STUDIES ON SOIL ACTINOMTCETES IN RELATION TO POTATO SCAB AND ITS CONTROL by Glenn KgpKnlght Thesis Submitted In partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Graduate School, Michigan State College Department of Botany and Plant Pathology June, 1939 ProQuest Number: 10008349 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008349 Published by ProQuest LLC (2016), Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ACKhOWLEDGEiViElMT The writer wishes to express his indebtedness to Dr. J. H. Muncie for advice and aid in carrying out this study, to the staff of the Farm Crops Potato Office of Michigan State College and of the Lake City Potato Experimental Farm for their cooperation, to Dr. W. D. Baten for aid in statistical analysis of data, and to Drs. J. E. Muncie and E. A, Bessey for criticism and correction of the manuscript. This investigation was carried out as a portion of a Bankhead-Jones Project on "Biological and physiological studies of Actinomyces scabies in relation to the scab disease of potatoes". 121499 TABLE OF CONTENTS PAGE 1 Introduction *........ *........ The use of antiseptics as soil treatments Review of the literature ........... 3 Materials and methods ............... 5 Results ........................ 7 Conclusions H ....................... The reason for failure of mercurials to control Review of the literature ......... Soil reaction ........ 20 * Oxidizing and reducing agents .......... Mercury placement tests 22 22 ............ Experiments with Long Island and New Jersey soils in 1937 ................ 24 29 Experiments with Long Island and New Jersey soils in 1938 .......... 38 Effect of calomel on yiel d 41 ...... Conclusions regarding the effect of calomel on scabbing ........ The causes of increases in scabbing from mercurial soil treatments ........... 43 55 Effect of mercurial soil treatments on the parasitism of Act. spp. on beets, radishes, and other hosts ........ Effect of mercurials on the host plant .. 58 63 PAGE Effect of mercurials and other disin­ fectants on the soil flora Technique ...... .......... • 67 68 Mercuric chloride in localsoil .. 72 Calomel in Long Island soil...... 72 Calomel in local soil.... ........ 75 Calomel in the pot experiment, 1938 88 Sodium fluoride In local soil ... 89 Studies on the tolerance of actinomycete-isolates to mercuric chloride 97 Conclusions ................ 100 Studies on biological control of potato scab Review of the literature .......... 103 Materials and methods ............. 114 Greenhouse trials in 1936 .... 117 Field trials in1936 ............ 118 Field trials in1937 .............. 119 Field trials in1938 .............. 120 Field trials with green and stable manures ............ 127 Studies on the associative action of Clark’s bacteria ..... 131 Discussion of biological control .... 138 PAGE Studies on the host range of phytopathogenic Actlnomycetes Review of the literature ....... 145 Field experiments and observations .. 150 Pathogenicity tests .... 154 Discussion of pathogenicity tests ... 164 Observations from isolations of Actinomycetes and dilution-plate counts...... 166 Discussion Summary Literature cited ..................... .......... 177 193 ....... 205 Studies on Soil Actinomycetes In Relation to Potato Scab and Its Control INTRODUCTION Measures which have been recommended for the control of scab include the use of resistant varieties, clean or disinfected seed, crop rotation, green-manures, potentially acid fertilizers, sulphur soil treatments, and soil disinfection by chemical means. Apparently any one of these control measures may be effective under certain conditions, but none has general appli­ cation. It seems probable that the scab problem will eventually be solved by the production of resistant verities, but until varieties are developed which are suitable to Michigan and which show a higher degree of resistance than those now grown, other control measures must be employed. The main purpose of this investigation was to de­ termine, if possible, the reason for failure of practices, which have been effective elsewhere, to control scab in infested soils under Michigan conditions. Special emphasis was placed on the cause of conflicting results from mercurial soil treatments* from several angles; This problem was approached (a) the re stilts of numerous soil treatments were compared, (b) the effect of mercurials - 2 - on scabbing of potatoes and other hosts of Actinomyces was compared, (c) investigations were made on the in­ fluence of different soil actinomycete populations on the effect of mercurials on scabbing, (d) on the effect of mercurials on predisposition of various hosts plants to scabbing, and (e) on the effect of mercurials on the soil flora. Attempts also were made to obtain con­ trol of potato scab through associative action of various fungi and bacteria. - 3 - THE USE OP ANTISEPTICS AS SOIL TREATMENTS FOR SCAB CONTROL Review of the literature The chemicals which have previously been tried by various investigators as soil treatments for the control of potato scab are aluminum sulphate, benzene, bleaching powder, Bordeaux mixture, carbon disulphide, copper sulphate, creosote, formaldehyde, hexamethylenetehramine, tetrachloroethane,„and various mercury compounds* Of these aluminum sulphate was partially effective in western New York but reduced yield (13^), and was of no value even at 1080 lbs. per acre in Wisconsin (32) and reduced scabbing at low rates in one instance (158) but not in others (42,43) in Mich­ igan. Copper sulphate was fairly effective in one trial in New Jersey (54), and in Michigan (158), but gave no control in Vermont (77). the soil was of Bordeaux mixture applied to value in New Jersey in some instances only (54), while frequent heavy applications of Bordeaux mixture to the foliage also tended to reduce scabbing. As a foliage spray Bordeaux mixture also reduced scabbing under New York conditions (86,87). Formadehyde as a soil treatment has been tried in New Jersey (54.|s, - 4 - England (63), Holland (126), and in Czecholovakia (112), and has been reported ineffective in controlling scab except in Czechoslovakia where it gave partial control at high rates of application. Carbon disulphide, ben­ zene, and kerosene were ineffective in New Jersey (54). Cresote and bleaching powder were of no value in England (96). In limited trials tetrachloroethane and hexamethylene-tetramine gave no promise of scab control in western New York (139). Various mercury compounds have given partial to complete control of scab in New Jersey (52,53,89,91-93), Long Island, New York (26), Main^93), Canada (83), England (63) Holland (126), Germany (138), and Hawaii (104). Conversely, mercurial soil treatments have failed to control scab in western New York (t 3 Fft\S % s * Mich­ igan (42,43,68), Ohio (106), and in some soils in New Jersey (27,54,93). In a previous study (6 8 ) the author failed to control potato scab in Michigan with various antiseptics applied as soil treatment. These are here listed with the highest rate per acre of each employed: aluminum chloride, 500 lbs.; borax, 50 lbs., cerium oxalate, 500 lbs.; coThalt chloride, 250 lbs.; copper sulphate, - 5 - 250 lbs*; cuprous oxide, 50 lbs.; lead acetate, 250 lbs*; mecuric oxide, 20 lbs.; mercurous chloride, 150 lb.; ”Wew Improved Ceresan (5$ ethyl mercury phos­ phate), 150 lbs.; nickel nitrate, 250 lbs.; potassium permanganate, 500 lbs*; potassium dichrornate, 250 lbs.; sodium fluoride, 500 lbs.; zinc oxide, 50 lbs.; zinc sulphate, 250 lbs.; nFormacidew, 650 lbs.; gentian violet 10 lbs.; malachite green, 150 lbs.; coal tar creosote, 20 gallons,; and wood creosote, 20 gallons. In a greenhouse trial, nickel cyanide controlled scab at 500 lbs. per acre, but not at 150. It therefore would be of little value because of the quantity re­ quired. Materials and methods Except where otherwise designated, all soil treatments were made in randomized, replicated, field plots. To facilitate application, the chemicals were diluted with air-dried soil from the field in which they were to be tried, and were applied in the plant­ ing furrow. The plots at East Lansing consisted of 10 hills each; those at Lake City of 40 hills each In 1937 and 80 hills in 1938. In all cases each treatment was repeated three times. The plots at East Lansing were planted to formadehyde-treated Katahdin tubers, and those at Lake City to untreated certified seed of the same variety. Figure 1 m m m I m m United States Department of Agriculture chart for estimation of the percent of the surface area of tubers scabbed. - 6 - In applying statistical analysis to the data, the percent of the tubers scabbed was not a satisfactory figure, since at East Lansing this was often 100$. The crop was sorted into grades according to the per­ cent of the surface area of the tubers scabbed, using the photographs supplied by the United States Depart­ ment of Agriculture as a basis of estimation (figure 1). The range for each grade was: clean, 0-2$ of the sur­ face area scabbed; light, 3-20$; medium, 21-50$; heavy, 51-100$. The percent of potatoes by weight or by nump ber falling into each class was then determined, and an arbitrary figure was taken as the average percent of the surface area of the tubers scabbed in each class. For example, in 1937 at East Lansing these figures were* light, 10$, medium, 30$; and heavy 70$. In other cases different figures were taken, an attempt being made to closely approximate the actual percent of the tuber surface area scabbed in each class in that par­ ticular trial. The same arbitrary figures were used for all treatments in an experiment, and in this manner the scabbiness of the tubers for each plot is expressed in a single figure. Thus the severity of scabbing in the various plots is readily compared. - 7 - Results In 1957 at East .Lansing in soil of ph 6 .9-7.5 (Table 1), mercury compounds aggravated scabbing, in some cases causing a four-fold increase. A similar increase in scabbing occurred v/ith combinations of calomel (6 and 20 lbs. per acre) with zinc (50 Tbs.), with sodium nitrite (1 0 0 and 300 lbs.), and with potassium permanganate ilOO lbs.). Sulphur alone at 300 lbs. and combinations of sulphur with calomel and with sodium nitrite had no significant effect on scabbing. Likewise zinc (50 lbs.), lead sulphide (50 lbs.), lead acetate (100 lbs.), and aluminum sulphate (50 lbs.) had no significant effect on scabbing. In another experiment in the same year and place (Table II), hydrochloric acid and sulphuric acid, each at 1500 lbs. per acre, and the same treatments in combination with mercuric chloride (15 lbs.) and copper sulphate (30 lbs.) respectively, as well as the two latter compounds applied separately, showed no tendency to reduce scabbing. The combination of mercuric chloride with hydrochloric acid caused a n gpp increase in scabbing. In a third experiment at East Lansing in 1937, sodium chlorophenylphenate, sodium orthophenylphenate, sodium tetrachlorophenate, sod^ium 2,4,5, tricialo- - 8 - rophenate, and tribromophenol each at 310 and 930 lbs*, per acre of the 1 0 $ preparation on both diatomaceous earth and bentonite, and 10 $ tetrachloroethane on diatomaceous earth at 310 lbs. all had no effect on scabbing. The scabbing in these plots was unusually uniform, running about 20 $ of the surface area of the tubers in control and treated plots alike. Fairly acid soil (pH 5.2-5.8 ) at the Lake City Potato Experimental farm was artificially infested with scab in the spring of 1937 by sprinkling in the plant­ ing furrows fresh horse manure mixed with macerated peelings (2 bus.) from scabby potatoes and manurecultures of half a dozen isolates of Actinomyces from potatoes (a two-quart jar of each). In this artifically infested acid soil, mercury compounds (calomel, corro­ sive sublimate, yellow oxide of mercury, and ethyl mercury iodide) tended to aggravate scabbing, although only yellow oxide of mercury gave an increase in fW tferecf scabbing that reached statistical significance. 4 Zinc at 75 lbs. per acre, and sulphur, both as seed and soil treatments, apparently reduced scabbing almost to nothing, although the reductions in scabbing were not statistically significant. The mixture of mercury compounds with sulphur in seed and soil treatments was In no case better than sulphur alone. m■and'-150 lbs . )-gave* cofr- copper oxide at all three rates of application (50, 100, and 150 lbs.) gave considerably less scab than the controls but not significantly less. Lead arsen­ ate was of no value as were also combinations of zinc (50 lbs.) with yellow oxide of mercury (6 , 20, and 50 lbs.) and with calomel at the same rates, and combina­ tions of both calomel and corrosive sublimate with red copper oxide, and corrosive sublimate with oxalic acid (Table III). In the fall of 1937 scabby potatoes were broad­ cast over this field and the soil was limed at 600 lbs. per acre. Ammonium thicty^yiate and the soil treatments that had given some reduction in scabbing in 1937 were tried in 1938 on the same land that had been used for soil treatments in 1937, the rows running at right angles to those of the previous year. soil reaction at harvest was pH 6 .6 -6 .9. The If zinc, red copper oxide, and sulphur were of value in the more acid soil, they certainly were not after the soil had been limed. Ammonium thiocyanate depressed yield without affecting scabbing. At 500 lbs. per acre only a few plants emerged, and these yielded poorly (Table V). In 1938 at East Lansing ammonium thiocyanate was tried at 500, 1000, and 2000 lbs. per acre in the plant­ ing furrow and the potatoes were planted both two and four weeks xater. Not a plant in the treated plots - 10 - emerged. Dowieides A,B,G,F,G,1,2,4, and 6 were tried each at 25, 100, and 250 lbs. per acre, hut it was so obvious that none of them had any appreciable effect on scabbing that the crop was not graded except for the controls which gave 21 % of the surface area of the tubers scabbed. In these field, trials, the percent of the sur­ face area of the tubers scabbed was calculated (as described under "Materials and methods”) on the basis of the weight of the tubers falling into each class at Lake City, and on the small plots at East Lansing. Bo yield data were taken on the small plots at East Lansing, but very obvious reduction in yield was caused by "DuBay #1155HH” at high rates of application. Except in the case of ammonium thiocyanate, there were no significant reductions in yield at Lake City with the treatment tried in 1958, but in 1937 ’’DuBay #1155BH" at 75 and 100 lbs, per acre, red copper oxide at 150 lbs., combinations of both calomel (2 0 lbs.) and corr­ osive sublimate (2 0 lbs.) with red copper oxide (50 lbs.), and some of the seed treatments with mixtures of mercury compounds with sulphur caused significant reductions in yield. If the plots had been larger, it is possible that some of the other treatments also would have shown significantly reduced yields. (Tables LV & V ). - 11 - Conclusion Of all the chemicals and combinations of chemicals that have been tried in Michigan at various rates of application, in this and previous experiments, none have shown any appreciable value under field conditions, except sulphur in some instances. Of all these chem­ icals, none have shown a consistent^wfe tendency to aggravate scabbing except mercury compounds. - 12 - TABLE 1 Effect of Soil•Treatments on Scabbing of Potatoes at East Lansing, 1957 ______ Planted May 18; harvested Sent. 7. 57 & 58 1 2 3 4 Treatment lbs. per % of surface T X acre area scabbed None Sulphur roll Sulphur KMh04 Zinc Alg(S04)2 PbS Pb (C2H 3 O2 )5 Pb(As03)2 NaNOg & sulphur HgCl HgCl EgCl & sulphur HgCl & sulphur HgCl & zinc HgCl & zinc HgCl & NaxiQg HgCl & NaNOg HgCl & KMn04 DuBay #1155EH DuBav #1155HH 300 100 50 50 50 100 50 300 & 6 20 6 & 20 & 6 & 20 & 6 A 20 & 6 & 50 100 500 300 300 50 50 100 300 100 15 39 8 9 11 10 29 28 17 18 21 29 4 4 45 25 36 53 43 68 70 7 21 10 20 25 18 12 10 45 18 14 33 10 12 7 10 12 15 23 19 31 22 17 24 24 13 27 10 20 22 52 38 44 28 41 41 3 2 17 5 21 34 45 32 33' 40 48 47 65 55 63 36 58 34 50 23 8 51 59 59 33 40 32 13,,25 25,.00 18,,75 18,.50 10,,00 11,.75 25,,50 22,.75 20,,25 17,,50 33,.75 34,.75 6 ,.50 16,.00 38,.75 40,,00 49,.75 45,.25 31,.00 59,.25 43,.75 582,547, 544,,655 ,2328 B ST2 - 324,340 SB2 - 1,362,894 G - (Sx)2 ♦ n - (2328)2 * 84 - ^ Sx2 - G 53 100 75 74 40 47 102 91 81 70 135 139 26 64 155 160 199 181 124 237 175 Sx2 - 88,262 J S7/ (ST2 * 4) - C f (SB2* 21) - C f sum of squares due to error. - 13-' - TABLE 1 (continued) Analysis of variance Variation due to Degrees of freedom Sum of Squares 83 23,743.143 3 380.857 Treatments 20 16,466.143 Error 60 6,896.143 Total Replication Mean Error Square 823.307 114.936 .10.7208 t =. 2 .0 0 0 F s 5.51** Standard error of difference between means ^ 10.7208/2% / T - 7.580. Difference between means required for statistical signifiance - 2.000 x 7.580 - 15.160 - 14 TABLE II Effect of Soil Treatments on Scabbing of Potatoes at East Lansing 1937 Planted Hay 2 2 ; harvested Sept. 10, 1937 2 1 Treatment lbs. per acre None T 3 4 % Of surface area T X 34 49 39 32 154 38.50 HgCl2 15 38 32- 66 44 180 45.00 CuS04 ' 30 37 49 36 27 149 37 .25 HC1 1500 28 35 42 49 154 38.50 H 2S04 1500 34 32 47 33 146 36.50 HgClg & HC1 15 & 1500 59 57 70 15 201 50.25 CuS<}>/ & H SO, A SO & 1500 31 45 48 33 157 39.25 261 299 348 233 1141 B 37.3 X SB2 = 532 ,915 ST^ r 188,399 (Sx) 2 + n - C - 42 .7 49 .7 33.3 (1141)2 Sx2 - G j(ST2 * 4) - O f due to error. 