The effect of anionic and nonionic detergents on soil microfungil

BENNY K. H, LEE* Department of Botany, University of Wyoming, Laranzie, Wyotning

Received September 10, 1969

LEE, B. K. H. 1970. The effect of anionic and nonionic detergents on soil microfungi. Can, J. Bot. 48: 583-589.

Surveys of the microfungal populations in potting soil watered with tap water, a solution of anionic 15-S-3A detergent (Tergitol, 15-S-3A), and a solution of nonionic 15-S-9 detergent (Tergitol, 15-S-9), revealed a greater reduction in propagule densities in the anionic detergent watered soil than in the nonionic detergent watered soil. In the anionic detergent study, 39 species, including six dominant species which contributed 58y0 of the isolates, were obtained from the treated soil. In the nonionic de- tergent study, both sample populations contained 29 species and the four to six dominants contributed 67-68yo of the 100 isolates.

Twenty-two isolates representing 10 dominant species were tested in vitro for sensitivity to the two detergents. All grew at the lowest concentration (10 p.p.m.) of both detergents. The growth rates of nine isolates increased with the addition of 10 p.p.m. of the anionic detergent; four isolates showed in- creased growth with 10 p.p.m. of the nonionic detergent. With increasing concentration of detergent from 100 to 1000 p.p.m., the growth rates of 21 of the 22 fungi decreased. Isolates recovered from de- tergent-treated soil grew better in the medium containing detergent than the same species recovered from the control soil. In concentrations of 1000 p.p.m., complete inhibition was displayed by four species in the anionic series compared to five species in the nonionic series. Both detergents affected pigmentation, exudate formation, and rigidity of the sporangiophore in several of the fungi.

Fourteen of the 16 isolates tested used anionic detergent and 12 isolates used nonionic detergent as a carbon source for growth. Dry weight differences indicated that the anionic detergent was more avail- able than the nonionic detergent.

Introduction

Since the time of Robert Koch, 1881 (ll) , the germicidal action of soaps has been known. Eggerth (2, 3, 4, 5) described the germicidal ac- tion of several types of soaps. He demonstrated that the bactericidal and bacteriostatic action of soaps is chiefly a function of the length of the hydrocarbon chain, its chemical configuration, and the hydrogen ion concentration. In the last 40 years, synthetic detergents have been devel- oped. These currently are favored for certain advantages over soap. For example, the sodium alkyl sulfates retain their efficiency in hard water. The bactericidal action of synthetic detergents was initially emphasized by Domagk in 1935 (9). He demonstrated that Zephiran (alkyldimethyl benzyl ammonium chloride) and other cationic, quaternary ammonium detergents possess ex- cellent germicidal properties. By 1941, more de- tailed investigations of the action of synthetic detergents on bacterial metabolism had been re- ported by Baker et al. (1). Initially they observed that a very low concentration of Zephiran inhib- ited the glycolysis and respiration of some micro-

lsubmitted as partial fulfilment of the requirements for the M.S. degree in Botany at the University of Wyoming, Laramie, Wyoming.

2Present address: Department of Botany, University of Hawaii, Honolulu, Hawaii.

organisms in pure culture. Later they used cationic and anionic synthetic detergents to study their effect on respiration of Gram-positive and Gram-negative microorganisms. The results ob- tained with anionic detergents indicated a much lower level of inhibition of bacterial respiration and glycolysis than that of cationic derivatives. The cationic detergents showed their maximum inhibitory effect under alkaline conditions, whereas the anionic detergents were most inhib- itory in acid solutions.

