1960. Aeration Fumigation. Nitrogen ‘_¥ FWTT NHL} - N + Nng -No treatment1 treatment2 carriers3 May 21 June 6 June 20 July E Jun nl9 A F (NHh)ZSOh 3th 25h 168 261 27h NHhNOB 388 2h8 152 297 19b Ca(NO3)2 320 220 1&6 228 180 'NO N 113 116 112 122 71 A F (NHu) so 291 2&6 130 181 137 o 2 h NHhNOB 233 256 122 2L7 169 Ca(NO3)2 2h6 25h 138 160 201 NO N 91 67 52 95 S7 A F (NH ) so 2h8 192 175 311 205 o h 2 h NHhNOB 309 29h 22h 398 208 Ca(N03)2 278 278 188 291 2&1 NO N 10b 101 83 91 68 A0 F0 (NHh)ZSOh 279 251 181 306 202 NHhNOB 193 235 105 301 106 Ca(N03)2 2th 161 161 253 16h NO N 70 67 63 S2 52 LSD .05 (A within FN) NS NS 62 NS NS LSD .05 (F within AN) 76 NS 58 NS 72 LSD .05 (N within AF) 76 95 L7 126 72 LSD .05 (F101 within A) 76 96 52 NS 72‘ 1 - All plots compacted after fumigation on October'Qb, I959. A0 9 Soil undisturbed except for planting operations in 1960. A a Plowed May 2, 1960, prior to planting celery on may 5. 2 - Fo a no fumigant. F 8 32 gpa Telone. 3 - SO poufids per acre N (100 ppm) applied May 7, 1960, and again on June 2 . 54 A.pmumwaabwas msafifi pwumwfiabm nom n mv .ucofiummuu aowumumm paw cowouuw: pmpnm mo moMDOm %n poocmsamca mm x058 scuswsom 5H cowouufic Hmumcfie Hmuou mo aowumasasoom co cowumeBSM mconH mo muommmm a .NH muswwm . wkmoH sthoz .HomH cw x055 couswaom Aahv woumeESMSa no asouw choc umosm mo mo>mmH :fl m60udEhm hocmaofiwww mmoamwcmz s .0H muswwm 73 .Hmo. .aseaasaa “moo. .asemenaaoa “Noe. .uaau “Loo. .aouon “Hoe. .wmaaou “Huo. .asaeom "consumaasm oa emumaau maueauaoaemam use muemaausa guano mo unaueoo mmwumsa - H NOS e .mamm, .' .1: .U..v anfll. ill-II "I‘l If m2 m2 so. we. m2 m2 ha. ma ma. new coo. omoo. owa. mq.o acm.m ama. «m.~ mma uneasy «am me One. aooo. mom. em.o m~.e ama. as.~ aea macama mam an oHo. mooo. nmm.. Ho.o ~w.o mmH. ww.~ OMH maoz unmouma unouuma unwouoa unmoumm unwouoa unnamed unwouon .uBu couH omecawdwz. .83fimoquz. asHonU eaMmMMuom wnwwmmwafim rewouufiz m~\m ammfiawmmm flea uHsh coxmu mowmfimm nfiupwmlwo mfimwwmnm .. mpaofiw coaumwwabm cuoo .HO0H .3058 acufiwdom .fiOHuwMHgm OU muwuwHwh mm mUd—muflou ufimeHuDG HQHHOW Guam cod—.00 U®w3m NO mfiHGHV I .WH 0H£MH .mooo. .eacoeaaaoa “moo. .oaau “moo. .aouon “coco. .ummaou ”coaumwfiasm cu pouwamu haugmofiwqdwwm no: mucmfiwuaa Huguo mo ucouaoo mmmuw>< n H 7h woo. woo. m2 m2 no. Nfi. mz mz mo. om no. 9mg coo. NNo. aoo. Loo. cow. so. wa.o «as. ~o.a was meoame «am we moo. «No. Nae. Hoe. Nam. am. an.“ ems. Hm.e Hes vacate «am an moo. mmo. sac. Hoo. mmm. Ho.H o¢.n «he. om.¢ mmm maoz unmowmn unmouma unwouoa unmouma unmopma usuowwm unmouma unmouwa unmoumn .muao 85c .anwpom couH one: Shaman Sawmwwo .Hmwmwmu may new mH\n uaoSummuB uwesa< semen: ummz aom nonamogm nouufiz mpHoH» aofiumwfiabm Hom mama coxmu mwflmaww pwupwe mo mfimmwmw< mosuqu .HcaH .xoaa nouawsom .GOauwwHEsm ou pwumamu ms muamucoo ucowuusa umfifiom new moduuwa mo meaoaw s .0H manna 7S deficient range. Even though there were no changes in manganese con- tent, it appears extremely likely that the major factor in the response of lettuce to fumigation was the increasing availability of manganese. Decreasing contents of the major and secondary nutrients, as well as of iron, sodium and aluminum, were associated with increasing yields. Although the content of iron was high, it is well within the reported ranges for this crop (5). While the basic response to fumigation appeared to be related rather simply to manganese availability, the yield reSponse to forms of fertilizer nitrogen noted in Table lb involved a rather complex sequence of deveIOpmental responses. On June 5, root browning report- edly characteristic of ammonium toxicity (22 and 26), was wide spread, even in plots where negligible accumulations of ammonium were found. The severity of root discoloration increased with fumigation level, and was most marked where ammonium sulfate and ammonium sulfate plus N-Serve had been used. Ammonium levels in excess of 300 ppm were found in some of these plots. Calcium nitrate plots were least affected. However, the levels of root injury observed were not reflected by visible differences in t0p growth. . On June 20, growth.was remarkably uniform on all plots, except that fumigated plots tended to be darker green and slightly slower in forming heads, particularly where ammonium sulfate, — with or without N-Serve, - was used. On the fumigated areas, calcium nitrate plots were distinctly'lighter green in color than those receiving ammonium sulfate. The plots which received ammonium sulfate plus N—Ssrve were still darker green and not fully ready for harvest when yields were taken on July 13. 