40.75 Sx2 - 50,543 * 28 - 46,495 ,7500 (SB'8 3 7) - C ■f sum square Analysis of variance Variation due to Total Degrees of i’reedom Sum of squares Aean square error 27 4047.2500 Replication 3 1063.5357 354.5119 Treatments 6 604.00 100.6667 Error E - 18 2379 .7143 1.3133 (not significant) 132.2064 11.4931 - 15 TABLE III Sbil effect of Treatments on Scabbing of potatoes at Lake City Potato Experiment Station, 1937 Planted May 27-28; harvested Sept. 21, 1937 ________ 1 2 3 4 Treatment Lbs. per % of surface area Sum Mean acre scabbed T X 2 14 3-50 4 5 None 3 1 14 3.50 2 3 50 8 DuBay #3155HH 5-25 1 21 11 4 5 DuBay #1155HH 75 7.75 31 0 1 19 1 0 0 11 DuBay #1155HH 9 39 % .1 5 6 8 16 6 HgO 68 17-00 10 • 6 13 20 39 HgO 50 12.50 10 21 14 HgO 50 5 15 3.75 7 2 2 6 4 HgCl 5.50 6 22 3 2 20 11 HgCl 4 3 23 5.75 1 50 15 HgCl 9 7 34 8-50 7 20 11 HgClg 0 2 6 1.50 0 HgO 8c'zinc 6 & 50 4 8 5 31 7.75 5 HgO & zinc 20 & 50 13 3 2 31 7-75 1 HgO & zinc 50 8c 50 25 4 0 I 8 2 .0 0 6 & 50 3 HgCl & zinc 7 0 7 18 4.50 20 & 50 4 HgCl & zinc 4 1 50 & 50 7 HgCl & zinc 7 19 4-75 7 21 5.25 1 3 10 20 & 50 HgCl & CugO 0 8 2 .0 0 5 2 HgClg & CugO 20 & 50 1 5 29 7-25 12 1 HgClg & HgC204 2 0 & 20 11 6 2 38 9.50 16 HgClg 8c HgCg04 20 & 50 14 0 4 1 .0 0 2 1 HgClg & sulphur 20 8c 100 , 1 0 0 .50 0 2 HgClg & sulphur 20 & 300 2 0 9 1.75 0 1 HgClg & sulphur 20&600 8 2 3 3 11 2 - 15 HgClg & sulphur*> 1/9 3 0 1 .75 1 3 HgCl & sulphur# l/9 1 3 2.75 HgCl & sulphur# 1/4 1 1 11 6 0 0 2 5 1.25 Sulphur# 3 Sulphur 2 2 1 6 1-50 100 ^ 1 0 Sulphur 0 .50 1 2 300 1 7 0 0 7 1.75 Sulphur 600 0 0 25 Zinc 1 30 7.50 25 4 Zinc 0 2 12 3.00 50 2 .8 0 0 Zinc 0 1 .25 75 1 0 -50 CugO 1 1 2 50 0 CugO 5 0 0 5 1.25 0 10 0 0 CugO ‘ .50 150 0 1 1 11 4 11 2.75 4 50 1 mAso-),, 2 Pb(AsOgJg 2 4 11 2.75 100 2 3 Pb(As03 )g 2 18 4.50 5 5 150 6 Sum B 270 157 140 125^92 Mean X_______________ 6.75 5.93 5.50 5.12 # Chemicl applied as dust seed treatment only. l/9 and l/4 indicate 1 part of mercural (by weight) to 9 parts of sulphur and one part of mercurial to 4 parts of sul phur respectively. - 16 - ST2 r 20440 SB2 = 132774 Sx2 - 7900 0 = (Sx)2+n = (692)2+ 160 = 478864+ 160 = 2992.900 Sx2 - C r (ST2+ 4)-C 4(SB2+40)-C 4sum of squares due to error. 7900-C s (20440+4)-C + (132774+40)-C *f s.s. due to error. 4907.1 - 2117.1 * 326.45 + 2463.55 Analysis of variance Variation due to Degrees of freedom Sum of squares Mean square 159 4907.1 30.8623 Total Replication 3 Treatments Error 39 117 P = 54.2846+21 *0560 s 2.578’ vKr Error 326.45 108.8167 2117.1 54.2846 2463.55 21.0560 4.5886 t z 1.980 Standard error of difference between means = 4.5886/*£+ /? z 6.4883+2 : 3.2441 Difference between means required for significance = 3.2441 x 1.980 « 6.4233 - 17 TABLE IV Effect of Soil Treatments on Yield, of Potatoes at Lake City, 1937 Planted May 27-28; harvested. Sept. 21, 1957 Treatment Lbs. per acre None 50 DuBay #1155HH DuBay # )> 75 100 DuBay # n 6 HgO 20 HgO HgO 50 6 HgCl 20 HgCl 50 HgCl 20 HgClg 6 & 50 HgO & zinc HgO & zinc 20 Sc 50 HgO & zinc 50 Sc 50 6 Sc 50 HgCl & zinc 20 & 50 HgCl & zinc 50 Sc 50 HgCl & zinc 20 & 50 HgCl & CugO 20 & 50 HgClg & CugO HgClg & HgCpOA 20 & 20 HgClo Sc H2 C0 O; 20 & 50 HgCl0 & sulphur20 & 100 HgClg & sulphur 20 & 300 HgClg & sulphur 20 Sc 600 HgClp & sulphur* 1/9 HgCl & sulphur# l/9 1/4 HgCl.& sulphur# Sulphur# Sulphur 100 Sulphur 300 Sulphur 600 Zinc 25 Zinc 50 Zinc 75 CugO 50 CupO 100 CugO 150 PbTAs03 )2 50 Pb (A SO3 )2 100 Pb(AsO*)2 150 1 2 3 4 Yield in lbs. per 35-ft. row____________ T ti.e 24.4 224.4 18.8 89.2 24.9 18.1 22.7 24.9 90.6 60.0 2 2 .1 1 2 .6 8 .2 17.1 2 0 .2 1.3 8.7 1 . 1 9.1 92.1 23.0 2 1 .1 19.9 28.1 9.0 29.5 15.2 24.1 77.8 13.2 16.3 13.6 2 1 .8 64.9 17.6 25.3 18.8 30.5 92.2 25.1 28.1 16.1 89.3 2 0 .0 24.4 27.1 16.4 16.6 84.5 23.8 25.1 18.1 15.7 82.7 16.8 17.8 34.8 90.4 2 1 .0 27.0 2 2 .6 25.0 18.9 93.5 20.7 13.2 15.5 20.9 70.3 26.0 14.3 23.2 2 0 .8 84.3 21.5 13.7 17.2 16.6 69.0 17.5 2 2 .2 11.5 18.6 69.8 8.4 11.7 23.2 55.3 1 2 .0 13.4 57.5 2 2 .2 10.5 11.4 22.4 83.9 2 1 .1 18.1 22.3 15.9 71.8 21.5 13.1 21.3 25.8 31.3 102.4 18.0 27.3 28.8 20.9 17.0 33.3 1 0 0 .0 26,9 25.9 27.5 31.3 111 §6 8 .1 7.6 9.1 32.2 7.4 15.2 15.7 1 2 .0 66.3 49.2 16.7 19.7 15.5 1 2 .8 64.7 16.0 29.7 13.7 10.5 69.9 27.9 2 2 .0 24.6 31.5 106.0 19.0 23.5 33.6 98.9 2 2 .8 13.3 2 0 .8 18.7 30.9 83.7 28.0 17.6 17.8 28.2 91.6 27.5 21.7 15.3 31.0 95.5 24.3 16.8 19.0 24.5 84.6 20.4 26.8 24.5 91.9 2 0 .2 2 0 .2 13.3 16.9 18.1 68.5 11.5 13.7 14.6 16.4 46.2 24.1 22.7 17,8 27.2 91.8 25.2 17.0 25,1 24.4 91.7 24.5 2 2 .1 2 2 .6 24.5 93.7 22.30 22.65 15.00 £.05 23.03 19.45 16.23 23.05 22.33 21.13 2 0 .6 8 22.60 23.38 17.58 21.08 17.25 17.45 13.33 14.38 20.98 17.95 25.60 25.00 27.90 8.05 12.30 16.18 17.48 26.50 24.73 20.93 22.90 23.88 21.15 22.98 17.13 14.05 22.95 22.93 23.43 787.4 775.1 742.1 8 6 8 .8 3173.4 - 18 - Sx - 5173.4 ST2 - 266,602.8© SB2 - 2,526,304.62 Sx2 - 69,640.64 C - (Sx)2 * n - (3173.4)2 * 160 - 10,070,467.56 * 160 - 62,940.4223 Sx - C - (ST2 4 4) - C f (SB2 i 40) - C + sum tof squares due to error. 69,640 - C 66,650.72 - 0 4 63,157.62 - C f ss. due to error. 6,700.21 - :3,710.29 + 217.23 \ 2772 .69 Analysis of variance Variation due to Total Replication Treatments Error Degrees of freedom Sum of squares Mean square 159 6700.22 3 217.20 39 3710.30 95.1359 117 2772.72 25.6985 E * 95.1359 * 23.6985 - 4.0144 Error 72.40 4.8681 t - 1.980 Standard error of difference between means 4.8681 [ z “ 4 /4~ - 3.4417 Difference required for significance ^ 5.44 x 1.980 6.811 - 19 - TABLE V Effect of Soil Treatments on Scabbing and Yield of Potatoes at the Lake City Experiment Station, 1938#-”(a) Effect of scabbing Treatment > Hone Zinc Zinc Cu20 CugO NH4 CNS NH4 CNS HH4 CNS Sulphur Sulphur*”- Ibs, per acre 4 3 2 scabbed surface area $ of 1 'mA 4.3 10.9 50 1 .8 8 .1 .0 100 100 1.3 5.0 ' 5.3 5.7 6.4 500 600 - 10.4 2.4 25 150 25 1 0 .8 13.5 10.5 6 .1 4.8 16.7 9.2 6 .8 1 1 .0 14.8 6 .0 7.7 .7 1 2 .0 1 2 ^8 7.0 .4 2 .6 10.3 10.3 23.7 16.2 3,9 6.9 4.5 18.5 Mean 6.15 6.97 6.60 8.70 11.80 7.18 6.78 10.17 9.65 7.63 By inspection : no significant differences (b) Effect on yield 1 Treatment None Zinc Zinc CugO CugO NH4CNS n h 4cns n h 4cws Sulphur Sulphur* * lb s. per acre 50 100 25 150 25 100 500 600 2 3 yield in lb s. 60.0 54.5 48.5 55.5 54.0 55.5 50.0 5.5 53.5 82.0 53.5 33.0 55.5 53.5 54.0 80.0 55.5 .0 78.5 65.0 37.0 37.5 66.5 53.0 55.0 53.0 51.0 1.3 6 8 .0 61.0 4 Mean 71.5 55.50 41.67 61.13 53.25 56.63 55.50 56.00 3.70 63.75 70.13 — 74.0 51.0 63.5 33.5 67.5 8 .0 55.0 72.5 Seed pieces rolled in sulphur. By inspection: no significant differences except a marked reduction in yield from 500 lbs. per acre of ammonium thiocyanate. Planted May 24; harvested Oct. 3«*4, 1938 - 20 - THE REAS OH FOE FAILURE OF MEECUEIiEli TO GOETHOn SGa B Review of the literature In certain areas in the eastern part of the United States and Canada and in Europe mercury compounds as soil treatments have given partial to complete control of potato scab in sub-infested soil. In Germany 1133) and in Hew Jersey (93( mercurial soil treatments have controlled scab only in certain soils. In one test in Ohio 1106) mercurials had no appreciable effect on scabbing, in western Rev/ lork and in Michigan (42,43,68, 137-159) the application of mercury compounds to scabinfested soil generally results in a marked increase in scabbing. In a paper presented before the Potato Association of America in 1936, Daines and Martin (27) presented data showing that zinc in combination with calomel con­ trolled scab in a Hew Jersey soil where calomel alone failed. They also found that mercuric nitrate leached more readily from soil in which mercurials were effect­ ive in controlling scab than it did from soil in which they were not effective. They concluded that mercurials are effective in controlling scab only in soils where they are permitted to migrate sufficiently to afford protection, but that in certain soils mercury is ren­ - 21 - dered ineffective. They considered this inactivation of the mercury related to the oxidation-reduction potential of the soil. More recently Stormer (133) obtained adequate control of potato scab and Hhizoctonia with applications of superphosphate containing Vfo corrosive sublimate at 357 lbs. per acre. She concluded that an acid medium (represented in this case by superphosphate in sandy soil of pH 5) is essential for the release of the fungi­ cidal properties of the mercury, which is immobilized in the presence of alkaline fertilizer, such as calcium cyanamide• MacLeod and Howatt (83) found that mercuric and mercurous chloride applied in the dry form at the rate of 10-15 lbs. per acre could be depended upon to control scab and black scurf in heavily infested soils at Fredericton, New Brunswick, Canada. From repeated tests they concluded that the efficiency of this type of treatment depends largely upon the uniformity with which the space to be occupied by the tubers is impregnated with the fungicidal agent. In field trials in Michigan mercury compounds have been applied in the planting furrow. In view of the findings of other investigators, the failure to control scab there might be due to not mixing the fungicide - 22 - through the soil, or to the soil having a hydrogenion concentration or an oxidation-reduction potential which is unfavorable for the release of the fung­ icidal properties of the mercury. Soil Reaction Mercurial soil treatments are not ineffective in Michigan; they generally cause a marked increase in scabbing. As previously reported (68^329 in­ dividual hill records of soil reaction and severity of scabbing in soil that had been limed and sulph­ ured in strips at East Lansing showed that mercury compounds aggravated scabbing over the entire pH range of 6 .0-8.4. The results of this experiment are summarized in Table vl. The apparent trend towards increased scabbiness with rise in pH was not statistically significant. At Lake City in 1957 (Table 111) yellow oxide of mercury caused a pH significant increase in scabbing in soil of^5.2-5.8 . No evidence was obtained that the effectiveness of mercurials in our soils is in any way related to the soil reaction. Oxidizing and Reducing Agents In the field trials of calomel and yellow oxide of mercury in combination with oxidizing and reducing agents and sulphur which were carried out at East -Lansing (Table 1) and at Lake City (Table 111) in 1937, neither oxidizing nor reducing ageits at the - 23 - TABLE VI Influence of Soil Reaction on the Effect of Mercurial Soil Treatments on Scabbing of Potatoes 329 observations, East Lansing, 1936 Soil pH fa of surface area of tubers scabbed control HgCl HgClg 6 ,0-6 .4 8 .2 19.9 21.5 6 .5-6.9 9.1 26.9 24.8 7.0-7.4 10.7 2 1 .6 29.9 7.5-7.9 1 1 .6 19.6 23.4 8 .0 -8 .4 11,7 38.0 1 2 .0 Mean: 1 0 .1 23.5 25.3 - 24 - at the rates employed had any marked effect on the tendency of mercurials to increase scabbing. In pot experiments described under the next heading (Table Vlll), calomel (340 parts per million) in combination with powdered zinc (8500 p,p.m.) caused a marked reduction in scabbing in both Long Island and local soils that had been infested with scab from Michigan potatoes. Potassium permanganate (8500 p.p.m.) in combination with calomel had no effect on scabbing in either soil. Lime (18,000 p.p.m.) with calomel caused a marked increase in scabbing in Long Island soil but none in local soil where the controls were too scabby to expect any increase from soil treatments. In local soil zinc alone at high rates of application caused a reduction in scabbing, and potassium permanganate apparently caused a slight reduction. In cases where combinations of zinc with calomel caused a reduction in scabbing while calomel alone did not, it seems apparent that it was the effect of the zinc itself rather than the combined effect of the zinc and calomel that caused the reduction. Mercury -placement tests If prevention of scabbing and aggravation of scabbing as a result of mercurial soil treatments are - 25 - both due to the effect of the mercurials on the soil flora, depending upon local conditions, then in mercury placement tests, the degree of control on the one hand and the amount of increase in scabbing on the other may both be taken as measurements of the thorough ness with vrhich the area in which the tubers develop became impregnated with the antiseptic. MacLeod and Howatt in eastern Canada (83) and Daines and Martin in New Jersey (27) found that mercury compounds were most effective in controlling scab when they were mixed thoroughly through all the soil in the space later to be occupied by the tubers. The latter authors con­ cluded that mercury compounds applied with the fertil­ izer are effective in controlling scab only in soils where they are permitted to migrate sufficiently to afford protection through^ the area in which the tubers develop. In placement tests v/ith yellow oxide of mercury at Lake City, Michigan, in 1938, the mercurial mixed thoroughly with the soil in the area in which the tubers later developed was no more effective in aggravating scab than where other methods of applica­ tion were employed. IN 80-hill plots with three replications, yellow oxide of mercury caused a marked increase in scabbing when (a) mixed thoroughly through the soil along the planting row to a depth of 5 to 6 inches, (b) applied on the surface of the soil in a band 6 inches wide after the tubers were planted and covered, (c) banded two inches from and on either side of the seed pieces and on a level with them, (d) applied in the planting furrow, and (e) applied in a band 2 inches directly below the seed pieces. However, when the mercurial was placed 4 inches below the seed pieces, the increase in scabbing was small and insign­ ificant. (Table Vll). If the cause of increased scabbing from yellow oxide of mercury soil treatments at Lake City were due to an effect of the mercurial on the soil flora, resulting in an increase in number of parasitic Actinomycetes. it must be concluded that a sufficient amount of the antiseptic migrated from the position of placement through the area in which the tubers developed to cause a significant increase in scabbing. Froaa this view point it appears that the yellow oxide of mercury showed^£ea%rtendency to migrate down in the soil than upward, since all of the placements gave approximately equal increases in scabbing except that the placement 4 inches below the seed pieces gave a smaller increase in scabbing. It should be noted here that two plots in this experiment (represented by x and y in Table Vll) were lost through an error in harvesting. For the purpose of statistical analysis, estimates for these missing plots were supplied by Baten's method (8)* - -2$ — T&B1M VII Placement test with HgO as a Soil Treatment fort Potato Scab at Lake City, 1938 ____________ Planted may 24. harvested Oct. 5-4. 1958_____ 1 2 5 4 _ Placement °/o of surface area scabbed T X sum mean 0. no treatment 11.9 11.7 14.0 12.8 50.4 12.60 1. mixed 38.0 30.3 35.4 37.5 141.2 35.30 2. surface 50.5 43.3 40.9 14.0 148.7 37.18 3. banded 12.6 41.3 31.5 37.8 123.2 30.80 4. furrow y - 29.8 25.2 54.6 38.9 98.7 32.13 4 y 128 .5 5. 2 in. below 27.6 28.4 6. 4 ini below 10.7 18.1 41.7 SK ■ » 20.8 28.1 21.2 125.8 31.45 50.0 17.70 4 Z 70. 8 sum B 151.3 r11*1 ’ 198.3 .98.1 i% 190.3 738.0 + y.+ 5 7 rs?.& Estimates for missing plots: Where r is the number of replications, siis the number of treatments, n is the total number of plots, y is the value of the missing plot in the first replication and P^ and are respectively the sums of the row and the column in which y falls, z is the missing plot in the third replication and Pj and Bk are respectively the sum of the column and the row in which z falls, and T is the sum of all the plots ex­ clusive of y and x: y -[(r-l) (s-1) (sih + rB^-T) - (sPjfrl^-T)j i Rr-l) (s-lj2-l1 ^ ( 9 8 . 7j f ^ ( 1 5 1 . 3 ; - 7 3 8 7 - / 7 1 5 0 ) f 4 (198 .1) -7 83 } i (3 x 6 ) 2- l - 29^. z -](r-l)(s-l) (sPj * rBk- T )-1sPi f rn^-T^ * l(r-l) (s-lj2-l18/7(50) -4(lS®.l)-758] - [7(98.7) -f 41151.3)-730 i f(3 x 6)-lm 20.8 -28- Using these estimates the fundamental summations are: ST2 = 97,118.06, SB2 = 156,251.40, Sx2 = 25,893*78, C = (Sx)2 t n s (788.6)2 f 28 s 22,105.04 Analysis of variance Variation Degrees of Sum of Mean due to sauare Error freedom sauares Total 3790.74 23* Treatments 6 2176.48 362.747 72.860 Blocks 218.58 3 Error 16* 1395.68 87.230 .9_O.40 . * 2 degrees of freedom lost by fixing the values of x and y. F - 5*88, which is significant. Omitting the values of x and y from the funda­ mental summations: Sx * 7 3 8 .0 , G = (S x )2 * 26 2 0 ,9 4 7 .8 4 6 , ST2 + T§) / SB2 / - i k = S (T § + T 2 }| ? . S (T | + t2 + T 2 4 = 1 2 ,2 4 1 .6 9 /3 - 7 5 , 5 9 3 . 1 7 / 4 = 2 2 ,9 7 8 .8 5 5 . a = S (B § + b | ) / 7 - 8 (B | ♦ Bp/ 6 = 7 5 ,5 3 6 .9 8 /7 6 2 , 1 3 5 . 3 0 / 6 - 2 0 9 .0 3 4 . Corrected analysis of variance Degrees of Sum of Mean sauare Error freedom sauares 25 3625.25 16 8 7 .2 3 0 1395.68 9.340 9 2229.57 Blocks 6 9 .6 7 8 199.03 3 Treatments 2030.54 ,...33,8...423 . Variation due to Total Error F (treatments) z 3 . 