The effect of detergents and other surfactants on fungal growth has been investigated recently. Sevag and Ross (13), after studying the mecha- nism of inhibitory action of Zephiran on yeast cells, reported that it inhibited the cytochrome oxidase of yeast cells. In 1960, Irwin and Hugh (7) demonstrated that the maximum toxicity to cells of Saccharonzyces pastorianus Hansen and conidia of Monilinia fructicola (Winter) Honey was exhibited by the homologous series of n- alkyl guanidine acetate compounds (dodine) with n-alkyl groups of 13-14 carbons. Forsyth (6) compared the activity of some cationic (Hyamine 3500, Cyprex or Dodine, Triton X-400), anionic (Atlox G-3300, Santomerse), and several non- ionic surfactants (including Tritons X-100 and X-114) which either inhibit respiration and spore germination of some test organisms, or act as

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584 CANADIAN JOURNAL OF BOTANY. VOL. 48. 1970

agents which promote the exudation of amino acids from the spores. Steiner and Watson (14) tested the inhibitory and toxic effects of cationic, anionic, and nonionic surfactants on fungi. The cationic agents inhibited more fungi than the anionic series tested, which required higher con- centrations for equivalent colony inhibition. The nonionic surfactants were inhibitory in some degree for several fungi above a concentration of 100 p.p.m.

Since little is known about the relationship of soil fungi to detergents, an investigation of the effect of detergents on the soil microfungi was undertaken. Two approaches were used: the determination of the effect of detergents on the fungal population of soil and sensitivity of the isolated dominant species; and the inves- tigation of the possibility that detergents, which are organic compounds, can be decomposed by fungi and serve as a source of carbon for fungal growth.

Materials and Methods Two specific detergents, namely Tergitol anionic

15-S-3A and Tergitol nonionic 15-S-9(16), were used in this study. The chemical compositions of these two deter- gents as described by the Union Carbide Corporation are "Tergitol anionic 15-S-3A is obtained by sulfation of the terminal alcohol group of a polyoxyethylene chain followed by neutralization to yield the ammonium salt. Tergitol nonionic 15-S-9 is derived by ethoxylation of linear secondary alcohols. The linear alkyl hydrophobe portion is a mixture of straight C-11 to C-15 chains. The hydrophilic portion is a polyoxyethylene chain attached to the linear aliphatic chain through an ether link (16)." Loam soil, collected from Laraniie, Wyoming, was dis- tributed in 3-in. pots. Radish plants were grown in the soil during the treatments, but effect of treatments on these plants is not discussed in this report. One set of pots was watered with Tergitol anionic 15-S-3A detergent (1000 p.p.rn.) for 90 days, and another sct with Tergitol nonionic 15-S-9 detergent (1000 p.p.m.) for 67 days. Each detergent treatment was watered daily with 50 nil of de- tergent. A control set for each series was watered with 50 ml of tap water daily for the same length of time. Two pots of each scries and its control were sampled a t the conclusion of the watering periods for the microfungi present in each soil using the dilution plate method (8). Soil samples were prepared by removing from each pot three or four cores of soil which were mixed thoroughly in a sterilized airtight plastic bag. Five grams of each mixed soil were used to make the initial 1 :10 dilution with distilled water. Soil extract agar (15) was prepared for plating each soil type. In a preliminary plating, it was found that a dilution of 1 5 0 0 or 1 :I000 contained about 3C300 microfungal propagules per milliliter. After a 3- day incubation period, hyphal tip transfers were taken from the isolation plates. One hundred random isolations were made from each soil. After 1C12 days' incubation,

the cultures were sorted into apparent taxononlic entities on the basis of cultural and niorphological features. The numbers of isolates for all apparent entities were recorded and the dominant species were selected and identifie.d Species with more than three isolates in one population were designated as dominant species. The number of or- ganisms per gram of dry soil was calculated according to a formula given by Waksnian (17).

After determination of the number of colonies per gram of dry soil, 10 dominant species were selected for sensitivity testing. If the species occurred in control soil and detergent-treated soil, isolates from both sources were used. Czapek solution agar (DIFCO 0339-01) was used as the base medium. The detergent solutions were sterilized by filtration through 0.22 p sterile membrane filters (MF-MILLIPORE, CS). Different concentrations of anionic and nonionic detergents were added aseptically to Czapek solution agar separately to yield final concen- trations of 10, 100, 500, 1000 p.p.m. of detergent. Petri dishes containing the test concentrations of detergents and one control medium were inoculated uniformly and aseptically with a piece of young mycelium about 1 mm in diameter. No replications were niade. After incubation for 8-12 days at room temperature (24-26 "C), colony di- ameters were measured and colony morphology noted.