76 Final yields were not related to the root browning observed in early'June, since maximum yields were obtained with ammonium sulfate in fumigated plots, - where the root discoloration had been most severe (Table l7-B). Preferential assimilation of ammonium rather than nitrate nitrogen was ruled out because of the unfavorable response to the nitrification inhibitor, N-Serve, which effectively maintained higher concentrations of ammonium in the soil through harvest. An inadequate availability of nitrate nitrogen might have been involved with this treatment, but not where calcium nitrate was used, since 100 to 200 ppm of nitrate nitrogen was maintained in the soil through harvest where the fertilizer nitrate was applied. Also tissue nitrogen was inversely related to the yield differences associated with nitrogen sources, indicating that growth was being limited by some other factor.. This limiting factor again appears to have been manganese. The tissue manganese values in Table 172A do not include any statistically significant differences. Significant differences among yields were found only among the averages for fumigation and for nitrogen sources (cf. Tables 13 and 1h). However, the close correspondence between manganese levels and yields indicates that the response to ammonium sulfate was a response to manganese expressed additively on the man- ganese reaponse associated with fumigation. The significantly reduced response to ammonium sulfate when com- bined with N-Serve suggests that the nitrification inhibitor counter- acted the solubilizing action of the sulfate on soil manganese. The solubilizing action of ammonium sulfate arises from its acidifying effect, half of which is expressed only as the ammonium is nitrified. It appears that the seasonal distribution of manganese availability closely paralleled seasonal patterns of nitrification as influenced 77 Table 17. - Manganese contents and yields of lettuce as related to fumigation and nitrogen sources. Houghton muck, 1961. Fumigation Nitrogen source Treatment NB'Nw CaKNO 7' (NH 7 36 (NH') SO Ave. 3 2 b 2 h h 2 h for plus fumi- ___ N-Serve Agation A. PPM manganese in leaf midribs on June 20 Unfumigated h 12 19 8 11 32 gpa Telone 19 h 16 h 11 h8 gpa Telone 12 12 8 8 10 Ave. for N source ‘ 12 9 1h 7 - B. Final yield, - ths. Unfumigated 23h 27o h33 36h 325 32 gpa Telone hlZ D93 511 hBO hél h8 gpa Telone hl9 D78 h79 hO9 hhé Ave. for N. source 355 hlh h7b hOl - 78 by fumigation and fertilizer sources of nitrogen. An optimal seasonal release of manganese was associated with fumigated ammonium sulfate plots. Where ammonium sulfate was treated with N-Serve, recovery of nitrifying capacity was additionally retarded. The associated delay in manganese release accounts for the delayed development and darker green color of lettuce on these plots on July 13 when lettuce on other plots had reached market size. If these plots had been left unharvested another week or ten days, it is likely that final yields would have equalled those where ammonium sulfate without N~Serve was used. The slow, but continuing release of manganese associated with retarded nitrification in fumigated soil accounts for the fact that lettuce yields four times greater than the state average of 100 to lhO cwt. per acre were produced, even though manganese in the plants was at critically deficient levels. Celery Of the two celery varieties used in 1961, Utah 5270 was the onxy one injured by fumigation. This was the same hybrid used in 1960. Early'injury was as striking as it had been on poorly drained repli- cations in 1960. The degree of injury increased with increasing rate of Telone application (Figs. l7eA, B, C). However, in contrast to 1960, there was no intensification of injury with ammonium sulfate and no beneficial effect of calcium nitrate. In fact, the latter material tended to intensify the injury (Fig. l7-D). Differences in final yields associated with~thaSeenitrogen sources were not significant. The fumigation injury was reflected in significant reductions in yield. The content of N, K, Mg, Mn, Fe and B in celery petioles on July 1 tended to increase as yield potentials associated with fumigation 79 Figure 17. - Utah 5270 ce1ery two months after planting in unfumigated soilLF and following fall fumigation with 32 gpaFg and 48 gpa F of Telone. Injury increased with fumigation evel where g 4372804 [N33 was used. Failure to respond to CaiNOfiz END was due to limiting Mn and P, and an interaction with increased availability of Fe in fumigated plots. 80 .NH muswam 81 levels decreased (Table 18). PhOSphorus content decreased. There were striking differences in the status of iron, manganese and phosphorus in celery tissue grown in 1961, as compared with.1960 (Table 19). The content of iron and manganese was much lower in 1961, but the ratio of iron to manganese was several-fold higher. Manganese sulfate was included in all insecticidal Sprays in 1960 but was not used in 1961. As a result, manganese was at a critically deficient level in celery tissue in 1961. During both years, the effect of fumigation was to increase iron concentrations in the plant and reduce the concentration of phosphorus. As a result, iron to phOSphorus ratios were wider in celery grown on fumigated soil. At the critically low manganese levels in 