8 8 Tabular value of t = 2.12. The standard error of dif­ ferences between means = (9.34 x /2 t A = 6 .6 0 3 The difference between treatment means required for statistical difference : 6,603 x 2.12 = 14.00 - 29 - Experiments with Long Island and New Jersey Soils m , 1221 In 1937 about 1200 lbs. of scab-infested New York (Long Island) soil were obtained from H. S. Cunningham, and a smaller quantity of New Jersey soil from R. H. Daines. Both soils had been fumi­ gated with carbon disulphide before shipment because of the Japanese beetle quarantine. The soils were stored in a dusty greenhouse until the odor of sarbon disulphide had entirely dissipated. It being feared that fumigation and desiccation of the soils might have eliminated most of the pathogenic Actinomvcetes. a portion of each soil was- reinfested by adding to it one gram of macerated potato scabs for each ten lbs. of soil. Calomel was applied at four rates which were intended to be 6, 15, 5 0 , and 150 lbs. per acre, but since there might be some disagreement as to how much chemical in a pot is equivalent to a given rate of application in the field, the rates of application are expressed in parts per million. The chemicals for soil treatments were mixed thoroughly through the soil. The pots were planted to clean, formaldehyde- treated Katahdin potatoes and placed out of doors. In Table VIII each figure under (t% scab" represents the percent of the surface are of the tubers scabbed in ten 8-inch pots. In the case of Long Island soil that had not been artificially infested, the controls gave 5.8% scab. Calomel at 340 p.p.m. had little, if any, effect on scabb­ ing. In Long Island soil that had been infested with scab' from Michigan potatoes, the percent of scab increased in proportion to the amount^ of calomel applied, from 7.6% for the controls to 38.7% for 2850 p.p.m. In local soil calomel had no appreciable effect on scabbing, a phenomen that frequently occurs at East Lansing when the controls are very scabby. In the case of New Jersey soil (Table VIII) there were only five 6-inch pots for each treatment and the potatoes were planted six weeks later, i.e., in mid-July. Beoausf. of weak seed and hot weather only a 50% stand was secured. Nevertheless, the results are very similar to those for Long Island soil, calomel causing an increase in scabbing in soil that was infested with scab from Michigan potatoes, but having no effect on scabbing in soil that was not artificially infested. In brief, In 1937 the effect of calomel on scabbing in Long Island and Jersey soils that had been infested with scab from Michigan potatoes was exactly what general! occurs under field conditions in Michigan, - calomel aggie, vated scabbing. In view of this it 31 it seemed likely that in Long Island and New Jersey where calomel controls scab the soil must be infested with strains of Actinomyces $fcat react differently to calomel from those that cause scab in Michigan* The failure to control scab in Long Island and New Jersey soils that had not been artifically infested might be explained by their having become contaminated with local strains of Actinomyces during their storage in the greenhouse, either from dissemination of the organ­ ism in dust in the air or by Arthropods such as sow bugs which were abundant in the greenhouse. That the Long Island and New Jersey soils that had not been artifically infested were contaminated with local Actinomycetes seems probable, not only because of the failure of calomel to control scab in those soils, but because natural contamination of sterilized soil has been of constant occurrence in the greenhouse. For example, in an attempt to test the pathogenicity of Actinomyces-isolates 114 6-inch pots of soil were sat­ urated with water and steamed for 15 hrs. Sixty of ft^ese were placed out of doors on a lawn and the others in the greenhouse. Duplicate pots were infested with actinomycete-isolates; planted with formaldehydetreated Katahdin potato tubers. At harvest, every tuber in the pots in the greenhouse was.heavily scabbed while only 3 out of 33 control pots out of doors showed any trace of scab. In the case of Long Island and New Jersey soils, that qalomel did not cause an increase in scabbing in the soil that was hot artificially infested, may have been due to its not having become so thoroughly contaminated with local strains of Actinomycetes as was the soil that had not been artificially infested. Evidence that soil would become contaminated with Actinomycetes (spores of bits of mycelium) settling down out of the air in the greenhouse in question is furnished in Table X. PA Petri dishes, (each containing 30 cc. of beef-peptone agar of pH 7.0) were placed on a bench in the greenhouse and the covers removed for varying lengths of time. The plates were then incubated for one week, one- half of them at room temperature and the rest at 37° G . Actinomyces colonies developed on most of the plates. 1he failure of the Actinomycetes to appear in proportion to the length of time of exposure, and the complete absence of fungi may have been due partially to the large numbers of bacteria (these acting as antibiotics at least by crowding , and possibly also through the production of toxins), and partially to the fact that the agar in plates exposed longer than 15 minutes became very dry. It is not known whether or not the Actinomycetes that developed on the plates were pathogenic, but it seems probable that, if sparophytic Actinomycetes are present in considerable numbers in the air of a greenhouse (with 33^ ventilators closed], parasitic species could be in that manner also. than one-half t 6 disseminated It will be noted in Table X that more out of 1 1 ) of the antinomycete colonies on plates exposed only 5 minutes turned the medium blue. This is a characteristic that is commonly associated with actinomycete-isolates from scabby potatoes from the Lake City Experiment Station. -34table VIII Effect of Soil Treatments with Calomel, Zinc and Potassium Permanganate on Scabbing and Yield of Potatoes in Michigan and Long Island Soils Treat­ ment parts per million Check 63,0 120 7.6 80 5.8 53 340 54.1 64 15.8 70 4.6 62 » 850 60.0 56 ro - .0o0 - Long Island soil infested with not Michigan afctifically infested scab yield* yield* yield* % in in in % % scab grams scab grams scab grams local soil 45 - w 2850 51.0 34 38.7 23 - tt 8500 70.0 13 - - - ' 340 18500 58.0 66 30.1 67 — HgCl HgCl & CaO _ j HgCl & Zn 340 8500 26.4 81 3'.8 26 &- HgCl KMn04 340 8500 65.8 118 8.4 27 — Zn 8500 41.7 100 - - - „ w 17000 28.0 93 - - ft 51000 24.2 70 ** - - KMn04 8500 47.3 74 - - - tt 17000 57.6 68 - - - v 51000 51.1 52 — ' — - -r\r\r* pot# ~k\ * per _ - - - 35 - TABLE. IX Effect of Soil Treatments with Calomel in New Jersey Coil* soil # nots p.p.m* HgCl io surface area of tubers scabbed artificially 4 0 55 infested 3 340 60 3 850 63 0 2800 artificially 3 0 23 infested 2 340 24 - not TABLE X Organisms on Plates Exposed to contamination in a Greenhouse in which the Soil was Infested with Potato Scab Jan. 5, 1958 Time of exposure, minutes Temp, of incubation of plates bacteria — Plate counts actinomycetes fungi "blue" total „ 215 260 2 2 2 7 0 0 15 560 80 200 0 1 0 5 5 0 0 0 0 30 560 #■ 0 27 0 room 0 80 1400 912 2 0 5 10 0 0 140 1200 1760 6 0 12 21 0 0 120 2 2 0 37 C. 5 V /% 15 78 0 1 0 030 128 840 0 0 9 0 0 0 1088 1000 0 0 2 1 0 0 2700 560 0 0 2 '48 0 0 # 0 15 0 # 2 36 0 # 3 19 0 - 80 140 24 hours ' ' 45-,count ruined by "spreader bacteria". $too many tc^sount - 57 - t able X continued Beef-peptone Agar, pH 7,0. The agar became very dry in plates from which the covers were removed for more than 15 minutes. Counts were made on the 7th day; bacterial counts of more than 200 are estimates arrived at by counting the colonies on a fraction of the plate. Experiments with Long Island Boil and "Long Island Seabn in 1958 In 1938 scabby potatoes as well as scab-infested soil was obtained from Dr. Cunningham of Long Island, N. X. As in the preceeding year, the soil was aired in the greenhouse until no odor of carbon disulphide could be discerned. lots. It was then divided into three One lot was not artificially infested. The other two lots were saturated with water and steamed for 15 hours. One half of the steamed soil was in­ fested with Long Island strains of Actinomyces by adding to it cultures of Actinomyces isolated from Long Island potatoes and 1% of unsterilized Long Island soil that had been protected from contamination from the time of its arrival by storing it in clean, sealed fruit jars. The other half of the steamed soil was infested with Michigan strains of Actinomyces by adding to it cultures of Actinomyces isolated from potatoes from Michigan and 1% of unsterilized scabinfested local soil. In like manner a lot of local soil was sterilized and infested with Long Island strains of Actinomyces: another lot was fumigated v/ith carbon disulphide ( .one lb. per cubic yard and sealed in garbage cans for 48 hrs.j ana desiccated to make it more comparable with the Long Island con- trol set; and a third lot was neither sterilized nor fumigated. Each lot was divided into four parts and treated with calomel as in 1937 except that the rates employed were 50, 350, and 1000 p.p.m. The pots were planted to clean, washed, formaldehyde-treated Katahdin tubers and placed on sod in an orchard, care being taken not to contaminate them. In order to make possible the application of statistical analysis, the pots were harvested separately The percent of the surface area scabbed and the weight of each tuber was recorded, and the percent of the sur­ face area scabbed of all the tubers in a pot was cal­ culated on the basis of the weights of the tubers. It is obvious that the surface areas of tubers do not vary in proportion to their weights, but this objection was counterbalanced by the fact that in these experiments the smaller tubers were less scabby than the larger ones In local, naturally infested soil the control gave 56.6% of the surface area of the tubers scabbed, while 350 p.p.m. of calomel gave 82.2%, an increase in scabbing x-nat closely approaches significance by analysis of variance (Table IX). In local soil that had been fumigated with carbon disulphide and desiccated to make it more comparable -40- with the Long Island soil, calomel caused a marked increase in scabbing, giving significant differences from the control at 50 and 350 p.p.m. (Table X). In Long Island soil that had been steamed and in­ fested with Michigan strains of actinomyces. 50 and 350 p.p.m. had no appreciable effect on scabbing, while with 1000 p.p.m. there was a highly significant in­ crease (Table XI), In Long Island soil that had been steamed to re­ move contamination and then reinfested with strains of Actinomyces from Long Island, calomel at 1000 p.p.m. gave complete control of seat (Table Xll). In local soil that had been sterilized and in­ fested with Long Island strains of Actinomyces, calomel caused a significant reduction in scabbing at all three rates of treatment, practically eliminating scab with luOO p.p.m. (Table Xlll), In Long Island soil that had not been artificially infested, but that had probably picked up some con­ tamination with local strains of Actinomyces, there was very little scab, but ealomel seemed to aggravate rather than control scab (Table XIV). Khizoctonia scurf occurred only in pots of local soil that had not been steamed, although the tubers from these lots were too scabby to leave much space for -41Rhizoctonia sclerotia to develop, 71 % of the tubers were more or less scurfy from the control pots of the lot of soil that had been fumigated with carbon dis­ ulphide. From, the control pots of the other lot of local soil 12fo of the tubers were scurfy, whereas there was no trace of scurf on any of the tubers from soil that had been treated with calomel at any of the three rates of treatment. Although partial to complete control of scurf with mercurials has been reported by several other investigators (15, 26, 83, 91, 92, 104), neither calomel nor yellow oxide of mercury controlled scurf in 1937 under field conditions at Lake City, Michigan, when applied at 6, 20, and 50 lbs. per acre in the planting furrow. Effect on Yield The effects of these soil treatments on yield in 1937 are given in Table Vlll, for Michigan soil in 1938 in Table XV, and Long Island soil in Table XVI. In the summarizing table (Table XVII) are given the yields of treatments within each set of pots. In 1937 calomel depressed yield in proportion to the amount applied in Michigan soil, but only at high rates in Long Island soil. The Michigan soil controls averaged almost double the yield of the Long Island soil controls, but whereas 340 p.p.m. of calomel cut in half -42** the yield in Michigan soil, it caused only a slight reduction in yield in Long Island soil. Hie results for 1938 are sharply in contrast with those for 1937. Long Island soil yielded tetter, hut calomel apparently depressed yield even at 50 p.p.m. while with 350 p.p.m. the decrease was statistically significant. Michigan soil yielded less, hut calomel caused an increase in yield reaching statistical sign­ ificance at 350 p.p.m. At 1000 p.p.m. there was a highly significant reduction in yield. The data on individual sets (Tahle XVll) show that all of the in­ crease in yield with calomel in Michigan soil came in soil that had heen fumigated with carbon disulphid®, whereas in the other sets 50 and 350 p.p.m. did not effect the yield. There was no recognized difference in the handling of the pots during the two years except in the moisture supply. In 1937 the pots were watered very sparingly. It was expected that this would insure heavy scabbing of the controls since there are many statements in the literature that potatoes scab most severely in dry soil 132, 88, 116). Since the potatoes in Long Island soil did so poorly under drouth conditions, the following year the pots were watered heavily early in the season. Later in the season heavy precipitation kept the soil wet. A decrease in yield was to be expected in treated Long Island soil since Cunningham (26) obtained a slight decrease in yield with low rates of application of calomel under field conditions. Under field conditions in Michigan mercury compounds have no effect on yield up to 501bs. per acre. Increases in yield from mercur­ ial soil treatments which have been reported from west­ ern New York (138), have never been observed under field conditions in Michigan. It is puzzling why there should be an increase in yield from calomel soil treatments in local soil that had been treated with carbon disulphide, and also why fum­ igation with carbon disulphide should cause an increase in Rhizoctonia scurf as noted above. Conclusions Regarding the Nffect of Calomel on Scabbing In pot experiments, calomel mixed thoroughly through the soil controlled scab in both Long Island and Mich­ igan soils that had been steam-sterilized and infested with strains of Actinomyces from Long Island. Conversely calomel aggravated scabbing in both Long Island and Michigan soils, infested with Michigan strains of Actinomyces, at the same time controlling Rhizoctonia scurf. In view of this it is concluded that calomel is not rendered ineffective as an antiseptic when used as a soil treatment in Michigan soil, and that differences in the effect of calomel on scabbing of potatoes are due to differences in the strains or species of paroaitic Actinomyces infesting the soil. - 45 - TABLE XI Effect of Calomel Boil Treatemtns on Scabbing in Michigan Soil, not Fumigated, Sterilized, nor Desiccated jo of surface area of tubers scabbed Calomel .UP .m. Fot#l. 2 . 3. 4. 5. 6 . 7. 8 . 9. 1 0 . - 0 93 ,1 85.0 ,v 90.0 95,.l 78.3 5.5 56 .3 5.0 57.4 Sum T 1304 C (Sx) 2 Sx 2 - C - Sx 2 .6 7 n - (ST2 f 828.2 82.82 x 116 ,994.98 (1394.6)2 1 0 19 Total Treatments - 1,00,6,724.2 97,245.46 Mean sauares Error 19,749.52 18 3426.96 20 Sum of sauares 3,426.96 3426.96 16,322.56 /r- 906.81 1 Error 7 ST 8 ) - C + sum of squares due to error. Degrees of freedom Variation due to F ^ 97.0 94.0 89.9 78.8 90.3 94.8 40.0 56.64 Sx ^ 6 8 . 0 566.4 X Mean 350 84.3 91.1 30.11 906.81 x 3.779, which is not significant. 7 The standard error of differences between means 30.11 x /§ The - ^ 13.46 From Fischer’s table, t-2.101 d i f f e r e n c e betY/een means r e q u i r e d for significance ^ 13.46 x 2.101 .7 27.607 statistical - 46 - TABLE XII ' Effect of Calomel Soil Treatments on Scabbing in Michigan Soil, Fumigated with Carbon Bisulfide and Desiccated. % of surface are of tubers scabbed 0 10.0 8. 10.0 50 30.0 90.2 54.0 70.3 60.0 26.0 91.7 46.3 9. 5.0 237'. 7 D .D alH• Pot # 1. 2. 3. 4. 5. 6. 7. Sum T Mean X 5.0 47 .8 17.0 8.1 41.6 93.2 . Sx - 1718.3 26.42 1000 v 350 88.5 75.4 13.8 68.4 95.0 20.0 65.3 68.9 82.6 13.0 62.4 65.0 3.0 50.0 50.0 7.6 50.0 65.0 488 .5 626.1 366.0 68.2 69.57 54.28 Sx2 - 113 ,405.19 ST2 - 0 - (Sx/2 * n - (1718) 2 ^ 36 - 40.67 821 ,090.75 82,015.4136 Sx2-C ^ (ST2 =. 9) -C * sum of squares due to error. Variation due to Total Treatments Error Analysis of variance Degrees of Sum of Mean squares square freedom 35 Error 31,389.78 3 9,216 .89 3072.296 32 22,172 .89 692.902 ** — 5072.£96 f 692.902 - 26.323 4.434, which is significant. The standard error of differences between means ^ 26.523 x /2 f /9 - 12.407 From Fischer’s table, t ^ 2.036 The difference between means required for statistical significance - 12.4 x 2.036 25.261 -47table XIII Effect of Calomel Soil Treatments on Scabbing in Long Island Soil, Fumigated with Carbon Disulfide, Steamsterilized, and Infested with Michigan Scab Organisms. % Calomel p.p.m. 0 Pot #1. of surface area of tubers scabbed 50 350 1000 9.2' 7.7 2.5 60.0 2. 4.5 4.4 6.7 65.0 3. 14.2 13.5 10.0 5.0 4. 4.5 10.0 17.2 3.0 5. 8.'3 8.6 10.3 75.0 40.7 44.2 46.7 208.0 Sum T Mean X Sx s 339*6 8.14 41.60 9.34 8.84 Sx2 = 14,867.6^/ St2 = 49,055.02 C = (Sx)2 * 20 = 5,766 .41 ] ‘ ' Sx2 - C = (ST2 * 5) - C ^-sum of squares due to error. Variation due to Total Treatments Error Analysis of variance Degrees of Sum of freedom squares 19 9,101.19 3 4,044.59 16 5,056.60 Mean square Error 1348.20 316.038 17.777 F r 1348.20 ■? 