Ten species isolated from control or detergent treated soils or both were used as test organisms to determine use of the detergents as a carbon source for growth. The fungi were cultured in liquid Czapek's medium, 8 ml per tube, modified in three ways as follows.

(1) With sucrose in concentrations of 10, 100, 500, and 1000 p.p.m.; this series served as the control.

(2) With each of the detergents in concentrations of 10, 100, 500, and 1000 p.p.rn, replacing the sucrose. By use of a Swinny filter holder (Millipore Corp., No. xx 30-012- 00) with a sterile 10 ml syringe, 1 rnl of detergent stock was transferred to 7 ml sterilized medium without sucrose to yield the desired final concentration. The Swinny filter adapter has the advantage of sterilizing without changing the chemical composition of the detergents. Before ad- dition of the detergent, the medium was tested with Benedict's solution after the addition of 1 ml of HCI to establish that there was no reducing sugar in the solution.

(3) Without sucrose. This also was tested for the ab- sence of reducing sugars.

All solutions had a pH of 7.7-7.8 measured with a glass electrode pH meter. Culture media were inoculated from 1 loop of spore suspensions prepared in sterile distilled water. Three replications of each isolate in each medium were prepared. After incubation for 25 days at rooni tern- perature (2426"C), notations on fungal growth were recorded. Then the mycelium was scparated from the cul- ture medium by suction filtration, washed in distilled water, dried at 80°C for 2 days, and weighed.

Results and Discussion

The Effect of Detergents on tile Number and Kinds of Fungi in the Soil Populations

A comparison of the fungal populations in the control soils with those in the anionic detergent treated soil and the nonionic detergent treated

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LEE: EFFECT O F DETERGENTS ON MICROFUNGI 585

soil populations is shown in Table I. Because the total number of fungi present in the two control soils differed greatly, the strict comparison be- tween the two treatments may be invalid. The results do indicate, however, that anionic 15-S-3A detergent effected a greater reduction in the fun- gal propagules in the soil than did the nonionic 15-S-9 detergent. The reduction in numbers ex- pressed as percentage was 237, for anionic series but only 167, for the nonionic. The difference in reduction was even more striking for species; 36% disappeared from the anionic series but none from the nonionic series. The detergent so- lutions were used in the pot tests at 1000 p.p.m.,

a level demonstrated to be at least 5076 inhibitory to growth for 17 and 11 of 22 isolates in the an- ionic test and nonionic test, respectively (Table 111).

In the anionic detergent series, 25 species in- cluding 4 dominant species were isolated from the anionic detergent treated soil compared with 39 species including 6 dominant species in the control soil. The four detergent-treated soil dom- inants accounted for 75y0 of the isolates in that population, while the six dominants in the con- trol soil accounted for only 58% of the isolates from that soil (Table I). Three species were dom- inants in both soils; Sporonsna sp. was a fre-

TABLE I Some characteristics of the soil and the fungal populations in the untreated control soils

and detergent-treated soils

Anionic series Nonionic series

Anionic Nonionic detergent detergent

Control treated Control treated soil soil soil soil

Water content (%) fresh weight 30 26 24 28 Numbers of microfungi per gram of

dry soil, in thousands 131 101 46 38 Reduction in numbers of microfungi

as a % of control 23 16 Numbers of species in total population 39 25 29 29 Reduction in Yo of species 36 0 Number of dominant species 6 4 4 6 % of total isolates contributed by the

dominant species 58 75 68 67

TABLE I1 The number of isolates and the dominant fungi in the control soils, Tergitol anionic 15-S-3A detergent treated soil and

Tergitol nonionic 15-S-9 detergent treated soil -

Number of isolates

Anionic Nonionic detergent detergent treated Source treated Source

Control soil, of Control soil, of Svecies soil 90 days dominance soil 67 davs dominance

Actirzotnucor harzii Penicillium lilacirzrrm Pet~icilli~rm steckii Sporotzen~n sp. Penicillium stoloniferlmz Aspergillrrs rrstrrs I Aspergillus ~rs t~ts I1 Penicillilrm simplicissim~rm Chnetophon~a sp. Aspergillus sp.