1961, these widening FezP'ratios were closely related to declining yields (Fig. 18). This apparent immobilization of phosphorus by iron in fumigated plots was most injurious to yields where calcium nitrate was used and least where nitrogen was supplied as ammonium sulfate. Iron in the tissue was much higher in 1960, and Fe:P ratios were 2 to S-fold greater (Table 19). Nevertheless, the relation to yield observed in 1961 was not obtained. Apparently, this was because of the higher manganese levels and much narrower FezMn ratios. Phosphorus contents were also higher in 1960. PhOSphorus levels in 1961 approached the minimum reported value of h3OO ppm cited for celery by Beeson (5). That phosphorus was in a deficient range in 1961 is indicated by the fact that yields declined with phosphorus content. However, the relationship between phosphorus content and yield was not a simple one. It was complicated by the interactions with tissue iron, fumigation treatments and nitrogen carriers which are depicted in Fig. 18. 82 .soo. .esfiezs menoo. .esssssbca 38. Jean “mooo. .ummnoo “sowuwwassm cu woumaou hfiuswoamwawfim uo: muswfipusa noSuo mo ucoucoo oweuo>< u H 4.28 newt macaw .3me - 1 Hooo. moo. mz . wz wz mz wz Ha. mm no. God moo. moo. mooo. mum. «w.H mo.n ammo. No.N Ham oaoHoH new we .4. moo. moo. mooo. mum. Hm.H om.n omq. oo.N mmm omoflea mow mm Noo. moo° mooo. oou. om.H «N.m owe. oe.~ one esoz uaoouoa uaoouoa ucoouoa ucooumo unmoumm . unmouwu “coupon unsouoo .mu3o couom couH omoawwcmzw.anfimmGMmz_ snfiowmo sawsmmuom mono;mwo£m smwouuwz ¢H\m umufiummufi H haso noxmu monEmm awofiuem.%o mmeHmu< noes“? moaumwwanm H mumaoo .HooH .xo=E.:ou:msom .Gowuowfiasm ou oouoaou mm muaoucoo uaowuus: umfiaom one huwfloo mo mpHmfiw n .ma wanes 83 .HooH .udofiumouu cmwouuwa one cofiumwflsnm manwfiuw> Sues zoos aouzwsom co umm>umn ouomon mxooB xwm moHoHuom mmoa cw monogamona ou song we owuou oau ou ooumflou no mpflowh muoaoo n .ma ouowwm mun—0:.ma 2. duo“. O_._.1" 231% ...—{:8 5 f .135...— :§:~.- -—_- 3...... .. n.+v+|00" .5. 0- v - IOOL l 1 l l I 1 I Days from treat. 48 63 82 92 97 HO Days at temp. 28 43 62 72 77 90 I l8 +500L TELONE VS. CHECK “ V r~ ::L——"""7————""’—""” 2 ”+300— ——“8 <7“ :33 o -_— .‘.'.‘.s .. ‘.'9. ..f" 2 2 g; :‘ :—-’—'°° ... :58; a. + / I" o~ 2! _ ~v -’I()() l l L I l l 1 Days from treat. 48 63 82 92 97 HO l28 Days at temp. 28 _ 43 62 7277 90 I I8 Figure 21. - Changes in total mineral nitrogen following chemical treatment of Houghton muck. Initially the soil contained 145 ppm mineral N. 100 ppm N was added as (NH )2 $04 when soils were placed in constant temperature rooms 48 days after adding4 chloropicrin and Telone. Prior to this time, soils were held at 180 to 20° C. 93 (cf. Fig. 19). This indicates that the major stimulus to resistant species in the recovery population had occurred during the earlier exposure and aeration periods at 18° to 20° Co At 10° C., the residual effect on total mineral nitrogen levels was maintained throughout the incubation period (Fig. 21). At 20° and 30° C., differences between treated and untreated soils had largely disappeared after 70 to 90 days at each temperature. In the case of Telone at the highest rate of application (6h gpa), significantly reduced accumulations of nitrate were observed only in the first sampling at 10° and 20° C. (Fig. 20). In later samplings, nitrate levels tended to be higher in the Telone treatments than in the checks, becoming significantly higher in the 10° room after the temperature was raised to 2&0 C. Differences in total mineral nitrogen between check and Telone treated soils were not statistically significant at any time (Fig. 21). However, there was evidence that a stimulus to net mineralization, similar to that expressed with chloropicrin had occurred following addition of Telone. Total mineral nitrogen was maintained rather consistently at levels higher than the check through 70 to 90 days of incubation at each temperature. Telone and Vidden-D were very similar in their action at all rates of treatment. Significant accumulations of ammonium occurred only in the first sampling at 100 C. and the quantities found increased with rate, reaching maxima of 100 to 150 ppm at the highest rate. At this same time, at 30° C., both total mineral nitrogen and nitrate nitrogen were significantly increased over the check at the higher rates of treatment only. .A similar enchancement of nitrate and total mineral nitrogen at higher rates, sometimes associated with significant 9b depression at the low rate, was observed after 62 and 72 days at 100 C. with these two materials. Generally, however, significant or con- sistent effects of rate were not observed. On the other hand, with Fumazone, significant effects of rate were expressed on net mineralization and nitrate accumulation on all sampling dates at one or more temperatures (Appendix Tables 21 and 23). Nitrate and total mineral nitrogen were consistently higher at the higher rates of