316.038 = 4.27, which is significant. The standard error of differences between means jr 17.777 x /2 * /5 = 11.24181 From Fischer's table, t» 12 The difference between means required for statistical significance = 11.2418 x 2.12 = 28.833 - 4 '8- TABLE XIV Effect of Calomel Soil Treatments on Scabbing in Long Island Soil, Fumigated with Carbon Disulfide, Steamsterilized, and Infested with Long Island Scab Organisms. fo Calomel P.P.m. Pot #1. of surface area of tubers scabbed 1000 3§0 0 50 .5 .0 1.0 .0 2.0 .5 .0 .0 .0 3.0 5.5 .0 .0 .0 4.0 12.0 .0 .0 5.0 .5 .9 .0 .0 T 19.0 .9 1.0 .0 Mean X 3.8 .18 Sum traces .20 .0 Sx a 20.9, Sx2 - 176.81 ST2 - 362.81, C = (Sx)2 +20-21.84 Sx2 - C - (ST2 * 5) - C ■f sum- of squares due to error. > Variation due to Total Treatments Error Analysis of variance Degrees of Sum of freedom squares Mean square 19 154 .97 3 50.72 16.907 16 104 .25 6.516 Error 2.5526 F - 16.907 * 6.516 - 2.59, which is not significant. The standard error of differences between means 2.5526 x /2 * & 1.61 From Fischer's table, t - 2..12 The difference between meaa s required for statistical significance - 1.61 x 2.12 « 3.41 -4 9 - TABLE XV Effect of Calomel Soil Treatments on Scabbing in Michigan Soil, Steam-sterilized and Infested with Long Island Scab Organisims. % Calomel p.p.m. Pot #1. 2. 3. 5. 6. 7. 8. 9. 10. Sum T Mean X Sx - 445.7 of surface area of tubers scabbed 0 85.0 7.8 2.6 16.1 60.8 10.8 60.4 51.9 25.1 61.4 50 1.6 4.0 .0 .5 2.4 2.9 1.8 .0 .0 2.5 381.9 15.7 1.57 38.19 „ 2 r 23, 115.49 Sx 350 .0 .0 trace 2.8 27.1 .0 14.5 .0 3.7 .0 1000 .0 .0 trace .0 .0 .0 trace .0 .0 .0 48.1 trace 4.81 trace St2 = 148,407.71 C - (Sx)2 * n r (445.7 )2 * 40 = 4966.212 Sx2 - C = (ST2 * 10) - C + sum of squares due to error. Variation due to Analysis of variance Degrees of Sum of Mean squares square freedom Total Treatments Error 39 18,149.28 3 9,874.56 3291.52 36 8,274.72 229.85 Error 15.161 P = 3291.52 ■i 229.85 = 14. 32, which is significant. The standard error of differences between means = 15.161 x /2 * / W a 6.780. prom Fischer’s table, t = 2.030. The difference between means required for statistical significance - 6.780 x 2.030 a 13.76 -50TABLE XVI Effect of Calomel Soil Treatments on Scabbing in Long Island Soil, Fumigated with Carbon Disulfide and Desiccated in a Dusty Greenhouse: probably Contaminated with Dust from Local Scab-infested Soil. jo of surface area of tubers scabbed Calomel 0 1000 350 50 Pot #1. .0 .0 .4 3.1 2. .0 1.5 1.8 .0 3. trace trace .0 .0 4. trace .3 .6 .0 5. trace .3 .0 .0 T .trace 2.1 2.8 3.1 Mean X trace Sum Sx = 8.0 •56 .42 Sx2 - 15.8 ST2 = 21.86 .62 C : (Sx)2 t 20 ; 3.20 Sx2 - C r (ST2 ■* 5) - C tsum of squares due to error. Variation due to Total Treatments Error Analysis of variance Sum of Degrees of squares freedom Mean square 19 12 .600 3 1.172 .3907 16 11 .428 .714 Error .8850 F r *301 * .714 - .547, which is not significant* The standard error of differences between means = .8450 x * / S - .534 From Fischer’s table, t = 2,12 The difference between means required for statistical significance ■ .534 x 2.12 = 1.13 -51table XVII Effect of Calomel Soil Treatments on Yield of Potatoes in Michigan Soil Wt. in grams Calomel P.p.m, Sums 0 50 350 Soil fumigated but not steamed Pot #1# 85 36 91 2# 21 88 113 3. 93 60 65 4. 90 64 55 5# 133 80 111 6. 38 139 66 7. 95 122 92. 8# 30 69 118 9. 21 58 180 606 716 891 Sums T Soil steamed and infested with Long Island scab1• Pot #1. 44 79 74 2# 94 77 40 3# 67 82 101 4# 94 66 157 123 65 120 6# 130 116 189 7# 96 124 123 8# 93 148 72 156 98 97 9# Sums T “ 912 869 944 Sums P 1518 1585 1835 Means '84',2> 88.1 101.9 C s 1000 13 102 11 28 38 21 51 40 40 344 71 52 42 30 71 85 34 109 33 62V Q X = 71.0278 Sx2 r 238,309 ST = 1,792,109 2557 X s 90.3333 Sx2 = 344,712 O ST s 2,755,770 3252 5869 48.4 80.'f f 871 (Sx)2 ♦ n s (5809)2 * 72 » 468,673.3472 Analysis of variance Degrees Sum of Mean Source of Formulae variation Freedom squares square Total 71 114,347.65 Sx2-C 36,646.54 Subclasses 7 (ST2*9)-C 1214.08 77,701.11 64 Within subclasses 6,708.68 6708.68 Between sets 1 (SQ2*36)-C 28,127.49 9375.83 3 Between treatments (SP *18)-G 1,788.94 596.31 Subclass discrepency 3 significant* Prom Fischer’s table, t = 2,002# The difference between treatment means required for statistical significance s(/l214.08 x /2 x 2*002) * /18 : 23#2244 P (sets) = 6708,68 * 1214,08 = 5,526, which is significant. The difference between set means required for statistical significance r (/l2#1468 x /2~ x 2,002) /2 = 6#9766 TABLE XVIII Effect of Calomel Soil Treatments on Yield of Potatoes in Long Island Soil Wt, in grams Calomel p.p.m. 0 50 350 Soil not steamed Pot #1. 46 25 60 2. 105 42 39 3. 98 50 101 4. 120 77 63 5. 105 72 50 Sums T 4*74 266 313 Soil steamed and infested with Long Island scab. Pot #1. 109 120 52 2. 99 81 92 3. 118 79 88 4. 80 180 41 81 5, 92 80 Sums T 487 480 353 Soil steamed and infested with Michigan scab. Pot #1. 141 48 65 2. 92 41 82 3. 84 91 34 4. 93 40 53 5. 21 104 21 Sums T 514 299 19V Sums P 1045 863 1475 Means 98.3 69.'7 57.-7 1000 35 62 35 15 71 218 33 74 46 88 80 321 28 29 28 38 8 131 670 ' 44.7 Sums Q X * 63.55 Sx p r 97,523 ST2 - 440,925 1271 X r 82.05 Sx = 145,715 p ST = 695,219 1641 X - 57.05 Sx2 = 88,445 ST2 = 409,567 1141 4053 C - (Sx)2 * n - (4053)2 * 60 - 273, 780.150 Analysis of variance Sum of Source of Deg. Formulae squar e s variation Free. Total 59 57902.85 Sxg-C Subclasses 11 35362.05 (T *5 )-C Within subclasses 48 22540.80 6730.00 Between sets (SQ *20)-C 2 Between treatments(SP^*15)-G 23641.11 3 4990.94 Subclass discrepency 6 Mean Error square 469.60 21.67 3365.00 7880.37 831*82 Standard «rror of difference between treatment means 21.67 x / 2 t /l5 s 7,9116 Prom Fischer's table, tr 2.01 The difference between treatment means required for statistical significance r 7.912 x 2.01 = 15.903 p (sets) a 3365 * 469.6 r 7.17, which is significant. The difference between set means required for statistical significance s (21.67 x /2 ~ * / z ] x 2.01 s 35.559 -54table XIX Summary of 1938 Results of Pot Experiments with Calomel as a Soil Treatment for Potato Scab Control in Michigan and Long Island Soils* % of surface Yield of area of tubers per tubers , scabbed scabbed pot gram % Soil Calomel p*p*m. Michigan, fumigated but not steamed 0 50 350 1000 Required difference for significance Michigan, neither fumigated nor steamed 100 100 100 100 26.42 54.28 69*57 40.67 25.26 67.3 79.6 99.0 44.3 33.5 0 90 56*64 88.1 350 100 82.82 90.6 27.61 Required difference for significance Long Island, steamed 0 and infested with 50 Michigan scab 350 1000 Required difference for significance 100 100 100 100 8.14 8.84 9.34 41.60 23.83 102.8 59.8 39.4 26.2 18.2 Long Island, steamed 0 and infested with 50 Long Island scab 350 1000 Required difference for significance 93 13 20 0 3.80 .18 .20 .00 3.41 97.4 96.0 70.6 64.2 27.4 Michigan, steamed & infested with Long Island scab 0 50 350 1000 Required difference for significance 97 48 27 22 38.19 1.57 4.81 trace 13.76 103.3 91.7 98.4 58.6 32.4 ' Long Island, not 0 steamed but con50 taminated with 350 Mich* scab 1000 Required difference for significance 10 37 37 60 trace .42 .56 .62 1.13 98.8 53.2 62.6 43.6 36.3 THE CAUSE OF INCREASES IN SCABBING FROM MERCURIAL SOIL TREATMENTS Except in cases where the controls are severly scabbed, mercurial soil treatments in Michigan nearly always give an increase in scabbing which is frequently statistically significant even when one deals with small numbers. Apparently the mercurial either stimulates the activity of Actinomvcetes in the soil, or it in some way predisposes the host to infection. By dilution plate counts, Frutchey and Muncie (43) showed that under low soil moisture conditions the presence of mercurials in a soil from East Lansing, Mich., increased the ratio of Actinomvcetes to other soil organisms. This increase In number of Aotinamyoetes took place in soil samples treated with calomel and yellow oxide of mercury held at BO$ and at 35$ of the moisture holding capacity while there was a decrease in the ratio of Actinomvcetes to bacteria and fungi in soil samples held at 55, 70 and 100$ of the moisture holding capacity. Under low soil moisture conditions the mercurials apparently either stimulated the development of soil Actinomvcetes or inhibited the growth of other soil organisms, so that the Actinomvcetes had less competition. These data lend weight to the theory that mercurials aggravate scabbing through their effect on the soil flora. However, seed -56treatment with corrosive sublimate sometimes causes a significant increase in scabbing ($59) • It is difficult to understand how so small an amount of mercury introduced into the soil could affect the soil flora through a sufficient volume of soil to cause a significant increase in scabbing. In this connection, it is of interest to note that Botjes (16) obtained an increase in blackleg (Erwinia carotovora) of potatoes from seed treatment with corrosive sublimate, and Tucker (l#$) reported an increase in Rhizoctonia in­ fection from soil treatment with yellow oxide of mercury. It has been shown that calomel at 50 p.p.m. con­ trolled Rhizoctonia scurf and also scab caused by Long Island strains of Actinomyces in Michigan soil. Although this is proof that mercury compounds can be effective in controlling scab in a soil in which an increase in scabbing would have resulted with normal soil infestation, it does not necessarily indicate that the mercurial migrated through the soil since the calomel was mixed thoroughly through the soil. The failure to control scurf with mercurials under field conditions at Lake City, while it was controlled in a pot experiment, might be taken to indicate that the mercurials, which v/ere applied in the planting furrow, did not migrate through the soil suffic­ iently to afford protection. However, as shown in Table Vll, -57*' yellow oxide of mercury at Lake City had the same effect on scabbing no matter whether it was applied in the planting furrow, or banded on either side of the seed pieces, or placed two inches below the seed pieces, or applied on the surface of the soil, or mixed through the soil in the area in which the tubers would later be formed. There was a much smaller and statistically in­ significant increase in scabbing where the mercurial was placed four inches below the seed pieces. Therefore, if the increase in scabbing from mercurials is due to their effect on the soil flora the mercury compound did migrate sufficiently to affect the soil flora except when the mercurial was placed too deep. Since the potato roots would reach the mercurial readily in the case of every placement employed except the one in v/hich only a small increase in scabbing v/as noted, these data do not detract from the plausibility of the hypothesis that the cause of increase in scabbing with mercurials is due to a predisposing effect on the host plant, and so do not prove that the mercurial migrated through the soil. In an attempt to throw more light on this subject, several experiments were designed to test each of the tv/o hypotheses as stated above. -58Effect of mercurial soil treatments on the -parasitism of Actinomyces sun. on beets, radishes, and other hosts. In 1937, in a study of the host range of phytopathogenic Actinomycetes, the test plants were planted in 100-foot rows in soil that had produced several consecutive crops of heavily scabbed potatoes. In 20-foot strips at right angles to the rows, the soil was treated respectively with calomel at 20 Ibe. per acre, lime at one ton, and a combination of 20 lbs. of calomel and a ton of lime. The chemicals were applied to the surface of the soil and raked in. The initial soil reaction of the plot was unusually variable; soil samples gave readings from pH 6.0 to pH 6.8. The results of these trials are recorded in Table XS. Since there was no replication except for potatoes and the controls, it is not known how much of the var­ iability was due to soil heterogeneity. However, the figures show certain consistent trends. It should be pointed out here that the failure of calomel to cause an increase in scabbing of potatoes in heavily infested soil is in no wise unusual. Calomel usually has no marked effect when the controls average over 50-55$ of the surface area of the tubers scabbed, but, when the controls are only lightly to moderately scabbed, calomel nearly always causes a marked increase in both -59incidence and severity of scabbing of potatoes under Michigan conditions. In this instance calomel did not greatly affect either the incidence or the severity of scabbing on the roots of radishes, turnips, and ruta6k bgas, but apparently greatly aggravated scabbing on the roots of table beets and eggplants. Lime caused no marked increase in scabbing on any of the hosts except table beets, and the increase in that instance was no greater than occasionally occurs between rep­ lications in a randomized system. The eombinntion of calomel with lime aggravated scabbing on all of the hosts except White Icicle radishes, and caused a greater increase in scabbing than did either calomel or lime applied separately except in the case of White Icicle radishes and eggplants. In 1938, the same hosts were again planted in scab-infested soil using yellow oxide of mercury at 20 lbs. per acre in replicated 20-foot rows. Although eggplants, and the crucifers were scabbed, as noted at mid-season, after heavy rainfall in August the roots were so damaged by fungus rots and maggots that it was not possible to determine the incidence of scab with an adequate degree of accuracy for comparison of the treatments. As shown in Table XXI, yellow oxide of mercury caused an increase In scabbing of beets that -60- fell Just short of statistical significance for perof j~h& 5 vr c e. a,r*- « cent4of the roots scabbed, but that was significant when perce*vfc calculations were based on the Kin'p^.wywaB of the roots scabbed. It is concluded that mercurials not only generally aggravate scabbing of potatoes in field trials at East Lansing, but that they also cause an increase in scabbing on the roots of table beets and eggplants. -61- TABLE XX The Effect of Calomel and Lime on the Incidence and severity'of Plant Actinomycosis Field trial. East Lansing, 1957 Control Calomel Lime Calomel Host 20 lbs. 1 ton & Lime per acre per acre________ % scabbed > <5f -f- >■ * «• H* t- 80 14 100 63 100 Crimson Giant Globe 15 100 I15 100 10 100 56 100 0 79 0 89 rf * Early scarlet Turnip 7 Radishes *> * ^ f- U 87 S O -o u I White Icicle 9 89 ! 0 I 90 j ; i Turnips Early Purple Top 0 22 j 0 20 j 0 27 0 5* 0 67 1 0 r I 47 ; 4 ! 50 S9 71 75 0 50 67 100 0 5 i 85 100 0 18 62 100: 59 100 63 100 Rutabagas Am. Purple Top Beets Burpee's Extra Early 0 27 , 59 Eggplants Burpee's Black Beauty 45 Potatoes Katahdin 7a 100 —62h TABLE XXI The Effect of Yellow Oxide of Mercury on Scabbing of Table Beets, Field Trial, East Lansing, 1938 Mercurial Control________ Treatment__________ Totals Jo of % surfac e % of % surface Jo Jo roots area roots area of roots area scabbed of roots scabbed roots __ . scabbed scabbed Sum 34 1.8 99 19.6 133 21.4 7 0.4 30 6.8 37 7.2 27 1.7 92 16.4 119 18.1 68 3.9 221 42.8 289 46.7 Mean 22.7 1.3 73.7 17-38* 14.3 i t D i f f e r e n c e h c im e e n ynetcns r e q u i r e d fo r Stjn i £ i 'c /iff c e . Variation due to Jo Deg. of freedom Sum of squares Mean square Error F of roots scabbed 5 7178.89 Treatments 1 2689.33 Blocks 2 3901.52 Error 2 588.04 294.02 5 1 2 2 342.17 237.94 55.25 48.98 237.94 Total 9.15 2689.33 13.998 of surface area scabbed Jo Total Treatments Blocks Error 24.49 9.72 4.949 The value of Hptf required for significance is Wf«' -63Effect of mercurials on the host x>lant Several attempts were made to determine whether or not mercurials predispose potatoes to scabbing. In one instance potato tubers were sprouted, and, when the sprouts were 10-14 inches long, they were detached and planted with each sprout having one end in one pot and the other end in another. Twenty pairs of pots were used for each of three treatments in scab-infested soil; la) one end of the sprout in soil treated with yellow oxide of mercury (350 p.p.m.) and the other in untreat­ ed soil, lb) each end in a pot of treated soil, and (c) each end in a pot of untreated soil. It was intended that each pair of pots should produce a single plant with two sets of roots, but one end of the sprout gen­ erally produced no roots when only one top was permitted to develop. Tne pots were replanted and one plant was permitted to gorw at each end of each sprout. After several replantings in some of the pots, an almost com­ plete stand of such pairs of connected plants was obtain­ ed, but the connecting sprout in every case died before the plants reached maturity. If the plants in untreated soil, but connected to plants in treated soil, had pro­ duced a scabbier crop than did the connected pairs of plants in untreated soil, it would have indicated that increased scabbing from mercurials is due to predisposition -64- of the host to disease. Unfortunately, in this trial the controls were too scabby for the mercurial to cause a large increase in scabbing. There were no significant differences in scabbing between the three sets of pots. With the same motive, another experiment of similar nature was attempted. Seed-pieces with one long sprout .each were planted in 8-inch pots and the sprouts threaded through the holes in the bottom of 6-inch pots. As the plants grew the upper pots were filled with soil. There were 40 such pairs of pots. Twenty of the lower pots were filled v/ith soil con­ taining yellow oxide of mercury at 350 p.p.m. soil in the other pots was untreated. The By means of wood blocks, a narrow space was left between the upper and lower pots so that the soil was not continuous. The upper pots were watered sparingly to discourage the production of roots there, and the lower pots were watered heavily. Out of 40 plants, 14 produced tubers only in the lower pot, 12 produced tubers only in the upper pot, and 7 produced tubers in both; consequently the numbers for