' C , control soil. D, detergent soil.

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586 CANADIAN JOURNAL O F BOTANY. VOL. 48, 1970

quent form in the detergent-treated soil but infrequent in the control soil; and two Penicillium species and one Aspergillus species, compara- tively common in the control soil, were absent or rare in the detergent-treated soil (Table 11).

In the nonionic detergent series, 29 species were found in both control soil and in the deter- gent-treated soil (Table I). Four dominant species contributed 68 isolates in the control soil. Six dominant species in the detergent-treated soil contributed 67 isolates. Actinomucor harzii, Peni- cillium lilacinum, and P. steckii were dominant species in both soils (Table 11). One unidentified Aspergillus species was dominant only in the con- trol soil. Aspergillus ustus, isolate 11, Sporonema sp., and Chaetophoma sp. were dominant only in the detergent-treated soil.

Three dominant species (Actinornucor harzii, Penicillium steckii, Sporonema sp.) were favored in the anionic 15-S-3A detergent treated soil and four species (Penicillium lilacinum, P. stolonije- rum, P. sirnplicissimum, and Aspergillus ustus, isolate I) were inhibited (Table 11). In the non- ionic 15-S-9 detergent treated soil, four species

(Penicillium steckii, Sporonerna sp., Aspergillus ustus, isolate 11, Chaetophoma sp.) were favored and three species (Actinomucor harzii, Penicillium lilacinum, Aspergillus sp.) were inhibited. Only Penicillium steckii showed an increase in num- bers for both anionic and nonionic soils (Table 11). Those species favored in the detergent-treated soil may have used the detergent as a carbon source; the detergent may have stimulated sporu- lation; the detergent may have inhibited certain sensitive species, thereby allowing tolerant spe- cies to become dominant; or a combination of these factors may have been involved.

The Sensitivity of the Isolated Dominant Species to Detergents

The sensitivity of the isolates to the detergents was tested in vitro and expressed as diameter of colony growth. Both detergents generally inhib- ited colony growth of the fungi with increasing concentration of the detergents (Table 111). Com- paring colony growth of the water isolates in anionic 15-S-3A and nonionic 15-S-9 detergents, the anionic detergent was more inhibitory than

TABLE 111 Growth of 10 fungi on media containingTergito1 anionic 15-S-3A and Tergitol nonionic 15-S-9 detergents after 12 days

(except Actir~omucor Izarzii after 8 days' growth)

Diameter as % of controlt Diameter

(cm) Anionic 15-S-3A detergent Nonionic 156-9 detergent on Czapek

without 10 100 500 1000 10 100 500 1000 Species Isolate* detergent p.p.m. p.p.m. p.p.m. p.p.m. p.p.m. p.p.m. p.p.m. p.p.m.

Penicilli~~n W 3.5 112 100 80 43 111 85 70 60 stolonifbruin A 3.2 120 110 53 47 120 97 72 63

P. simplicissim~rm W 4.8 104 79 52 40 104 42 31 30 P. lilacinrrm W 4.7 94 73 36 34 87 53 34 30

A 5 .O 90 68 40 36 92 50 40 40 N 4.6 91 63 43 39 98 57 50 46

P. steckii W 2.3 87 70 61 48 87 65 52 26 A 2.2 109 82 73 68 82 77 59 50

-. . - . -

CI~aetophoma sp. N 4.1 110 93 49 39 98 71 60 54 Aspergill~rs sp. W 5.0 70 66 52 40 74 70 56 52

N 4.8 100 7 1 60 54 100 71 58 54 Actinomucor harzii W 6.0 110 67 10 0 72 45 22 16

*W = water isolate (control soil); A = anionic detergent isolate; N = nonionic detergent isolate. tControl = 100%.