treatment (7 and 1h gpa) than at the low rate (3% gpa), at all temperatures (Fig. 22). Levels at the-low rate were frequently reduced significantly below those in the check. As was true with Telone and ViddenéD, accumulations of ammonium were found in the first sampling at 100 C. Sixty ppm were still present at 20° C. where the highest rate was used. There was a tran-- sient accumulation of NHh+ immediately following the temperature change in the 10° room. This was observed also where Telone and ViddenéD had been used, but not with NeServe. In no case was the ammonium accumu- lation associated with this temperature change as dramatic as in the check soil. Ammonium accumulations near the end of the incubation at 30° C. appeared to be related to drying of the soil. Since the soil was initially high in moisture, drying associated with this high air temperature promoted a generally increasing level of microbial activity, as reflected by 002 evolution. Microbial Numbers and Evolution of Carbon Dioxide The generally increasing level of microbial activity associated with drying of soil during incubation at 30° C. is apparent in Fig. 23. It was much less apparent, or did not appear, in connection with soils % |O° C O 600" 24 C a: 2: _ r1 I ‘4()C) I. - E r1 2 y- E = = = = /: a 200- : fig /: he“ 0- 5 %E as f5 = f: = E _ E 4% E = Days after treat. 48 63 82 92 97 IIO |28 Days at temp. 28 43 62 72 77 I3 4| NH4’N ' M NO -N 20° C ' o 600 _ 3 24 C Fl F}: l:3 I 2: .. I 400“ “ H 75 5 f5 /_ a. 5&5 200" I: a ?E Days'after treat. 48 63 82 92 IIO |I3 I28 Days at temp. 28 43 62 72 9O 93 25 I3()° (3 600~ flit - fl ” 7.. f 400 c z; 2% as /E :5 ~ :ggg €255 a 200L g:- ME 0. ,rgg $3? .. t a: L . ée Days after treat. 48 63 82 92 IIO l28 Days at temp. 28 43 62 72 90 I I8 Figure 22. - Levels of ammonium and nitrate nitrogen in unfumigated muck and in muck previously treated with three levels of Fumazone, during incubation under three temperature regimes. (F1=3% spa; F2=7 gpa; F3=14 gpa) 96 Figure 23. w Effect of ehlzripierin or the evalution of carbon dioxide and the numbers of beeteria {8) sad fungi (F) in Houghton muck at three incubation temperatures. 97 8 8 F “05) U (IOO) - z 40_ 0—0 CHECK [— «80 3 0—o CLPCN. I . Jyg <5 .. 24 C. /f o 30 / . ago (3 ¢ / IO° C 8 20 " ’0’.~.\ “‘ 4O ' .. -0, 0.8 ~ I. \ g '0‘ ." ° \‘N/z: ° \° 0/ =3~gv..... ‘0 ~20 9 ;—:\O\ ’.Oo B ’0 0a 0'0 F °_o’/°~o—o r. E _ Days from treat. 46 79 95 I38 a Days at temp. . 25 58 74 43 (2 x B ( ) '3 98 o 2 E40; ’ 2° C 24°C. i803 / 3 30— % / B -60 l 0 “z. 9 .a _ " \ 20 _ o .\./.‘.. .\ .’. 40 x 9 o. / ‘ f" /o-O’° / <3: 2 I O _ O ,o.°;85{0_;>6-..o:§\ ’0‘0/ w?\o- 8‘0‘0 _‘ 20 E (3: 00 B ‘_. \0-0 F l-IJ F (I ,_ II‘ [L .. a 0 Days from treat. 46 79 l I I I38 g Days at temp. 25 .58 90 27 > F 30° c A g 40 b B /O'O-O 80 \ o-O\ 3 so - /<\o .-"\ i 50 o .,0{ 9 /°~o‘o / \°\9' \o:// o? J \ 20‘/ O .‘o ‘0 ,0-3 40 U ./ ‘0 ./ ‘0' O o )3’ \0’ \ 8 F 2 :0—0 / «:922' e . —20 n. rl F Fi J r— . Days from treat. 46 79 I38 Days at temp. 25 58 I I7 98 incubated at 10° and 20° 0. Periodic readjustments in composition and size of the soil population were very likely responsible for the peaks and valleys in the rate curves for 002. At 30° C., these readjustments occurred at about the same time in treated and untreated soilso At 100 and 200 C., this coincidence was markedly disrupted with all chemical treatments. The degree of phase displacement and the maximum rates attained varied with the different chemicals and with rate of application. At the high rates of treatment with'ViddenéD, Telone and Fumazone, higher rates of 002 evolution than in the check soil were maintained over major portions of the incubation period at 100 and 200 C. In this reSpect their behavior was similar to that shown in Fig. 23 for chloropicrin at these lower temperatures. Total 002 evolution, accordingly) was higher for all of these treatments. With N—Serve and the low rates of ViddenéD, Telone and Fumazone, total 002 evolved was greater than the check only at 10° C. Chloropicrin treated soil exhibited a markedly increased activity following the temperature change in the 200 room (Fig. 23). There was no similar reaponse to the earlier change in the 10° room. In the case of N~Serve and the high rates of ViddenéD, Telone and Fumazone, these temperature changes were accompanied by enhanced 002 evolution in both the 10° and 20° rooms. At the low rates of the last three fumigants, these late temperature changes had no effect. The relative differences described above indicate that the com- position and general activity of the soil microbial population was altered in.varying degrees by different chemicals and by rates of application of a given chemical. The altered populations responded 99 differently when temperatures were raised or lowered 3 weeks after fumigation. Even a minor temperature change from 20° to 2h° imposed h months after fumigation produced differential reSponses in activity. The sequential