comparison are small. Only a few of the tubers were severely scabbed; the majority had less than 10% of the surface area scabbed. In the lower pots the tubers in mercury-treated soil were considerably -65- more scabby than those in untreated soil., The tubers produced in the upper pots ranged from moderately scabby to clean with no apparent difference between those above treated and untreated soil* Since none of the differences approached significance, the re­ sults are not tabulated. In a third trial with the same aim in mind, mercuric chloride solutions were swabbed on the leaves of potato plants with cotton. applied to the soil, controls. No mercurial was .alternate plants were left as Three concentrations of mercuric chloride, 0.2%, 0.1%, and 0.01%, were applied each to 20 plants. The first application was made three weeks after plant­ ing at which time all of the plants had emerged. Two- tenths percent mercuric chloride caused such severe burning that it was not repeated, while the 0.1% solution was applied three times and the 0.01% solution four times at weekly intervals, ^t harvest all of the tubers were severely and rather uniformly scabbed with no differences between treated and control plants. In these three trials no evidence was obtained that the increase in scabbing from mercurials is due to a predisposing effect on the host plant. However, the results were not at all conclusive since in two cases the controls were too scabby for mercurials to diow -66- any marked effect on scabbing. Furthemore, the possibility that mercurials predispose potatoes to scabbing through injury to the lentic^ls, through which infection is said usually, if not always, to take place (29, 41, 78, 118), was not investigated. However, studies v/ith this point of departure were discontinued because dilution plate counts of soil samples left little doubt in the mind of the author that the principal cause of increased scabbing from mercurials arises from the effect of the treatments on the soil flora. -67- Hffect of mercurials and other antiseptics on the soil flora During the summer of 1957 an attempt was made to determine the effect of various soil treatments on the soil flora by taking samples directly from the field. The results were unsatisfactory due to tremendous differences in plate counts from a small number of Samples from plots treated in the same manner. These differences were probably due to soil heterogeneity as regards moisture content, reaction, aeration, and supply of readily decomposable organic matter, hart of the difficulty arose from an unfortunate choice of medium,beef-peptone agar of pH 7.u, These platings indicated that mercurial soil treatments caused an increase in number of ” pin point" and "lens-shaped" bacterial colonies and in the total number of bacterial colonies, while "spreader" bacteria were reduced in number. The Actinomycetal counts were too variable to draw any conclusions from them. In later studies an effort was made to eliminate the factor of soil heterogeneity by treatment of aliquot samples of thoroughly mixed soil in pots, bottles, or test tubes. In the case of bottles and test tubes the effect of varying soil moisture was also greatly reduced by incubation of the samples in moist chambers. -68Teohniaue In greenhouse experiments the soil was thoroughly mixed by shoveling it over several times before potting. After a crop of potatoes had been harvested, each pot was dumped onto a clean platform and its contents again thoroughly mixed. A soil sample was then taken from each pot, and soil samples from pots with the same treat ment were poured together and mixed to give a composite sample. Pebbles and organic debris were removed from the samples with a forceps, but the soil was not screen­ ed, since in order to do so it would have been necessary to dry the soil, and unless the screens were cleaned after each sample and precautions were taken against raising dust, a considerable amount of contamination of one sample with another might have arisen from this procedure. In laboratory studies, the soil for each experiment was air-dried and sifted through a 40-mesh screen, thoroughly mixed, treated, and aliquots placed in large moist chambers for incubation. Care was taken to adjust the volume of samples to equality since it has been shown that the density of soil samples incubated in this manner greatly influences the actinoraycetal plate count. No water was added directly to these soil samples, but they absorbed water rapidly in a saturated atmosphere. -69To offset the effect of air-drying the samples at the start, long incubation periods were employed in some of the studies. For plating, 10-gram samples of soil were added to 90 cc. of sterile tap water in a bottle (or 11 grams to lOOcc.) In the case of samples in test tubes it was unnecessary to reweigh the samples and thus contamination with laboratory organisms at time of plating was largely avoided. In the case of samples incubated in bottles, sterile water was added directly to the sample to give a 1-10 dilution. One cc. of this dilution was then pipetted into 100 cc. of sterile w a t e r to give a 1-1000 dilution. In like manner dilutions up to 1-1,000,000 or higher w e r e prepared. The 1-10 dilution was shaken 100 times, allowed to stand for 15-30 minutes and shaken 100 times more. After each step in dilution thereafter the dilution bottles were given 100 shakes each. In most cases two or more dilutions were plated, usually 1-100,000 and 1-1,000,000, but usually counts were made from only one of these. One cc. of dilution was pippetted into each Petri dish and roughly 20 cc* of agar (at about 50-55 C.) added to it and the plate rotated a dozen times. The medium employed and temperature of incubation varied from one experiment to another. Colony counts for fungi -70- in dilutions of 1-10,000 or less were generally made on the third day, and those for bacteria and Actinomycetes (regardless of degree of dilution) on the sixth day and the counts checked on the tenth day. All colonies not definitely recognized as not being Actinomycetes were examined under low power of the microscope. Counts recorded in parentheses were from plates obviously damaged by "spreader" bacteria. Since on some plates Actinomycetes come up apparently uninhibited through spreader bacteria that blot out the bacterial count, a few actinomycetal counts are recorded in the absence of parallel bacterial counts. That this technique could be repeated closely enough to give comparable results from aliqouts of a single soil sample was demonstrated by plating three 10-gram portions of a thoroughly mixed, air-dried sample using Waksman’s egg albumin agar. The results of the 1-100,000 dilution after 10 days incubation at 25 C. are given in Table XXII. Possibly even more comparable results could have been obtained by adjusting the volume of the sterile vrater blanks after autoclaving and by measuring with precision the volume of agar added to each plate, and by holding the agar for plating in a water-bath so that its temperature would have been exactly the same for each sample; but it is questionable whether the additional accuracy accruing from such a practice would compensate for the additional time and labor required. -71TABLE XXII Plate Counts from Three Aliquots of the Same Soil Sample* ______________ at Dilution of 1-100,000-*_______________ Aliquot Plate Bacteria Actinomycetes 1 2 5 4 5 6 7 8 9 10 24 95 19 109 22 98 28 120 28 102 23 124 28 135 15 168 26 122 (overrun by fungi) Mean 119.33 1 2 3 4 5 6 7 8 9 10 20 115 16 76 28 117 39 149 23 128 13 90 15 152 40 86 16 101 (overrun by fungi) Mean 112.67 23.33 2.00 1 2 3 4 5 6 7 8 9 10 119 86 111 148 117 73 127 110 119 92 29 38 33 41 27 12 36 27 34 16 5 4 2 1 2 1 0 1 3 3 Mean 110.20 29.30 2.20 23.67 Fungi 2 1 2 3 4 1 0 6 2 2.33 1 2 3 0 3 2 2 3 2 By inspection: no significant differences# •K-Waksman’s egg albumin agar, incubated 10 dyas at 25 C* -72- Mercuric chloride in local soil. In a preliminary study of the action of antiseptics on the soil flora, duplicate 10-gram soil samples, un­ treated and treated with 10 and 100 parts per million of bichloride of mercury, w$re incubated in a moist chamber (placed in a 25 C . constant-temperature incubator) for two weeks and then plated with sodium asparaginate agar adjusted to pH 7.0. The results from the duplicate samples checked fairly well. The average number of Actinomycetes per plate for the 1-100,000 dilution increased from 5.12 for the controls to 7.13 and 8.78 for 10 p.p.m. and 100 p.p.m. of bichloride of mercury, respectively. The number of bacterial colonies increased from 7.4 for the control to 921 for 10 p.p.m. and 15.9 for 1000 p.p.in. The percent of plate counts ruined by spreading bacterial colonies decreased from 50$ for the control to 20$ and 10$ for 10 and 100 p.p.m. res­ pectfully (Table 2XL11). Calomel in Long Island soil (contaminated with local flora): In like manner scab-infested Sassafras Loam from Riverhead, Hew York (from a pot employed as a "not artifieally infested" control in the 1937 experiment on the effect of calpmel on scabbing in Long Island soil) was treated with calomel at rates up to one part -73by weight of calomel to 100 parts of soil. The treated samples were incubated in test tubes in a moist chamber at room temperature. Platings were made on beef-peptone agar (pH 7.0) after two, three, and four weeks. The plates were incubated at 25 C. except that for the third plating an entire set was also run at 16 C. Th© soil samples in the moist chamber contained about 12$ moisture at each plating. A few untreated samples 'were also placed in a rack outside the moist chamber as "air-dried controls". The results for the 1-1,000,000 counts for bacteria and Actinomycetes are given in Tables XXIV and XXV respectively. Since the order of recording of plate counts in the two tables is identical, one may compare the bacterial and actinomycetal counts for individual plates as well as for treatments. In analyzing these data one must proceed with caution since duplicate samples were not plated as was done in the experiment with corrosive sublimate. All of the plates at one plating were poured from a single batch of agar, but a new batch was made up for each plating. The increase in numbers of both bacteria and Actinomycetes in nearly all treatments including airdried soil in later platings might lead one to suspect that the medium was not duplicated and that this increase -74was not real. V/ith these short-comings in mind, it yet seems fair to conclude that: Calomel at high rates of application, and even up to 1$ of the weight of the soil, did not eliminate Actinomycetes from soil samples, and did not reduce the bacterial population as measured by the number of colonies on dilution plates. A small amount of cal­ omel in the soil (10 p.p.m.) caused a striking increase in number of Actinomycetes which was consistent at every plating, and a smaller increase in the bacterial population. Air-drying soil samples up to four weeks did not reduce the number of actinomycetal colonies developing on plates but reduced the number of bacterial colonies to about one half that of similar samples incubated in a moist chamber. The number of bacterial colonies and the percent of total colonies that were bacterial was much greater in plates incubated at 16 C . than at 25 C. There were few to no fungi on the plates at high dilutions, Hovrever, at the second plating, t#& 1-5000 dilution-plates were poured from each sample. One half of these were incubated at 16 G . and the others at 25 G . The results (Table XXVI) seem to indicate that the cal­ omel had very little effect on the fungal count. How­ ever, with high rates of treatment the fungi appeared to -75- develop much better at the lower temperature. Since f A* practically all the colonies counted were Penicill^a, no information was obtained on the effect of the treat­ ments on other fungi. Calomel in local soil: In the preceding experiment it was shown that in unsterilized soil actinomycetes and bacteria increased in number with small applications of calomel. In the 1938 pot experiment 1% unsterilized soil was added to sterilized soil in some instances* Parallel to this a soil treatment experiment was carried on in bottles held in a garbage can converted into a moist chamber, for comparison of the effect of calomel on the soil flora in unsterilized soil and in sterilized soil to which 1% unsterilized, scab-infested soil was added. Ko Actinomycetes cultures were added to the sterilized soil in the bottles as had been done in the pot exper­ iment. vVater was added to the soil in the bottles to bring it up to 50% moisture holding capacity (14% of the oven-dry weight of the soil) before placing the bottles in the moist chamber. Samples w e r e plated on glucose agar after one week and after three months of incubation. The plates were held at 25 C . The results for the counts of both the 1-1000,000 and the 1-1,000,000 dilutions after one week of incubation -76- of treated and untreated soil are given in Tables XXV11 and XXV111. In the unsterilized soil calomel had a marked depressing effect on the number of Actinomycetes even at 10 p.p.m, (a rate at which there was an increase in Actinomycetes after two-weeks in­ cubation in Long Island soil)* However, calomel did not eliminate Actinomycetes even at the rate 1 part of calomel to 99 parts of soil, Ho Actinomycetes developed in plates from samples of 1 part of calomel to 9 parts of soil, but this does not necessarily prove that the Actinomycetes were killed in the soil, they may have been prevented from growing through the transfer of a considerable amount of calomel to the agar in the dilution process. After one week’s incubation extremely heavy applications (10,000 & 100,000 p.p.m,) of calomel caused enormous increases in number of bacterial colonies, while 100 & 1000 p.p.m, had little to no effect on the number of colonies although it v/as obvious that the predominant types of colonies in the treated soil were not the same as those in untreated soils. After three month’s incubation of unsterilized soil in the moist chamber (Table XXIX) , the number of Actinomycetes in the control appeared to have decreased, -77In all of the treatment samples the Actinomycetes had increased in numbers since the first plating, with more than a 1200% increase in soil containing 1% (10,000 p.p.m.) of calomel. The number of bacteria was again greatest in the mixture of 1 part of calomel to 9 of soil. In this case there were nearly as many Actinomycetes in the soil contain'* lo% of calomel as there were in the control. At least theoretically there were only one percent as many of each of bacteria, fungi, and actinomycetes in the sterilized soil to which 1% of unsterilized soil had been added as there were in the unsterilized soil. How­ ever, after one vreek of incubation the bacteria had in­ creased so enormously that there were 18 to 56 times as many bacterial colonies from the initially sterilized soil as from the unsterilized soil not only in the un­ treated soil, but with all rates of treatments except the two highest. Although with 10,000 p.p.m. (1 part calomel to 99 of soil) there were as many or more bac­ teria than there were in the control in initially sterilized soil, there was with that rate of treatment just as large an increase in the unsterilized soil. At the first plating of initially sterilized soil there were no Actinomycetes on any of the plates at any dilution employed (including 1-10,000) except in the -78case of the control v/here there were 5 actinomycetes on four plates. This is slightly less than 1% as many as found in the parallel unsterlized sample, so there appears to have been little or no change in the actinomycetal flora (as measured in this manner) at a time when the bacteria were increasing at a tremend­ ous rate. After three months, however, the Actinomycetes had shown an increase, reaching fUO,000 per gram in the control as compared with 400,000,. 460,000 and 280,000 with calomel at 100, 1000, and 10,000 p.p.m. respectively. On the other hand, the number of bacteria in initially sterilized soil had decreased since the first plating in all samples. This was true for unsterilized soil also except in the case of the control where the differ­ ence v/as small and in the other direction. -79TABLE XXIII Effect of Mercurials on the Soil Flora as Determined by Dilution-plate Counts HgCl2 p.p.m. I bact. 0 x 10# II act bact. 9 5 it it 5 5 it Or \> 4* ft A# 6.0 5.0 8.3 5.3 670 1000 900 1350 6 11 8 act. n 11 3 4 4 $50 800 900 900 7 5 4 it 12 10 8 5 vt «v x 100 980. 