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TABLE I V Growth (mg dry weight) of 10 fungi in the presence and absence of Tergitol anionic 15-S-3A detergent and Tergitol nonionic 154-9 detergent after 25 days

Growth and dry weight (mg) of fungi in the modified Czapek liquid media* Czapek E

m medium Sucrose (pH = 7.7) Anionic detergent? (pH= 7.8) Nonionic detergent? (pH = 7.8) without v sucrose 10 100 500 1000 10 100 500 1000 10 100 500 1000

Species Isolate (pH=7.7) p.p.m. p.p.m. p.p.m. p.p.m. p.p.m. p.p.m. p.p.m. p.p.m. p.p.m. p.p.m. p.p.m. p.p.m. 4 0

Penicilliunz w$ 0 x 2.6 3.0 3.7 x 2.1 2.3 l.S(l.5-2.1) 0 0 0 0 v stolor~i&erum

P . siniplicissirnurn W 0 x x 0.2 0.3 0 x x 0.1 0 E x 0.1 0.1 ;;1 P. tilacinurn W 0 x x 0.3 1.8 x x 0.4 0.5 0 x x x

W 0 7

P . steckii x x 0.5 0.9 x x x 0.3 x x x 0.2 0 A 0 x x 0.9 1.4 x x 0.6 0.3 x x N 0 x x 0.7 1.3 x x 0.6 0.3 x x

Chaetophoma sp. N 0 x x 0.3 1.1 x x 0.2 x 0 0 x d3 8

W 2.3 2.4 x 0 0 0 0

Aspergillus sp. tr x 2.2 x 1.4(1.1-1.7) x x 21 A. ustus I W tr x x 0.8 1.4 x x x x 0 0 0 x A. ustus I1 W 0 x x 0.7 1.1 0 0 0 0 x x x x

R r(

Acti~lo~n~icor harzii W 0 x 0.4 O.g(O.6-1.2) 1.5 0 0 0 0 0 0 0 0 8 A 0 x 0.5 1.3 2.0 x x x x 0 0 0 0 % N 0 x 0.2 1 .O 1.5 x 0 0 0 0 0 0 0 c

Sporonema sp. W 0 x x 1 .O 1.9 0 x x x x x x x A 0 x 0.3 1.2(0.9-1.5) 2.1 x x x x x x x x s? N 0 0 x O.S(O.7-1.2) 1.5 x x x x x x x x

*O. no growth: tr, trace: x, dry weight less than 0.1 mg. Dry weights based upon three replicate cultures for each isolate, each medium. Weight ranges of less than 0.5 mg have been omitted. tczapek's medium without sucrose and supplemented with the indicated amount of detergent (see text). SW, water isolate; A, anionic detergent isolate; N, nonionic detergent isolate.

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588 CANADIAN JOURNAL OF BOTANY. VOL. 48, 1970

the nonionic detergent for Penicilliurn stolonife- rum, Aspergillus sp., Aspergillus ustus, isolate 11, and Actinon7ucor harzii. The nonionic detergent was more inhibitory than the anionic detergent for Penicilliurn simplicissimum, P. lilacinum, P. steckii, Sporonema sp., and Aspergillus ustus, iso- late I. In general the isolates from detergent- treated soils grew better than the water isolates on the media containing detergent. This could represent either a selection of tolerant strains or a physiological adaptation to the detergent- treated soil.