pattern of adjustment to the 100 temperature was markedly disturbed by all chemical treatments. Adjustments when the temperature was later increased to 2&0 C. were distinctly different for different chemicals, particularly at high rates of treatment. It appeared, however, that at 30° C. these residual effects on the soil population were rapidly dissipated. As a result, fluctuations in activity associated with normal population changes and with drying of soil followed similar patterns in treated and untreated soil. Attempts to characterize qualitative changes in the composition of the pOpulation were not highly successful because of the great variability encountered in plate counts for bacteria and fungi. Significant differences in geometric means for various treatment com- parisons were obtained principally in the first counts, made after 25 days of controlled temperature incubation (Appendix, Table 2b). The major differences at this time were associated with the chlorOpicrin treatment (Table 20). Bacterial numbers were dramatically increased at all three temperatures. INimbers of fungi were sharply reduced where the temperature had been lowered to 10° or raised to 30° from the pre-incubation temperature of 180 to 20° C. There were few significant differences for other treatments. Those that were ob- tained were consistent with the conclusion that chemicals other than chloropicrin had had a stimulating effect on bacterial numbers at 10° and 20°, similar to but less pronounced than chloropicrin. These other chemicals had had no effect on bacterial numbers at 300 C., 100 .acoumabm paw aconB .auamppfi> mo mcumu was mfiOfiumofiHQou wcwnwnaoo enema ugpuaaomw .mhce em umea Mom .0 oqN cu emawmu Boon com a“ annumumaEwH .mhap ow umma now .0 cam on woman“ Soon 00H 5“ auaumummSmH mum manuwumon name madam maomwuaaaoo mnowua> new mowumu m unmofiwwamwm n I m uom-z mm.ma am.H -.s o~.m o~.~ me.a mm.w Hm.m e~.e noseo x a m16 . . «ca . a m es.“ Ha.eH we.ea em.m AH.m mN.e mm.m ow.HH me.o~ Nmuemweasm Hague mm.o~ ma.o e~.am H~.mn He.H Nm.m Ho.ea oe.wa oo.eoH enuoaaoyonno ea.m mo.n ww.e mm.e ma.m an.m oo.m ea.oH om.mH w>pmm-z en.a oa.mm am.ooH NH.m mN.m mo.~ we.m ea.e sm.HH sumac 00H x mantuomm .< ucmfiumfiflm om tom -oH om om mm. on a~,-t 0H mesa.wsnewm NAH « we mm .aaeu om mwma mm 3038 counwsom mo nowuansocw magnum “wasm was «finances mo I. 1.1!! .auvuaumaamu coaumnsoaa paw mfimoflamno on pauaaau H mumnasc came owuuaaoau I .oN candy 101 and essentially none on numbers of fungi at any temperature. It is of interest that bacteria were more numerous in the first enumeration with all chemicals at 10° and 20° than at 30° C. After 68 days at each temperature, bacteria in these soils had declined to levels comparable to those at 300 C. In chloropicrin treated soil, bacteria at 20° and fungi at 10° and 20° were significantly fewer than in check soil or soil treated with other chemicals. Fbllowing the temperature increases in the 10° and 200 rooms, bacterial numbers increased rapidly in the check soil. Smaller gains were observed also for all fumigated soils, although differences in numbers among chemicals were non-significant. Few differences in fungal count were statistically'significant. However, it did appear that fungi were on the increase in check and chloropicrin treated soils at 300 C. This may have been a reflection of the drying soil conditions. Summary of Incubation Results Results of both field and laboratory studies indicate that, in organic soil, effects of chemical fumigation on patterns of nitrogen transformation are compounded of effects on both heterotrophic and autotrophic components of the soil microbial population. In the in- cubation study, partial sterilization effects on both components were expressed by all fumigant chemicals. Effects of ViddenéD, Telone and Fumazone were neither so drastic nor so prolonged as with chloropicrin. However, the intensity'and duration of effects of all fumigants were markedly different under different sequences of soil temperature imposed 3 weeks after initial exposure. At soil temperatures normal to the summer season (30° C., 86° F.), effects of the fumigants, including chloropicrin were rapidly dissipated. Heterotrophic p0pu1ations in both treated and untreated soils were characterized by relatively low numbers of bacteria, high ammonifying capacity (rapid net mineralization of nitrogen), and by synchronous papulation readjustments reflected in essentially parallel fluctuations in rate of 002 evolution. AutotrOphic nitrifiers had recovered full activity 7 weeks after initial exposure, except in chloropicrin treated soil, where an additional Biweek delay was observed. At soil temperatures normal to late autumn and spring (100 and 200 C., 50° and 58° F.), a slight delay in nitrification beyond 7 weeks was observed with fumigants other than chloropicrin, notably at recommended and higher than recommended rates. With chloropicrin, nitrification at both