14 16 15 19 it x 16.0 5.5 10 16 5 7 \t 4\ 9.5 863 20 16 14 18 11 ’15.8 8.8 8 6 8 7 12 8.2 Corrosive sublimate treatments in local scab Infested soil in test tubes, incubated 2 weeks at 25 C!. in a moist chamber • Plated on sodium asparaginate agar of pH 7.0. Plates incubated at 32 C. and counts made on 6th day and checked on 10th. Dilution: 1- 100,000. # 1/16 of plate counted and number of bacterial colonies estimated from this, entire plate counted for actinomycetes. it Count ruined by spreaderbacteria. -80TABLE XXIV Effect of Mercurials on the Soil Flora as Determined by-Dilution plate Counts ACTINOMYCETES COUNTS Soil incubated parts 0* 2 weeks X 122 117 101 108 120 39.3 113.6 6 7 3 7 3 5.2 6 7 6 2 4 5.0 18 44 31 25 22 48 36.5 21.5 25 25 15 29 28 x §4.4 34 27 39 57 50 48 41 41 50 x ?6.0 64 46 30 60 62 52.4 65 171 133 66 176 122.2 39 42 50 32 40.8 59 53 29 52 48.3 56 52 51 41 50.0" 248 263 269 261 279 26?.0 46 32 32 34 36.0 24 18 20 13 - 6 2 4 1 4 3 7 1 4 5.3 2.2 4 1 1 1 Q 1 36.5 1*6 39 36 27 44 57 24 40 40 33 38.8 13 8 4 12 9 9.2 36 36 40 42 37 38.2 44 36 56 45 47 45.6 8 9 11 3 6 71.4 to 4 weeks (plates incu­ bated at 16C;) counted after 2 weeks. x 15 16 17 17 22 17.4 of calomel 500 1000 10000 P. 4 weeks 15 15 10 15 13.8 million 100 10 H 3 weeks per 0 ”Air-dried control”, similar to the control except that the tubes were placed In a rack out side the moist chamber. All missing counts are due to ’’spreader bacteria.” Calomel treatments in Long Island soil (contaminated with local soil flora) in test tubes incubated at room temper­ ature in a moist chamber. Plated on beef-peptone agar (pH 7.0) agar; incubated at 2 & b ; counts made on 6th day and checked on 10th. Dilution: 1-1,000,000. -81TABLE XXV Effect of Mercurials on the Soil Flora as Determined by Dilution-plate counts BACTERIA COUNTS 1-1 ,000,000________________________ soil incubated parts per million of 0* 0 10 100 2 weeks 7 10 11 8 X 3 weeks 29 40 38 35 - 16 18 14 8 16 9 iff.4 X 35.5 101 105 122 108 105 86.5 108.2 X 43 43 32 35 37 3§'.0 116 41 106 85 56 102 103 85 #3.2 76.0 - 4 weeks 4 weeks 82 (plates incu75 b a t e d at 16 C ») 69 45 X 92 67 90 97 114 119 117 84 160 124 134 128 112 67.8 108.5 131.6 calomel______ 500 1000 10000 18 58 41 21 18 31 .a 11 23 16 39 32 24.*. 18 21 19 26 21.0 46 50 32 49 44.3 33 38 - 43 80 49 36 35 40 37 113 35 41 .0 60.8 - 35.5 54 24 17 59 47 38 47 - - - 47.8 186 26 22 101 - 8378 103 39 46 84 88 31.7 91 105 101 95 107 99.8 73 208 90 109 115 70 .0 118.. 131 180 131 176 119 165 125 168 121 172 125.4 173.0 # "Air-dried control", same as control except that the tubes were placed in a rack out side the moist chamber. x = mean. All missing counts are due to "spreader bacteria." (Soil and method the same as for Table XXIV) 20 27 21 19 18 21. -82- TABLE XXVI Effect of Mercurials on the Soil Flora as Determined by Dilution-plate Counts FUNGI COUNTS 1-5,000 plates incubated at parts 0* 0 25° C, (count on 3d day) per 10 million of calomelL 100 500 1000 10000 X 306 170 274 166 290 220 294 188 264 128 685. 6174,4 342 347 335 344 366 346.8 276 256 313 289 235 279.6 215 142 209 119 212 127 224 110 180 131 208.,0/25.8 3.23 117 .131 111 86 3.13.6 X 56 108 90 51 57 72.4 103 106 110 106 106 106. 2 91 82 97 64 82 83.2 78 82 92 104 89 106 84 79 98 87 86.8 93.0 118 112 115 107 114 113.2 16° C. (count on 4th day) 58 63 64 61 59 61.0 # ”Air- dried control”, same as control except that the tubes were placed in a rack outside the moist chamber. x - mean, (Soil incubated 3 weeks, plated on Cook’s agar, pH 4,8.) TABLE XXVII Effect of Mercurials on the Soil Flora as Determined by Dilution-plate Counts Lusterilized Soil; Incubated one Week p.p.m. EgCl 1- lOCpOp:/J* bact. act. fungi 0 28 17 16 24 __ 16 X 20.2 10 13 11 6 5 _ 8_____ X 8.6 100 10000 23 24 0 2 2 2 2 1.6 1 0 3 0 1 2 2 3 1_____ 0_______ 1.6 1.0 0 1 0 0 1_ 0.4 2 0 3 1 1 1 3 0 2 1 2.2 0.6 1 0 2 0 1 1 1 1 0_____ 0_______ 1.0 0.4 1 0 0 0 0_ 0.2 4 3 3 2 0 0 0 0 0 1 0 0 25 1 25 _ 2,1 x 23.6 1 0 1.8 3 5 1 1 1_________ I _____ Q_______ Q__ 2.2 1.4 0.4 0.2 17 19 . 18 29 _ 37 X 24.0 3 3 3 0 4 2.6 2 1 4 0 1: , 1.6 10000 _ x 100000 28 13 18 22 18 18.8 1-Ip00,000* act. fungi bact. 107 98 7 8 8 11 7 8.2 2 2 3 0 0 1.4 0 0 0 0 1 3 2 2 3 . . 2____ 2.6 42 44 43 40 _____ 42 42.2 15 6 0 1 1 0 0 1_______ 0.6 0 0 1 1 1__ 0.6 0 0 0 1 0_______ 0.2 1 2 1 0 0__ 0.8 TABLE V II (c o n tin u e d ) * D ilu tio n c o u n te d . C a lo m e l tr e a tm e n ts i n l o c a l s c a b - in fe s te d s o i l in c u b a te d i n d i l u t i o n b o t t l e s i n a m o is t chamber a t 2 0 -2 2 C . P la te d on g lu c o s e a g a r ; in c u b a te d a t 25 G . ; co u n ts made on 6 th day and checked on 1 0 t h . -85- lliKLcJ aa V111 iSffect of iaereurials on the Soil Flora Steam Sterilized Soil Mixed with 1>j Unsterilized, Scabinfested Soil; incubated one Week p.p.m. B-gCl bact. 1-1,000,000 act. fungi 0 38 42 32 40 _ 31 X 36.6 10 46 0 50 0 38 0 48 0 _ -_______ X 45.5 0 1 0 0 1 0 0 0 bact. 1-100,000 act. 0 0 0 1 0 0 0 0 0_______ 0__________ 0 0.2 2 2 1 2 4______ 1.8 10000 + 4 0 0 0 8 z______ 4 0 84 4.4 40 37 0 0 1 1 4 5 0 0 1 0 2 0 2____________ z______ 1.0 - 0 0 6 2 2 5 6 0 2 0 2 0 0 2 . 1.6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 _ _jQ_____o______ 1 _______ x 0 1 1 0 2 0 0 ______0 0.8 0.2 0 0 0 0 2 0 0 1 0 0 3 -_ 0 0 _______ 1 ____________=_______ 0 0 1.0 0 53 0 _ ______ Q_____ X 41.8 0 1 0 0 0 0 0 0 + f 0 4 0 2 -________ 0______ 2__ 0 2.74 108 0 1 106 0 1 89 0 3 96 0 0 __ 84______0_______ 0____________-______ x 96*6 0 1*0 142 144 136 139 ' 117 x 3.35.6 fungi 0 0 0.4 0 0 0 ______ 0 _____ 1 0 0 0.2 -86TABLE XXVIII (continued) 1-10,000 dilution: Actinomycetes from control: 2,1,1,1, and 5th plate overrun by fungi. No actimomyces at any dil­ ution from treated soil. poil and method the same as for Table XXVIl} TABLlPfm Effect of Mercurials on the Soil Elora as Determined by Dilutions-plate Counts Soil incubated three Months; Dilution 1-100,000 Unsterilized soil p*p *i HgGl 0 bact. 27 2 1 31 act. 4 7 5 2 1 2 15 2 4 1 2 8 1 23 14.6 9 5.4 2 2 1 2 18 24 13 6 14.6 90 8 6 84 77 81 83.6 4 2 1 2 1 8 1 2 8 . 2 4 4 3 3 5 3.8 8 1 104 99 1 1 2 114 1 0 2 . 0 4 4 5 if 4.0 3 2 1.4 0 1 1 307 315 285 300 1 . 0 0 1 0 4 0 2 0 4 — 301.8 0 2 . 8 47 24 32 43 65 . 42.2 4 2 1 1 98 93 96 5 2 . 0 6 1 0 1 . 0 4.6 5 4 4.6 226 209 238 4 2 0 0 106 5 4 3 4.6 .. 1 . 8 0 6 5 1 4 •a: 1 1 2 5 3 5 7 4.0 5 3 2 00 • o 4.6 9 0 + J . 2 . 2 2 . 2 1 00 2 _ _ fungi 1 • cn 2 6 4 7 100000 3 1 0 8 10000 0 1 107 93 1 13 4 7.0 X 1 1 . 2 8 1000 8 8 6.4 9 9 5 100 0 3 1 8 26 25 .2 10 bact. 91 act. 1 fungi 8 2 1 8oil sterilized and id' fested vith 1fo unsteri' lized soil. z & 2 1 2 P.33 223.6 3 4 .. .3 ... 2 . 8 1 0 . . 0 ___ 0.5 7 0 0 3 6 0 0 0 6 0 0 7 0 0 0 0 1 2 7.6 0 0 ...... _______ Q-, 0 (Soil- and method the same as for Table XXYII) ___ Q 0 . 6 -88- Qalomel in the not experiment of 1938: On July 17, 1938, a soil sample was taken from each pot in the experiment with Long Island and local soil, using a cork borer. Samples from pots treated in the same manner were mixed to form composite samples so that only one composite sample ti& s plated for each treatment. Five plates of each of two dilutions (1-200, 000 and 1-20,000} were poured, but in all cases there were too many fungi to permit bacteria and Actinomycetes to be counted at the lower dilution. An attempt was again made to use beef-peptone agar, but with disastrous results. In liable XXX missing counts are due to plates having been spoiled by "spreader” bacteria. Counts recorded in parenthesis were from plates obviously damaged by "spreaders” and were not taken into consider­ ation in deriving mean values. The plate-counts are summarized in Table XXXI. Apparently there was little, if any, relationship be­ tween the number of soil Actinomycetes as determined in this manner and the severity of scabbing of the tubers at harvest. However, it will be noted that there was a tendency for fewer "spreader” colonies in treated soil. 26.7$ of all plates (of the 1-200,000 dilution) from all control pots were free from spreaders, whereas 45.0$, 60.0$, and 58.0$ of the plates from all pots treated with calomel at 50, 350, and lOOOp.p.m., re­ -89- spectively, were free from spreaders. Sodium thiocarbonate in local soil: Soil from an orchard and on which potatoes had not been grown in years, if at all, was thoroughly shoveled over to avoid soil heterogeneity and then potted in 12-inch pots in the greenhouse. Two pots were used for each treatment, but one of each pair was waxed and the basal hole plugged with a tight-fitting stopper while the other was neither waxed nor plugged: consequently, there was no real replication. All of the soil except enough for two pots was artificially infested v/ith scab by adding to it one gram of macerated potato peels from scabby tubers for each pound of soil. The next day two pots each were treated with sodium thiocarbonate at rates of 0.001, 0.01, 0.02, 0.05, 0.10, 0.20, 0.30, and 0.50 lbs. of the 50$ solution per square foot of soil surface. The soil was kept saturated for eight days and then planted v/ith formaldehyde-treated Katahdin tubers (March 10, 1938). The crop was harvested nine weeks after planting. The number of scab lesions were counted for each tuber, and the weight of the tuber taken. summarized in Table 200.1, These results are It will be noted that there was no tendency for the chemical to control scab except at rates that caused a sharp reduction in yield. At -90high rates of application there was a marked reduction in number of lesions per tuber. As usual the soil that was not artificially infested became contaminated in the greenhouse. Two days after harvesting the tubers soil samples were taken from six pairs of the pots, the soil in each pair of pots being first thoroughly stirred. These were plated with sodium asparaginate agar of pH 6.0. The results of counts of the 1-1,000,000 dilution plates are given in Table XXXL11 along with a statistical analysis. Such an analysis is an experiment of this sort is objectionable on the grounds that all of the readings for a given treatment are for a single (com­ posite) soil sample and not from separate samples from individual pots. In Table XXX11 a comparison is made between the number of lesions per tuber and the number of Actinomvcetes that developed on the dilution plates for pairs of pots. It will be noted that the correlation be­ tween these two sets of observations is fairly good, that is, reduction in scabbing was accompanied by a reduction in the total number of soil Actinomycetes as determined in this manner. Bacterial numbers were less affected by the treatments, but there was a "significant" reduction with the highest rate of treatment. Eungal numbers ran roughly parallel to the actinomyeetal numbers. -91TABLE XXX Comparison of Severity of Scabbing with Dilution-plate Counts of Soil Microorganisms in the Pot Experiment with Long Island and Local Soils in 1938. Plated July 18 on beef-peptone agar, pH 6.0; incubated ---- --- / Soil HgCl Bacteria Actino­ mycetes p.p.m. Michigan; 0 not fumigated. nor steamed, nor artifically infested. 56 (28) (15) Mean*- 56 350 Mean Michigan; 0 fumigated, not steamed, nor artifically infested 2 2 51 53 42 50 * o 38 42 41 46.3 25 15 14 18 % scab. Severity Inc idenc e 13 (7) (3) 13 56.64 go 8 18 1 2 “ .T2.7 82.82 1 $ 6 26.42 166"' 3 1 2 6 ~T 1 2 9 17 12-.7 ' ” 54.28 ' -- T U a .. 350 19 18 19 2 0 14 18 32 32 69 33 41.5 1 0 0 0 0 Michigan; steamed and infested with "Long Island Scab" 50 ------- • Mean** 8 7 5 6 12.3 I00~~ 40.67 1 W 38.19 §7 8 8 11.3 0 227 0 69 69.57 15 14 262 (164) 192 76 62 (42) Mean*- 1 1 0 0 2 0 (0 ) "52TABLE XXX Soil HgCl p.p.m. Michigan; 350 steamed and infested with wLong Island Scab” 1000 Michigan; air dried from planting date 0 (continued) Bacteria Actinomycetes 54 38 62 52 Mean 51.5 0 0 0 0 0 4.81 27 68 82 48 Mean 66 0 0 0 0 trace 22 27 38 Mean 32*5 6 8 7.0 trace 100 0.42 100 Long Island; 0 all spreaders fumigated, 15 not steamed, 50 51 nor artifically (3) (1) infested; probably (4) (1) (6) contaminated (1) 15 Mean-*51 "Mich. scab”. 350 1000 Long Island; 0 fumigated. steamed, and 50 infested with wLong Island 350 tr scabw. 1000 % scab Severity Incide 122 152 Meanl27.5 13 19 16 0.56 100 88 79 (1) (1) (1) Means-83.5 19 17 (5) (3) (2) 18 062 100 all spreaders 0.80 93 all spreaders 0.18 13 6 4 (2) 5 0.20 20 2 0.00 0 80 72 (1) Mean-”-76 130 -93- TABLE XXX Soil Hgoi Long Island; scab Incidence Severity Bacteria Actino­ mycetes 48 46 (35) 47 4 11 *3) (6) >.5 100 8.14 Mean 150 138 157 140 125 142 4 4 3 8 10 5.8 100 8.88 Mean 82 132 112 3 2 2.5 100 9.34 Mean 181 72 194 180 200 145.4 2 9 8 0 12 6.2 100 41.60 0 fumigated, steamed, and infested with "Mich. scab". (continued) - Mean* 50 350 1000 Long Island, 0 air dried from date of shipment Mean 2 5 7 4.7 3 1 3 (3 ) (2.5) % -94table XXXI Summary of the Plate-Counts from the Pot Experiment with Long Island and Local Soils. -- ____________ Soil _______ Dilution 1-200,000______________ " ave. count % plates Calomel per plate free from % p.p.m.____ bact. act.____"Spreaders" scab Michigan: airdried from planting — date. Mich.; not fumigated nor steam­ ed Mich: fumigated, not steamed 32.57.5 40 0 56.0 13.0 20 56.64 350 40.0 12.7 60 82.82 42.0 7.0 18.0 12.7 18.0 12.3 41.5 11.5 60 60 100 80 26*42 54.28 69.57 40.67 0 50 350 1000 Mich.: steamed 0 and infested with 50 ’"long Island Scab" 350 1000 227.0 0 69.0 1 51.5 0 66.0 60 40 40 80 60 88*19 1.57 4.81 trace Long Island: not 0 steamed or artif- 50 ically infested buil50 probably contami-1000 nated with local scab. 51.0 15 127.5 16 83.5 18 0 20 40 40 trace 0.42 0.56 0.62 76.0 130.0 0 0 40 10 3.80 0.18 0.20 0.00 Long Island: steam- 0 ed, infested with 50 "Long Island Scab"350 1000 5.0 2.0 Long Island: steamedO 47.0 7.5 40 8.14 infested with 50 142.0 5.8 100 8.88 "Michigan scab" 350 112.0 2.5 40 9.34 1000 145.4 6.2_____ 100______41.60 Long Island: air dried from 4.7 2.5 60 --date of shipment _______________________ -95XXXII *" t able Effect of Sodium Thiocarbonate on Scabbing, Yield, and Dilution-plate Counts of soil Actinomyeetes in a pot Experiment. NaCS3, lbs. per sq. ft. Scab: Tubers ____ no.________ lesions no. wt. total per feuber _______grams pot 04C- 0 u. p. 6 2 u, P« 11 1 1 2 59 1030 57' 136 18-8 17.0 3 97 124 387 > 674' 76 u< P< 5 6 52 48 385 , 253' 58 u. P* 8 4 124 44 418 422 70 u< P< 4 7 55 46 143 158' 27 u, P< 2 4 16 70 123. 245 ' 61 u. P. 4 7 28 80 26) 260 26 u. P* 2 2 0.30 u. P. 7 4 0.50 u. P* 0 0.001 0.01 0.02 0.05 0.10 0.20 8 0.8 11 16.4 17.0 . O' 5.2 29. 24' 42 19 O' O' 0.8 2 Actinomycetes l,000,000*s per gram m.o 0 3.8 Pots: u. s hole in bottom of pot not plugged; p.^= hole plugged with a cork. 0# — soil not artifically infested; soil from an orchard was used in this experiment and all the soil except that for this set of controls was infested by means of macerated potato scabs. £ •S \ oJ- i H $ )***&*•£ x)i 1 0 * J ^ i i * \ 31 £ * > r j j ' -96TABLE XXXIII Effect of Bodium Thiocarbonate on the Boil Flora as Determined by Dilution-plate Count (Plated with sodium asparaginate agar, pH 6.0; incubated NaCB^, lbs. per sa. ft. None* None 1 0 2 Bacteria 3 4 5 7 5 2 3 4 12 .0 1 5 2 0 2 1 .05 .20 .50 0 2 0 None* None 4 4 Fungi 4 3 4 4 .0 1 3 5 5 .05 .20 .50 1 0 0 8 2 0 0 6 0 1 0 None* None 0 2 6 T 14 23 14 3 3 3 1 0 2 0 2 2 3 4 12 11 1 3 3 3 3 17 20 20 20 16 .0 1 5 9 14 12 .05 11 4 .20 5 2 .50 Sx^ - C = (aT^**5) ____________ 1 1 0 0 1 0 0 20 23 5 1 0 I 2 .8 4.6 2 .8 2.4 2 .2 Sx.. 8 x2 = 8T2 C - 77 391 1195 2.5667 .6 3.4 4.0 4-.6 1 .0 .2 .0 Sx> ■ Sx2 8 T2 s: C 66 284 1244 145.2 Actinomyeetes 14 20 94 18. 8 Sx - 691 18 15 16 85 17. 0 8x2 = 7197 82 16. 4 16 38 14 ST2 = 31,047 30 17 12 85 17. 0 5 3 3 26 5. 2 C 5096.0333 6 4 2 19 3. 8 — C + sum of squares due to error. 20 Analysisofvariance_________ _ Mean Error Variation Degrees of Bum of freedom due to Bacteria 388.433 29 Total 236.433 47.2866 5 Treatments 6.3333 2.5166 24 152.000 Error Fungi 138. 8 29 Total 20.7200 103.6 5 Treatments 35. 2 1.4667 1 .2 1 1 1 24 Error Actinomyeetes 2100.967 29 Total 5 1113.367 222.6734 Treatments 987.6000 41.1500 5.4113 24 Error Standard error Difference required difference between for significance F means 1.5914 Bacteria 7.47 3.2847 .9275 1.9143 Fungi 14.13 Actinomyeetes 5.41 4.0566 8.3728 * Boil from an orchard was used in the experiment and all the soil except for this set of controls was in­ fested by means of macerated potato scabs. -97Studies on the tolerance of Actinomyeetes to mercuric chloride It was concluded from pot experiments that the parasitic Actinomyeetes in scab-infested Long Island (New York) and New Jersey soils must be less tolerant of mercury compounds as antiseptics than are those in scab-infested Michigan soils. To test this hypothesis numerous Actinomyeetes were planted in duplicate tubes of glycerine synthetic solution (2 0 g. glycerine, 2 g. NaN03, 1 g. KH2 PO4 , k S* each of KC1 and MgS0 4 *7 H0 H and a trace of FeClg in a liter of distilled water; pH 6 . 6 after autoclaving) with four concentrations of mercuric chloride: 250,000 500,000, and 1,000,000* 1 to 1 0 0 ,0 0 0 Isolates that did not grow well in the control tubes were discarded. Of the remainder, 5 (out of 14) were growing in one or more of the mercury dilutions after two weeks of in­ cubation. After 4 weeks one more isolate had made appreciable growth in a mercury dilution. At that time 4 of the strains that had shown no growth on any of the dilutions were discarded, while some of the others were saved. It will be noted in Table XXXIV that Act. #20 which showed no growth in any of the dilutions in 4 weeks was growing even at 1-250,000 in 8 weeks, and that Act. #10 which had made no noticeable growth in -98- four weeks even at 1 -1 ,0 0 0 ,0 0 0 , was growing at 1 250,000 after 8 weeks, and at 1 -1 0 0 ,0 0 0 after 12 weeks. This preliminary experiment showed that the range of dilutions,(1 -1 0 0 ,0 0 0 to 1 -1 ,0 0 0 ,0 0 0 ) was not suffic­ iently broad, that it was necessary to draw a time limit to observations, and that a medium more favor­ able to the growth of a large number of actinomvcetes was to be desired. The medium was modified by substituting 10 grams of glucose for glycerine, leaving 10 g. glycerine and the same basic salt solution as employed in the first trial. The reaction was adjusted to pH 6 . 