Some fungi exhibited increased growth rates in Czapek agar with 10 p.p.m. of detergent (Table 111). Six detergent isolates (Penicilliz~m stoloniferunz, P . steckii, Clznetophoma sp., Actino- mucor harzii, and two isolates of Sporonema sp.) and three water isolates (Penicilliurn stoloniferu~n, P . simplicissinzum and Actinonzucor harzii) grew better on a medium containing anionic 15-S-3A detergent than on the medium without detergent. Two detergent isolates (Penicilliurn stoloniferum and P. simplicissimum) exhibited better growth on media containing 10 p.p.m. nonionic deter- gent. At all concentrations in excess of 10 p.p.m., growth was inhibited in both series except for one anionic isolate, Penicilliunz stolonifer~lm, which exceeded 100yo in the anionic series when used at 100 p.p.m. At 1000 p.p.m., though, 11 isolates in the nonionic series achieved 50y0 or better of the control growth. In the anionic series only five isolates showed a comparable growth level. Cl~ae- toplzon~a sp. was recovered only from the non- ionic detergent soil, suggesting that the nonionic detergent specifically favored its active growth, an assumption which was not supported either by colony growth or dry weight. It was more tolerant, though, of 500p.p.m. and 1000p.p.m. of nonionic than anionic detergent in the growth series. As the original nonionic detergent treated soil was watered with a solution of 1000 p.p.m., this may explain its recovery from the nonionic detergent treated soil.

The detergents affected the morphology of

lates and even when the detergent isolate from detergent-treated soil was grown in the non- modified Czapek medium for 12 days, the ex- udate was less than that formed by the isolate from water-treated soil. The rigidity of the spo- rangiophores of Actinomucor hwzii decreased when the fungus was grown in the detergent media. Some sporangiophores collapsed after 12 days growth which was not characteristic of growth in the control.

Tlze Use of Detergents as a Carbon Source for Fungal Growth

At low concentrations the detergents generally were used as a carbon source for fungal growth (Table IV). Eight species (Penicilliurn stolonife- rum, P. simplicissimunz, P. lilacinum, P. steckii, Clzaetophoma sp., Aspergillus ustus isolate I, As- pergillus sp., Sporonema sp., and the detergent isolates of Actinonqucor Izarzii) used the anionic 15-S-3A detergent. Aspergillus sp., Aspergillus ustus, isolates I and 11, Chaetoplzoma sp., Sporo- nema sp., Penicilliutn lilacinum, P, steckii, and P. simplicissimutn used the nonionic 15-S-9 deter- gent.

In comparing use of the anionic and nonionic detergents as a carbon source for the 10 fungi tested, it is evident that the anionic detergent is more available than the nonionic detergent. An- ionic 15-S-3A detergent is an ammonium salt and may supply nitrogen for the fungal growth. As early as 1914 McLean and Wilson (12) reported that when ammonium salts are found in the soil, some of the soil fungi quickly used them, and converted them into fungal protein. Later studies by Robbins (1937) and Steinberg (1939 and 1950) showed that fungi fall into fo~ir categories of nitrogen use; the fiingi in three of these can use aminonium nitrogen (10). Another possible ex- planation may be that nonionic detergent is more inhibitory than the anionic detergent to the 10 fungi tested. The mechanism of the inhibition was not determined. Further study of the deter- gent effect of the fungi is necessary.

some species~~porulation in ~enicilliim lilacinum and P. steckii was obviously reduced. Pigment Acknowledgments diffusing from the mycelium was conspicuous The author expresses his sincere appreciation when P. lilacinum grew on detergent medium. to Dr. Martha Christensen and Dr. Henry T. Exudate production in Penicilliurn stoloniferum Northen for their generous advice, comments, also was affected by the detergents. Less exudate and encouragement throughout this study, and was formed in the detergent media by both iso- thanks Dr. Gladys E. Baker and Dr. Roger D.

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LEE: EFFECT OF DETERGENTS ON MICROFUNGI 589

Goos for their time and generous help in prepar- ing this manuscript. The cooperation of the Union Carbide Chemical Division for providing the chemical descriptions of the detergents is also greatly appreciated.

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13. SEVAG, M. G.: and 0. A. Ross. 1944. Studies on the mechanism of the inhibitory action of Zephiran on yeast cells. J. Bacterial. 48: 677-682.

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