temperatures was delayed for about 3 months after treatment. Heterotrophic bacteria were greatly increased in numbers 7 weeks after exposure to chloropicrin; increases with the 102 103 other three fumigants were much less, though statistically signif- icant in some instances, approaching significance in others. Fungi were significantly suppressed by chloropicrin but little affected by the other three fumigants. These changes in the proportion of major taxonomic groups gave only a very meagre indication of the extent to which heter- otrophic populations were altered in their physiological composition. This is indicated by the very marked chronological diSplacement of sequential phases of increase and decline in rates of CO2 evolution in treated and untreated soils. This was apparent at both 100 and 20° C., but it was most prominent at the lower temperature, and with chloropicrin and the higher rates of ViddenéD, Telone and Fumazone. Heterotrophic activity at these lower temperatures was char- acterized by enhanced net mineralization in soils treated with chloropicrin and higher than recommended rates of the other fumigants. At lower than recommended rates, net mineralization was reduced. Carbon dioxide evolution was similarly affected by dosage of these three chemicals. These differences in efficiency of retention of carbon and nitrogen indicate that microbial papulations which were able to survive intensive levels of fumigation were qualitatively different than those which survived treatment at lower dosage or with less toxic materials. Since the nitrifiers are very sensitive to fumigants, it may be assumed that toxic quantities of chemical had been physically removed from the soil prior to recovery of nitrifying capacity. Differential reSponses in heterotrophic numbers and activities were, nonetheless, observed when temperatures were raised in the 10° and 200 rooms near 101: the end of the incubation period, - long after effects on nitrification had disappeared with most treatments. These residual effects of fumigation treatment parallel those observed in the field where temperature sequences following fall fumigation were somewhat similar to those imposed on soils incubated in the 100 room. It was observed in the field that nitrification was not completely suppressed in the early spring months following fall fumigation, although it was retarded. Recovery of full nitrifying capacity was associated with rapid warming of the soil in June. Enhanced mineralization of nitrogen was observed in fumigated soil in the field during the spring and summer, paralleling the dif- ferential stimulus to microbial numbers or 002 evolution or net mineralization associated with different chemical treatments when temperatures were raised during the later stages of incubation in the 10° and 20° rooms. Much larger accumulations of NHh+ were encountered in the field with fall fumigation with Telone than with the same material in the laboratory. A substantial portion of this was already present at the time of the earliest samplings in April. These early accumue lations had very likely accrued during the late October exposure period and remained unchanged during the late fall andwinter months. when soil temperatures were near freezing or below. Such temperatures were not reproduced in the laboratory, - nor were the completely saturated moisture levels which develop normally at the field location during late winter and early spring. LOW'nitrate levels in the early spring in the field were very likely due to leaching of nitrate which may have been present in the 105 fall. The slow accumulation of nitrate in fumigated soil during the spring months suggests that nitrification was limited by low numbers of nitrifiers rather than by any specific residual inhibition by the chemical. The early recovery of nitrifying activity in Telone treated soils in the laboratory supports this conclusion. If this conclusion is correct, then the delays in rapid nitri- fication observed in fumigated soils in the field were due to factors which restricted growth and reproduction of the nitrifiers without hampering the activity of those present. Antagonism by heterotrOphic components of the recovery population may have been involved. In- hibition by excessive NHh* is suggested by the synergism observed between fumigant and (NHh)280 _fertilizer. However, the very marked reduction of the delay periodhby nitrate fertilizers (both the ammo- nium and the calcium salt) suggest that nitrate itself may have stim- ulated the growth of the nitrifiers. It may be relevant to note that the incubation study was conducted with soil which contained initially lh2 ppm of nitrate nitrogen. In the case of the non-fumigant fertilizer additive NQServe, specific inhibition of nitrification was observed h weeks after treatment only at 20°C. At this time, some stimulus to bacterial numbers was expressed at 10° and 20°, and CO2 evolution remained at a higher