8 with NaOH. 23 Actinomyeetes were planted in duplicate tubes of this solution using 9 dilutions of mercuric chloride beginning with 1 -1 0 ,0 0 0 ,0 0 0 and doubling the concentration of mercuric chloride for each successive dilution. The organisms all grew fairly well in the control tubes. Readings were taken after 2 dnd 4 weeks of incubation at a constant temperature of 25°C, Tne results of the tw o trials are given in Tables XXXIV and XXXV. In all 31 i-ictinomycetes were employed. These included two from England (Act. Setonii and Act. viridis from the National Type Culture Collection), two from Maine (C#23 and C#66 from L. A, Schaal), three from Long Island, New York (isolated by the writer from scabby potatoes sent to him from Riverhead, N.Y. by H. S. Cunningham), and 24 others isolated from various plant hosts and from soil in Michigan. Two of the Long Island strains were very similar if not identical (Act. #40 and net. #41). Possibly some of the others also should be considered as the same species. The Actinomyeetes from England and from Long Island were all less tolerant of mercuric chloride as an antiseptic than were 12 out of 13 Actinomyeetes isolated from plant hosts in Michigan. Two of the Michigan Actinomyeetes (Act. #43 isolated from a radish scab-lesion and net. #38 isolated from a potato of the Warba variety) tolerated more than 100 times the con­ centration of mercuric chloride than did Act, viridis from England and Act #40 & 41 from Long Island. Act. #6 , isolated from a scabby eggplant root, was the only Actinomycete isolated from a plant host in Michigan that was less tolerant of mercuric chloride than any of the isolates from plant hosts from other areas. Since a mercurial soil treatment aggravated scabbing of eggplant at East Lansing (Table XX), this would appear to be evidence that either Act. #6 was not the organism causing the scab lesions on the egg­ plant root, or more than one strain or species of Actinomyces is capable of scabbing eggplant roots. -100- 0 onelusions Not only was there an increase in numbers of pathogenic Actinomyeetes in mercury-treated scabinfested Michigan soils as shown by the effect of the treatments on scabbing of potatoes and other hosts, but in some instances there was an increase also in the total actinomycetal count of treated soil as measured by dilution-plate counts on various media. Mercurial soil treatments apparently gave an initial reduction in humber of Actinomyeetes. but those that could tolerate the mercurial multiplied. Extremely high rates of application of mercuric chloride and mercurous chloride to the soil did not eliminate iictinomycetes. A test of the value of mercuric chloride as an antiseptic in a synthetic solution demonstrated that in general Actinomycetes isolated from plant hosts in Michigan were more tolerant of the mercurial than were Actinomyeetes iso* lated from soil in Michigan or from plant host in Eng­ land and Long Island. These findings bear out the be­ lief that increased scabbing of potatoes in Michigan as the result of mercurial soil treatments is caused by the effect of the chemicals on the soil flora, re­ sulting in an increase in number of parasitic Actinomycetes. -101. TABLE XXXIV Tolerance of Actinomyeetes to HgClg in glycerine Syn­ thetic Solution ______________ _____ ____________ Actinomyces ~ ' time" growth; ref, no. hahatat source in dilution (x 1000) or name________________________ weeks 1000 500 250 100 A. viridis potato England 11 It 2 * 12 J* .. 4 0 0 0 0 L.I.,N.Y. 4-::- 0 0 0 0 L.C.,Mich. 2 Hh + ■■ .... ir---- +■ 0 0 0 0 40 0 0 0 0 0 18 soil n R 8 4 19 tt n 4 0 0 0 0 20 tt tt 4 8 0 tt Tl 2 4 2 0 -f +■ 0 0 21 *- 0 f .f .-tir 4 0 i~ 7 10 lb turnip air Solanum nigrum 48 4 8 12 E.L.,Mich. 4 8 1! 16 E.L,,Mich tt Amaranthus graecizans ti ,f 0 0 •4* 0 0 * 0 0 2 8 + + 4 ~h + + 2 4 4* + ■4* 4+ 0 0 0 0 0 0 0 0 0 -t L.l. s hong island, JNew yorK. L.C.,Mich. - Lake City, Michigan. E.L. .Mich. = East Lansing, Michigan 4 weeks was growing at 1:5, 000,000 but not at 1:2,,00 o #• At Abbreviations: -102TABLE XXXV ! Tolerance of Actinomyeetes to HgOla in Glycerineglucose synthetic solution____________ lowest dil. highest dil time giving Actinomyces giving ref. no. habitat source in no growth: growth: or name weeks 1 to 1 to Act. viridis potato Eng. 2,600,OOO 5,000,000 2 tt tt 4 Act. Setonii potato Eng. 1,250,00(5' 2.500.000 2 4 625.000 1.250.000 C#23 156,250 312,500 potato Maine 2 it tt 4 0#66 625,000 312,500 potato Maine 2 tt it 4 40 potato L. I. , 2 5,000,000 2, 500, 00(5 625,000 4 1,250,000 N. Y. 5,000,000 41 2,500,000 potato L. I., 2 312.500 4 156.250 N.T. 156,250 IB potato L. 0. , 2 312,500 tt it Mich. 4 78,125 35 potato L. 0., 2 39,063 39,063 Mich. 4 78,125 37 2 156,250 potato L. C. , 39.063 Mich. 4 39,063 38 potato L. C. , 2 tt Mich. 4 156,250 78,125 39 potato L. C. , 2 tt it Mich. 4 625,000 312,500 5 beet E.L. , 2 78,125 39.063 Mich. 4 10,000,000 6 E. L. , 2 egg5.000,000 2.500,000 Mich. 4 nlant 7 turnip E. L., 2 78,125 39,063 _______Mich. 4_______ I*__________ "____ 625,000 312,500 Solanum E.L., 2 13 156.250 78.125 nigrum Mich. 4 312,506 156,250 Solanum E. L., 2 14tt tt nierum Mich. 4 78,125 39,063 Solanum E.L. , 2 15 tt it nigrum Mich. 4 39,063 radish E.L., 2 43 it ----Mich. 4 78,125 39,063 Amaranthus E.L., 2 48 — ---39.063 graecizans Mich. 4 625,000 312,500 E.L. , 2 soil 4 78,125 39,063 Mich. 4 1,250,000 625,000 E.L. , 2 soil 3 156,250 Mich. 4 312,500 156,250 78,125 31 soil E.L. , 2 78,125 Mich. 4 39,063 78,125 39,063 10 air E.L. , 2 ------39,063 Mi ch. 4 Abbreviations: L. I. sl Long' Island, L.0. ^ Lake City; E.L . -s- East Lansing; Eng. - England. -103- STUDIES IN BIOLOGIC a L CONTROL OF POTATO SCAB It has been observed repeatedly by various in­ vestigators dealing with divers organisms that soilborne plant pathogens in general, when inoculated into sterilized soil, usually produce much more prompt and severe disease symptoms on a susceptible host than when the same organism is inoculated into unsterilized soil* Presumably, the other organisms in the unsterilized soil inhibit the pathogen to a certain extent. is known as biological control or antibiosis. This The complexity of the soil population makes it difficult to study the interrelationships of the various groups of soil microorganisms as a whole* However, the interreact- ions of a great many isolates from the soil population have been studied by various investigators under labratory conditions* Those workers have shown that the inhibiting effect of one organism upon another may be due to one or more of several factors: Production of toxins; change in the reaction or oxidation-reduction potential of the medium rendering it unsuited to the growth of the other organism; and competition for nut­ rients * Review of the literature Although the inhibiting effect of one organism upon another is readily demonstrated in the case of many -104- microorganisms on artificial media, relatively few attempts have been made to control plant diseases by antibiosis under field condition* In the case of potato scab, Millard (96, 97} in England obtained a reduction in scabbing by adding organic matter to the soil in the form of green grass or spent hops at the rate of 10 to 20 tons per acre. Later, Millard and Taylor (100) found that grass alone exerted no in­ hibitory action on scabbing, but that when ActinomycetBfes •praeoox (a saprophytic species) and Act, scabies were both inoculated into -soil mixed with grass, less scab occurred than when Act, scabies employed alone. Goss (48), who tested the value of ActinomycefeBrs nraecox for biological control of scab in Nebraska, failed to confirm the findings of Millard and Taylor. Sanford (118, 120) considers the soil flora an important factor in determining the pathogenicity of Act, scabies. He suggested that when scab is controlled by green rye crops the antibiotic qualities of certain predominant soil microorganisms influence the development of the pathogen. However, he found that green rye plants, applied in the field at the rate of 50 tons per acre showed no tendency to reduce scab in thoroughly in­ fested soil of pH 5.0-5.4 in Minnesota. Although green manuring has been widely recommended -105- as a control measure for potato scab, there are relatively few experimental data on the subject. Vi/hite (160) found that green manuring reduced the amount*scab in Kansas, and that green manure in com­ bination with sulphur was more effective than alone. Dippenaar (32) got no control of scab in Wisconsin with green pea vines as a manure, now with sulphur in com­ bination with the manure. Riha (112) reported negative results with green manure for scab control in Czechoslo­ vakia. As pointed out by Huisman (59) it is well known that the disease is prevalent on plowed up grass lands although the contrary might be expected from the large quantity of plant remains in the soil. However, he reported that it had been noted that infection had been reduced by a green manure of oats. As intimated by Mill­ ard and Taylor (100) and Sanford (118), it may well be that green manuring is effective in reducing the scabbing of potatoes only where the green manure serves as a medium for the growth of organisms that are antibiotic to the pathogenic Actinomyeetes. Kiessling (70), in Germany, infested soil under field conditions with a mixed culture of bacteria an­ tibiotic to Act. scabies and obtained a crop of tubers free from scab except for a small amount of a type of scab due to a different species than Act, scabies, where­ -106- as the controls were heavily scabbed. He concluded that the bacteria were not antagonistic to all types of scab, but that biological control would be practical. Wieringa and Wiebols (161) isolated a polyvalent bacteriophage for species of Actinomyeetes. including Act, scabies, and they suggested the possibility of using bacteriophagy in controlling potato scab. How­ ever, as yet no bacteriophage for any plant pathogen has been utilized in controlling a disease under field conditions. Weindling (151, 152, 155) and Weindling and Emerson (154) found that Trichoderma lignorum and Gliocladium produce a substance lethal to certain fungi. That Trichoderma lignorum and other species of Trichoderma markedly effect the growth of various other fungi has been frequently reported (6, 57, 66, 67, 102, 151). Butler (19) found that a species of Trichoderma caused a marked reduction in the amount of Texas root rot caused by Phymatotrichum omnivorum. Bisby, lames, and Timonin (12) showed that T. lignorum suppressed the the virulence of Eelminthosporium sativum and ffusarium cuitnorum on v*rheat. However, Christensen (23) found that the addition of Trichoderma lignorum and several other fungi and bacteria to naturally infested barley seed or to sterilized or nonsterilized soil did not -107- inhibit or delay the parasitic action of seed-bone parasites; but the addition of T. lignorum and cer­ tain other fungi and bacteria to seed or sterilized soil inoculated with Helminthosporium sativum in­ creased the stand, decreased the number of deformed and stunted plants, and suppressed seddling injury. Bliss (13) failed to prevent the infection of seedling palms vtfith Omphalia by means of infesting potted soil with T. lignorum. In laboratory, greenhouse, and field experiments, Weindling and Fawcett (153) successfully controlled Rhizoctonia damping-off of Citrus by acid­ ifying the soil layers next to the seed to about pH 4 by application of aluminum sulphate or acid peat moss, but the treatment was effective only in the presence of Trichoderma spp., and the evidence obtained indicated that the decisive factor was a change in the soil microflora. Falck (3f) stated that the well-known fact that structural timber that has become water­ logged by floating it in water during its transpor­ tation is less liable to decay from dry rot (Merulius lacrymans), Coniophora. and the like is due in part to heavy infection of such wood by T. lignorum and viridis. the enzymes of which are poisonous to the wood-destroying fungi while they themselves cause little injury. His experiments showed that such -108- Trichoderma-infected wood is resistant to the attack of C. cerebella. Allen and Haensler (4) reported t. .at a species of Trichoderma was antagonistic towards the damping-off fungi, Rhizoetonia and Pythium, when the former was added to cucumber seed beds contamin­ ated with the latter. However, Daines (28) obtained no reduction in potato scab on the resulting crop bydipping the potato seed pieces in a suspension of Trichoderma spores. In addition to the claims for control of scab by soil infestation with bacteris by Kiessling, and with Act, praecox by Millard and Taylor, and the intimation that inhibition of scabbing'! green-manuring is due to biological factors, there are other indications that biological control of scab does occur under field con­ ditions. For example, Goss (48) noted that: "Soil sterilization before inoculation resulted in the most severe scab. This effect could be greatly reduced by the addition of filtrates of unsterilized soil or of organic matter in the form of manure and by delaying inoculation until soil saprophytes had become establish­ ed in the soil". Again, the sptimum soil temperature for scabbing of potatoes has been reported as 17-21 C. (32, 65), whereas the optimum temperature for growth of most soil Actinomyeetes on artificial media falls between -10928 and 37 C. That this discrepancy is explainable by the inhibitory action of other soil microorganisms at the higher temperatures is indicated by the re­ sults of an experiment conducted by Goss (48) who sound that: "Sterilized soils inoculated with A. scabies and incubated at temperatures below 22* C . did not give rise to as much scab as those incubated at temperatures from 22° to (All were held at a temperature of 22 during the infection period). XJnsterilized soils did not show this effect of temper­ ature upon the development of the pathogen"• Sanford suggested that antibiosis may play an important role in natural control of potato scab. However, in the case cf natural control sometimes brought about by a wet soil condition, he theorized (116-118) that the reduction in scabbing was due to the exclusion of air from the soil, thus inhibiting Act. scabies which is said to be strongly aerobic. Futhermore, Moore (101) observed that shallow plant­ ing and deep covering (ridging) tended to make scabbing worse. That soil aeration does have an effect on the soil Actinomvcetal flora was shown by KenKnight and Muncie (69) who incubated aliquot soil samples in test tubes in a moist chamber and found a marked correlation between the ratio of soil volume to soil weight and the -110- numbers of Actinomyeetes as determined by dilutionplate counts. Dippenaar (32), however, obtained no increase in scabbing of potatoes by artificially aerating soil, and, consequently, he doubted that exclusion of air was the cause of inhibition of scabbing in wet soils. Using the Cholodny slide methos (22), Conn \25) found that by increasing the moisture content of the soil, the natural flora of fungi and Actinomyeetes quickly became changed to one in which bacteria predominated. Dippenaar (32) con­ firmed Conn's findings and came to the conclusion that "The inhibitive effect of a high soil moisture con­ tent on the potato scab organism appears to be in­ direct. Abundant soil moisture favours the activities of the bacterial flora in the soil and this seems to be chiefly responsible for the lack of spore germina­ tion and of development shown by the scab organism in wet soils". Dippenaar1s explanation that control of scab in wet soils is biological in nature, appears to be more probable than that of Sanford. The conflicting litera­ ture on the effect of soil moisture on scabbing of pot­ atoes could be explained on the basis that in some soils there are microorganisms vjhich are antibiotic to the parasitic Actinomyeetes in that soil, whereas in other -111- wet soils either these antibiotic organisms are few or lacking, or are themselves inhibited by other organisms, or certain parasitic Actinomyeetes in the soil are tolerant of them* In regard to this conflict of opinions concerning the effect of soil moisture on scabbing, it will be noted that although Sanford (116-118), Martin (88), and Dippenaar (32) all demonstrated experimentally that scabbing decreased with increased soil moisture in their soil samples, Schacht (123), Caspri (33), Frank (41), Sorauer (129), Humphrey (60), and Lutman and Cunningham (77) all considered that a wet soil condition tended to increase scabbing, while Voekel and Klemm (142) re­ corded very severe scabbing during an unusually wet year. MacMillan (84) observed in potatoes under irri­ gation that steadily growing plants, maintained free from excess of drought or moisture, appeared to escape the disease a longer time than where improper application of water had occurred. Although severe scabbing of potatoes occurs in Michigan in rather wet as well as in dry years, the writer has noted, in three years of green­ house studies, that when soil in pots is kept distinctly wet throughout the period of tuber development, little or no scabbing occurs. However, when the soil is per­ mitted to dry out for a brief period at any time during the development of the tubers, scabbing is generally -112- severe. Further, it will he noted that in contrast with the results of Conn and of Dippenaar reviewed ahove, Lutrnan, Livingston, and Schmidt (80), from monthly plate counts over a period of eight years, found that hoth Actinomyeetes and bacteria increased with increase in soil moisture with no antagonism observed between bacteria (rods and cocci) and Actinomyeetes in the plate counts, the two running reasonably parallel. It may we11 be that in the absence of soil organisms antagonistic to Actinomyeetes the latter increase in numbers with rise in soil moisture, as reported by Lutrnan e t al, and in such a situation an increase in scabbing of potatoes might .be expected from an increase in soil moisture. On the other hand, an increase in soil moisture in some soils may favor the development of organisms antagonis­ tic to parasitic Actinozayoetes to the detriment of the latter with