level than in untreated soil through most of the incubation period at 10° 0. Significantly reduced levels of nitrate and total mineral nitrogen at all three temperatures appeared to be due to immobilization of the ammonium nitrogen added with the chemical. It has recently been rqported that this nitrification inhibitor 106 is inactivated by sorption on organic matter.5 The results reported here for an organic soil suggest that this sorption of N-Serve may be accompanied by a simultaneous irreversible complexing of NHh+° S Goring, C.A.I. 1962. Control of nitrification by 2-chloro-6- (trichloromethyl) pyridine. Soil Sci. 93:211-218. GENERAL DISCUSSION The significant effect of fumigation on the nitrogen economy of soils and crops is to alter the seasonal distribution of ammonium and nitrate. This is a conclusion reached in earlier investigations in Michigan by Wolcott, Maciok et al (83). It was substantiated by the field and laboratory studies reported here and is consistent with results reported by numerous investigators down through the years (1:8) (68) (69) (71) (82) - These effects on the seasonal pattern of nitrogen transformations are mediated by partial sterilization effects on both heteotrophic and autotrophic components of the soil microbial population (78)(31)(32). The results of the present incubation experiment demonstrate that these partial sterilization effects may be projected over long periods of time where soils are subjected to reduced temperatures after initial exposure and aeration to remove volatile fUmigant chemicals. It appeared that these residual effects were generated by heterotrophic components of the recovery population, and were not due to retention of toxic quantities of the chemicals themselves in the soil. In field studies, high concentrations of ammonium nitrogen (200 to 350 ppm) and wide NHL+:N03- ratios (up to 6:1) were encountered in May in organic soil fumigated in the fall with 32 to h8 gpa of Telone (mixture of dichloropropenes). Such abnormally high proportions of ammonium to nitrate may seriously impair metabolism in some plants(23)(51) (55)(h7)(h9)(50)(83). Celery appears to be a crop which is sensitive to such imbalances. Early injury from fumigation was corrected in 1959 and 1960 by sidedressings of nitrate fertilizers. However, the residually'enhanced levels of heterotrophic activity in fall fumigated organic soils give rise also to reductive soil con- 107 108 ditions the following season. As a result, soil fumigation increases the availability of reducible nutrients, notably manganese (lS)(38)(h8). Such increases in manganese availability were responsible for dramatic increases in yields of sweet corn and lettuce in fumigated soil in 1961. With celery, increased availability of iron was reflected by significantly increased concentrations in tissue samples taken in 1960 and 1961. Under conditions of limiting manganese and phosphorus nutrition in 1961, immobilization of phosphorus in the soil or in the conductive tissues of the plant by increased levels of available iron in fumigated soil resulted in significantly reduced yields of celery. Under these conditions, there was no response to early sidedressings of Ca(N03)2. The oxidation state and the solubility of iron and manganese are strongly influenced by soil reaction. Various fertilizer nitrogen sources differ in their direct and indirect effects on soil pH. For this reason, responses to different nitrogen carriers cannot be ascribed indiscriminantly to differences in availability or assimi- lability of the nitrogen. The early reSponse of celery to nitrate fertilizers in 1959 and 1960 appeared, however, to be specifically a response to the nitrate form of nitrogen. No such re3ponse to nitrate was obtained in 1961 with celery, - nor with sweet corn or lettuce. The latter two crops reaponded to (NHh)ZSOh’ but leaf symptoms and tissue analysis revealed that this was a response to manganese rather than nitrogen. Although the early response of celery to nitrate in 1959 and 1960 appeared to have been a specific response to the nitrate form of 109 nitrogen, the possibility remains that it was also a reflection of the much earlier rapid disappearance of toxic concentrations of ammo- nium. There was evidence in each of the three years that fertilizer nitrate greatly accelerated the recovery of full nitrifying capacity in fumigated soil, whereas (NHh)2SO extended the delay period. b The basis for stimulation of nitrification by nitrate is not readily apparent. The possibility presents itself that the Nitrosomonas group of nitrifiers may be able to use nitrate as a terminal electron acceptor in growth metabolism. In 1960, almost complete disappearance of applied nitrate immediately preceded full recovery of nitrifying capacity in fumigated plcts which had been compacted in an area subject to impeded drainage. The nitrate lost in plots which had received Sa{N03)2 or NHLNO3 appeared almost quantitatively as NHu+. If reduction of nitrate by nitrifiers under poorly aerated con- ditions actually contributed to the observed transformation, the Nitrosgmcnas group, rather than Nitrdbacter, appears more likely to have been implicated. Qualitative tests revealed no accumulations of nitrite in fumigated plots, w even when traces were occasionally found in unfumigated soil in the Spring of the year. This would suggest that nitrification was blocked at the first step, leading from NHh+ to NOZ'. The fertilizer additive, N~Serve, appears to have no appreciable direct effect on the heterotrophic population. Its inhibitory effect on the autotrophic nitrifiers is reported to be greatly reduced in soils high in organic matter.5 However, a significant effect on both NHh+ and NO - levels in Houghton muck was observed in the field, and 3 5 See footnote, P.106 110 this effect was additive when combined with fumigation. The retarded nitrification of (NHh)ZSOh delayed the development of the residual acidity associated with this carrier sufficiently to reduce manganese availability and yields of lettuce in 1961, under conditions of critically deficient manganese nutrition. Laboratory studies with NuServe suggested a simultaneous com— plexing of the chemical and NHh+ by organic matter. Appreciable release of the complexed nitrogen was not observed during nearly four months of incubation at 30° C. If this observation is borne out by further work, it suggests the use of this chemical as a direct soil additive to retard subsidence of organic soils and to reduce accumu- lations of nitrate late in the season. Crops such as sugar beets are reduced in yield and quality on organic soils by reason of excessively high nitrogen nutrition duIing the fall. SUMMARY The effects of chemical fumigants and a nonwfumigant nitrifi- cation inhibitor on nitrogen transformations in soil and on availability of nutrients to crops were studied in the field and in the laboratory. An organic soil known to be free of nematodes was used. Marked effects on the seasonal distribution of NHh+ and N03“ were observed following fall application of a fumigant chemical in the field. Laboratory studies confirmed field observations which indicated that these effects were due to readjustments in the heterotrophic and auto- trophic components of the recovery population rather than to retention of toxic concentrations of the fumigant in the soil through the winter. The non—fumigant chemical, applied as a fertilizer additive on (NHh)2SOh in the laboratory, inhibited nitrification without appreciably influencing heterotrophic activities. Simultaneous irreversible com- plexing of the chemical and NHh+ by organic matter appeared to be responsible for the uniform reduction in total mineral nitrogen which was maintained for long periods after addition of this material to the soil. Enhanced mineralization of organic nitrogen and retarded nitri- fication in fall fumigated soil resulted in large accumulations of NHL*, reaching maxima of 200 to 350 ppm in May or early June. Wide ratios of NHh+ to N03~, ranging up to 6:1, appeared to be injurious to early growth of celery. Application of nitrate fertilizer corrected this early injury during two seasons. In the third season, under con- ditions of limiting manganese and phosphorus nutrition, there was no response to fertilizer nitrate. Foliar analysis indicated that in- creased availability of iron in fumigated soil had interferred with 111 112 uptake or translocation of Phosphorus and significantly reduced the yields of celery. In the same season, and in the same experiment, yields of sweet corn and lettuce were significantly increased by the increased availability of manganese in fall fumigated soil. These increases in availability of iron and manganese were attri- buted to the reductive conditions associated with enhanced heterotrOphic activity in the recovery population. In the case of lettuce, an additive response to (NHM)ZSOh at all levels of fumigation was shown by foliar analysis to be due to an additive effect on manganese availability resulting from the acidifying action of this nitrogen carrier. Leaf symptoms in sweet corn showed a similar manganese response to (NHh)230h prior to silking, but addi- tional increases beyond the fumigation response were not significant. 10. ll. 12. 13. lb. BIBLIOGRAPHY Alexander, Martin. Introduction to Soil Microbiology. John Wiley and Sons, Inc., New York, 1961. Arrington, L. and Shive, J. Rates of absorption of ammonium and nitrate nitrogen from culture solutions by tenuday-old tomato seedlings at two pH levels. Soil Sci. 392b31-b35. 1935. . Baslavskaja, S. 5. Influence of chloride ion on the content of carbohydrates in potato leaves. Plant Phys. 11:863-871. 1936. . Bear, Firman E. Cation and anion relationships in plants and their bearing on crop quality. Agron. J.h22176—178. 1950. . Beeson, Kenneth C. 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