the result that an Increase in soil mois­ ture would inhibit scabbing. These antagonistic organ­ isms would not necessarily be bacteria, since species of Trichoderma (which are antibiotic to many organisms) abound in wet soils (145). #n. M a r t in and Person ( 9 4 ) w ere a b le to r e p r o ­ duce th e d is e a s e w it h v a rio u s i s o la t e s A ctinom yces fro m pox l e s io n s , s im ila r ity t o fr e q u e n t r e p o r t s of in s t r ik in g r e g a r d in g scab o f w h ite p o ta t o e s , P o o le fo und t h a t th e pox d i ­ sease was w orse i n d r y th a n I n w et y e a rs t h a t s o ils (1 0 9 ), t h a t p a ck h a rd a f t e r r a in s a re f a v o r ­ a b le t o i t s d e velo p m en t ( 1 0 7 ) , and t h a t th e m alad y c o u ld be c o n t r o l le d b y s u lp h u r s o i l t r e a t ­ m ents ( 1 0 8 ) . A c t . T o ts o h id lo w s k ii S e rb in o v a tta c k s th e fr u its o f c h i l e p ep p er ( Capsicum annum) i n E u ro p e, sometimes c a u s in g c o n s id e r a b le damage ( 1 2 1 - 1 2 2 ,1 2 5 ) . A c t , b r a s i l i e n s i s S pen cer r o t s B r a s i l n u ts ( B e r t h o lle t ia n o b ilis and B . e x c e ls a ) ( 1 3 0 ) . Root nodules of Elaeagnus angustifolia and various species of Alnus in Europe are said to be caused by Act, alni ( 1 8 , 5 6 , 7 1 , 1 0 5 , 1 1 4 ) , which, it is claimed, is capable of fixing atmospheric nit­ rogen. I n addition, Beijerink ( 1 0 ) isolated Act, ohromogenus in practically pure culture from the roots of various plants (including ferns, shrubs, and trees), although he did not consider -149- th e o rg a n is m p a r a s i t i c . F u rth e rm o re Banga ( 7 ) i s o l a t e d an a c i d - f a s t a c tin o m y c e te fro m l i v i n g t is s u e s ( le a v e s , p e t i o l e s , r o o t s ) o f s tra w ­ b e r r y , p o ta to , to m a to , b ean and v a r io u s o th e r p l a n t s , - th e organism s o c c u rin g i n a p p a r e n tly h e a lt h y as w e l l as i n d is e a s e d t is s u e s . The r e l a t i o n s h i p o f A ctinom yces scab o f v a r io u s h o s ts to t h a t o f p o ta to e s has lo n g been r e c o g n iz e d , due to th e f a c t t h a t c e r t a i n o f th e s e h o s ts f r e q u e n t ly show symptoms o f th e d is e a s e when grown I n s o i l i n f e s t e d w it h th e p o ta to scab o rg a n is m s . C o n s e q u e n tly th e scabs o f b e e ts ( 5 , 7 7 , 1 6 7 ) , t u r n ip s d is h e s ( 1 6 8 ) , ru ta g a g a s ( 1 6 9 ) , r a ­ (1 7 0 ), c a rro ts ( 1 7 2 ) , and p a rs n ip s h ave b een c o n s id e re d to be caused b y A c t, (1 7 1 ) s c a b ie s , a lth o u g h I n th e case o f b e e ts and c a r r o t s o th e r s p e c ie s o f A ctinom yces a ls o have b een Im p lic a te d (6 1 ,9 9 ,. H ow ever, r e l a t i v e l y few r e p o r t s have b een made o f a tte m p ts to p ro ve th e i d e n t i t y o f th e c a u s a l organism s by c r o s s - in o c u la t io n s . The r e l a t i o n s h i p o f p la n t -p a th o g e n ic A c t- anim a l d is e a s e s has n o t b een e x t e n s iv e ly In v e s t i g a t e d . H ow ever, i t in o m y c e te s to th o s e c a u s in g is w o rth y o f n o te t h a t Gordon and Hagan ( 4 6 ) -150- r e p o r t ed t h a t an a c id f a s t a c t in o m y c e t e - is o la t e fro m a p o ta t o scab l e s io n was h i g h ly p a th o g e n ic to r a b b it s . F i e l d e x p e rim e n ts and o b s e rv a tio n s S in c e s u s c e p tib le p la n t s o th e r t h a ^ p o ta to e s p r o b a b ly p la y an im p o rta n t p a r t i n th e m a in te n ­ ance o f sea|? i n f e s t a t i o n i n s o i l s , s ir a b le it seemed d e ­ to o b ta in in fo r m a tio n r e g a r d in g th e sus­ c e p tib ility o f th e v a rio u s r e p o r te d h o s ts o f scab t o th e s o il- b o r n e A c tin o m y c e te s i n M ic h ig a n . I n o r d e r to do t h i s , e x a m in a tio n s w ere made o f th e r o o t s o f a l l crops grown on s c a b - in fe s te d s o i l I n th e p l a n t p a th o lo g y f i e l d L a n s in g , p lo t s a t E a s t in 1937 1 0 0 - f o o t rows o f each o f th e f o llo w i n g cro p s and weeds w ere p la n t e d on h e a v i l y in f e s t e d s o i l : Crim son G ia n t G lo b e , W h ite i c i c l e and E a r l y S c a r le t T u rn ip ra d is h e s s a tiv u s ) , E a r ly P u rp le Top t u r n ip s A m erican P u rp le Top ru ta b a g a s ( Raphanus ( B ra s s ic a r a p a ) , (B . n a p o b ra s s ic a v a r . s o l i d i f o l i a ) , G olden A cre and D a n is h B a l l Head cabbage (B * o le r a c e a v a r . e a p i t a t a ) , B u rp e e 's B la c k B e a u ty e g g p la n t (Solanum m e lo n g e n a ), J im son weed (D a tu r a s tra m o n iu m ), S a v o y -le a v e d -151- s p in a c h ( S p in a o ia o l e r a c e a ) , C hantenay c a r r o t s (Daucus c a r o t a ) , H o llo w Crown p a rs n ip s ( P a s tin a c a s a t i v a ) . Ohio Y e llo w G lobe and Y e llo w S w eet S p a n is h V a le n c ia onions ( A lliu m o e p a ) , and r e d r o o t ( Am aranthus r e t r o f l e x u s ) . s in c e lim e and m ercu ry compounds as s o i l tr e a tm e n ts g e n e r­ a l l y cause a m arked in c r e a s e i n b o th In c id e n c e and s e v e r i t y o f sc ab b in g o f p o ta to e s i n M ic h ig a n , a p o r t io n o f each row i n th e h o s t ran g e e x p erim en ­ t a l p l o t was t r e a t e d w it h lim e a t one to n p e r a c re and c a lo m e l a t 20 l b s . p e r a c r e , s p e a r a t e ly and i n c o m b in a tio n . The r e s u l t s o f th e t r i a l s f o r r a d is h e s , t u r n i p s , r u ta b a g a s , b e e t s , and e g g p la n ts a re g iv e n i n T a b le XX i n w h ich i t is shown t h a t th e r e was a c o n s id e r a b le amount o f scab on a l l o f th e s e h o s ts . S cab b in g o f e g g p la n t r o o ts is n o te d h e re f o r th e f i r s t t im e . a p p a r e n tly The le s io n s ap­ p e a re d s i m i l a r to th o se on th e r o o ts o f t u r n ip s and ru ta b a g a s and i n v a r i a b l y y ie ld e d a lm o s t p u re c u lt u r e s o f A c tin o m y c e te s on p l a t i n g . case o f p o ta t o As i n th e scab , th e in c id e n c e and s e v e r it y o f s c a b b in g o f e g g p la n t r o o ts was m a rk e d ly i n ­ c re a s e d b y s o i l tr e a tm e n ts w it h lim e and w it h c a lo m e l. T h a t w hat appears to be scab le s io n s may o c c a s io n a lly be o b serve d on th e r o o ts o f Am aranthus r e t r o f l e x u s was f i r s t c a l l e d to th e a t t e n t i o n o f th e a u th o r by D r . Muncie who b ro u g h t two scabbed r o o ts i n t o th e la b o r a t o r y i n 1 9 3 6 . B o th y ie ld e d an A ctinom yces o f th e a lb u s group on p l a t i n g , i n a 1 0 0 - f o o t row o f t h i s p l a n t on s c a b - in f e s t e d s o i l i n 1937 th e r e w ere o n ly a fe w p la n t s t h a t ap p eared to have scab le s io n s on th e r o o t s , A few o th e r s i m i l a r l y d is e a s e d p la n t s w ere fo u n d as weeds i n th e p o ta to p l o t s . A po o r stan d o f p a rs n ip s was o b ta in e d , th e r e b e in g o n ly a b o u t 20 p la n t s in th e 1 0 0 - f o o t ro w . The roots of these were free from lesions resemb­ ling scab, as were also the roots of the carrots, spinach, and cabbage, and the bulbs of the onions. Examination of roots of other plants on scabinfested soil revealed no scab on red peppers (Capsicum annuum), tomatoes (Lycopersicum esculentum), wild ground cherry (physalls spp.), nor on any weeds except Amaranthus retroflexus and 153- Solanum n ig ru m . The o c e u rre n c e o f w hat ap p eared t o be t y p i c a l scab le s io n s on th e r o o ts o f th e l a t t e r was commonplace b o th I n th e f i e l d and g re e n h o u s e , and th e le s io n s alw ays y ie ld e d A c t in o m yce tes on p l a t i n g . D r . muncie c a l l e d to th e a t t e n t i o n o f th e w r i t e r t h a t th e n u ts o f a few h ills o f p e a n u ts p la n t e d on s c a b - in fe s te d s o i l b y D r . G rig s b y w ere co vered w it h s m a ll r a is e d le s io n s w it h a p r o l i f e r a t i o n o f t is s u e t h a t r e ­ sem bled s c a b . Many o f th e le s io n s had a w h ite pow dery a p p ea ra ce due to f r u i t i n g Ac t inomyc a te s.. I n 1938 p o ta to e s E x tra E a r ly ) , t u r n ip s ( K a t a h d in ) , b e e ts (B u rp e e 's ( E a r l y P u rp le T o p ), r u t a ­ bagas (A m e ric a n P u r p le T o p ), e g g p la n t (B u rp e e 's B la c k B e a u ty ), two v a r i e t i e s o f r a d is h e s (F re n c h B r e a k f a s t and Crim son G i a n t ) , and Amaranthus r e tr o fle x u s were p la n t e d i n 2 0 - f o o t rows i n s c a b - i n f e s t e d s o i l , w it h and w ith o u t y e llo w o x id e o f m e rc u ry as a s o i l tr e a tm e n t, and w it h two ra n d o ­ m ize d r e p e t i t i o n s o f each tr e a t m e n t . A lth o A . r e t r o f l e x u s , e g g p la n ts , and th e c r u c if e r s w ere scabbed, as n o te d a t m id -s e a s o n , a f t e r h e a v y r a i n ­ f a l l i n A ugust th e r o o t s were so damaged b y fungus r o t s and maggots t h a t i t was n o t p o s s ib le to -154- d e t erm in e w it h a c c u ra c y th e in c id e n c e o f th e d is e a s e * The e f f e c t o f th e tr e a tm e n t on s c ab b in g o f b e e ts i s in shown i n T a b le X X I and is d is c u s s e d th e t e x t p re c e d in g th e ta b le • The r o o ts o f r e d peppers p la n t e d on scab- in f e s t e d s o i l i n 1938 showed a h ig h In c id e n c e o f le s io n s t h a t somewhat rese m b led p o ta to scab and w h ich y ie ld e d A c tin o m yc ete s as w e l l as d iv e r s o t h e r organism s on p l a t i n g * A lth o u g h many r o o ts o f th e s e p l a n t s w ere examined i n J u ly , no tr a c e o f scab was n o te d a t t h a t tim e * The a u th o r doubts t h a t th e A c tin o m y c e te s were i n t h i s case p r im a r y in v a d e r s • P a t h o g e n ic it y t e s t s The r e s u l t s te s ts o f s t r a in s o f th e w r i t e r ' s o f A c tin o m yc ete s w ere r a t h e r u n s a t is f a c t o r y due t o d i f f i c u l t y c o n ta m in a tio n o f s t e r i l i z e d A c tin o m y c e te s * p a t h o g e n ic it y o f p r e v e n tin g s o i l w it h p a r a s i t i c F u rth e rm o re , i n most cases K och's p o s tu la t e s w ere n o t c a r r ie d o u t to th e e x te n t o f r e i s o l a t i o n a n d , i d e n t i f i c a t i o n o f th e o rg a n is m s . N e v e r th e le s s , n o te * some o f th e r e s u l t s a r e w o rth y o f -155- On May 1 3 , 1937 s p ro u ts fro m p o ta to tu b e rs (K a t a h d in ) w ere washed and la y e r e d i n steam s te r iliz e d s o il in s ix - in c h p o ts * T h ree o r f o u r s p ro u ts w ere la y e r e d i n each p o t i n o rd e r t o i n ­ s u re a good s ta n d , b u t o n ly one p l a n t was p e r ­ m it t e d to m a tu re * One h a l f o f th e p o ts ( 8 2 ) w ere p la c e d o u t o f doors on a law n and th e o th e rs w ere le ft i n th e g ree n h o u s e* On J u ly 8 , d u p lic a t e p o t s , b o th in d o o rs and o u t d o o rs , w ere in f e s t e d w it h each o f 8 i s o la t e s o f A c tin o m yc ete s on a g a r . No a g a r was added to th e c o n t r o l p o t s . o f p o ts w ere h a rv e s te d August 3* B o th s e ts A l l tu b e rs pro d u c ed i n th e p o ts i n th e g reen h o u se, in c lu d in g th o s e i n th e 30 c o n t r o l p o ts t h a t w ere n o t a r t ific a lly in f e s t e d w it h scab, were s e v e r e ly scabbed* O ut o f doors t h e r e was scab i n o n ly th r e e o u t o f th e t h i r t y c o n t r o l p o ts whereas th e r e was scab I n b o th p o ts f o r each o f 7 o u t o f 8 o f th e A c t­ in o m y c e te s te s t e d * The r e s u l t s o f th is t e s t w ere d is c o u n te d b y th e w r i t e r because o f th e ap pearance o f contam­ in a tio n in 10% o f th e c o n tr o ls and because a to o -156- k ig h p e r c e n t o f th e A c tin o m y c e te s t e s t e d ap p eared t o be p a th o g e n ic # A lth o u g h hands and im plem ents w ere r in s e d i n 0 ,1 ^ b i c h l o r i d e and a f t e r o f m ercu ry b e fo r e s o i l in o c u la t io n i n th e case o f each a c tin o m y c e te te s t e d f o r p a t h o g e n ic it y , lik e ly it seems t h a t clo u d s o f spores w ere r e le a s e d in t o th e a i r w henever a c u l t u r e and t h a t one p a th o g e n ic (a g a r p l a t e ) was opened sp e c ie s i n th e group m ig h t h a ve c o n ta m in a te d a l l o f th e p o ts * About 20 cc. o f a g a r was added to th e s o i l i n each p o t w ith th e in o c u lu m , b u t no a g a r was added to th e c o n t r o ls * A p a th o g e n ic c o n ta m in a n t p ro b a b ly c o u ld become more r e a d i l y e s t a b lis h e d i n added th a n i t s o i l to w h ich ag ar was c o u ld i n th e u n in o c u la te d s o i l , c o n s e q u e n tly i t seems l i k e l y and t h a t scabbing i n some o f th e p o ts w it h in o c u la te d s o i l may have been due t o c o n ta m in a tio n . t u r e to be opened. A c t. #31 T h a t t h is was th e f i r s t c u l ­ a c tin o m y c e te ap peared to be n o n p a th o g e n ic (w h ereas a l l th e o th e rs ap p e a re d to be p a th o g e n ic ) may have been due to th e f a c t t h a t t h i s o c u la te d in t o th e von p a th a g e n ic c u lt u r e was i n ­ s o i l and th e a g a r co v e re d o v e r -157- b e fo r© a p a th o g e n ic c u lt u r e was opened. On J u ly 2 0 , 1 9 3 8 , a number o f p la n t s o f s e v e r a l weeds w ere tr a n s p la n te d i n t o s o i l i n th e g re e n h o u s e . s c a b - in fe s te d The s o i l was n o t s t e r i l ­ iz e d , n o r w ere an y p la n t s l e f t as c o n t r o ls , th e t e s t b e in g one t o d e te rm in e w h eth er th e weed r o o t s w ould scab r a t h e r th a n a p a t h o g e n ic it y t e s t o f s c a b - is o la t e s * The s o i l I n one h a l f o f th e p o ts was h e a v i l y in o c u la te d w it h c u lt u r e s o f A c t. # 4 2 ( i s o l a t e d fro m an Amaranthus r e t r o f l e x u s r o o t ) and th e o t h e r h a lf jw it h A c t . # 2 ( i s o l a t e d fro m a p o ta t o s c a b - le s io n ) • The p o ts w ere w a te re d h e a v i l y and th e s o i l th e n a llo w e d t o d ry u n t i l th e p la n t s began to w i l t and th e n w a te re d h e a v i ly a g a in , t h i s p ro c ess c o n tin u in g 5 w eeks. Many o f th e p la n t s d ie d , b u t o f th ose t h a t l i v e d e v e ry one had w hat ap p eared to be scab le s io n s on th e ro o ts * A fe w le s io n s w ere p l a t e d b u t th e y y ie ld e d s e v e r a l ty p e s o f A ctinom yces in s te a d o f o n ly th e one in o c u la t e d i n t o th e s o il# The h o s ts in c lu d e d w ere Am aranthus r e t r o f l e x u s , A . g r a e o iz a n s , Solanum nigrum, and Brassica a r v e n s is . -158- I n 19 38 an a tte m p t was a g a in made to t e s t th e p a t h o g e n ic it y o f A ctin o m y c e te s in th e g re e n ­ ho u se* I n o rd e r t o a v o id c o n ta m in a tio n th e f l o o r o f th e greenhouse was c o v e re d w it h a s p h a lt: r o o fin g . The s o i l was steam ed i n 6 - in c h p o ts f o r 15 h r s . an d*w ere p la c e d f o u r each i n g a lv a n ­ iz e d pans s p e c i a l l y c o n s tru c te d f o r th e purpose and w ere w a te re d fro m th e b o tto m . The s u rfa c e o f th e p o ts were c o v e re d o v e r w it h r o c k wool w it h th e hope t h a t t h i s w o uld p r e v e n t c o n ta m in a tio n w it h A c tin o m y c e te s fro m th e a i r * . The p o ts were p la n t e d w it h fo r m a ld e h y d e -tr e a te d K a ta h d in tu b e r s , and th e s o i l ( s t e a m - s t e r i l i z e d 1!§ h r s . b e fo r e I n ­ fe s ta tio n ) i n th e p o ts was k e p t r a t h e r w et th ro u g h ­ o u t th e e x p e r im e n t. The s o i l was in o c u la te d w it h s p o re -s u s p e n s io n s o f th e A c tin o m y c e te s . The r e s u l t s a re g iv e n i n T a b le XLV. It w i l l be n o te d t h a t tu b e rs i n 5 o u t o f 16 o f th e c o n t r o l p o ts w ere somewhat scabby. r e n d e rs th e r e s u l t s o f th e e n t i r e tio n a b le . Ho?/ever, A c t . v i r i d i s T h is , o f c o u rs e , e x p e rim e n t ques­ and n o s . 5 , 7 -159- and 38 p rod uced much more scab th a n d id any o f th e c o n t r o ls * N o. 43 gave no i n d i c a t i o n o f p a t h o g e n ic it y a t a l l , yet it is th e one t h a t ap p ea re d to be p a th o g e n ic t o r a d is h e s u n d e r f i e l d c o n d itio n s (T a b le X L V ). -160TABLE XLV A. v ir id is 1 Nfe> o c+ . P a t h o g e n ic it y T e s ts o f A c tin o m y c e te s on P o ta to Tubers so urce p o ta to (E n g la n d ) s e v e r ity n f s c ab b in g 2 p o ta t o s e v e r e , up t o 50 le s io n s p e r t u b e r . tra c e 5 beet s e v e re 6 e g g p la n t tra c e 7 tu r n ip severe 8 s o il tra c e 9 s o il tra c e 10 a ir m oderate 12 p o ta to tra c e 20 s o il m oderate 35 p o ta t o m o d erate 36 p o ta t o tra c e 38 p o ta to s e v e re 43 r a d is h none 59 ru ta b a g a lig h t 63 a ir lig h t A. S e to n ii p o ta t o (E n g la n d ) m o d erate C o n tr o l ( 1 ) C o n tr o l ( 2 ) tra c e m o d erate C o n tr o l ( 3 ) none C o n tr o l ( 4 ) none Each tr e a tm e n t c o n s is te d o f 4 6 - i n c h , p o t s . T h e re w ere 16 c o n t r o l p o ts o f w h ich a l l th e tu b e rs i n 11 w ere f r e e fro m s c a b . P la n te d N o v . 22 to D e c , 1 2 ; h a r v e s te d F e b , 2 8 , 1 9 3 9 . -161- R e f e r r in g b a ck to T a b le XXXIX i t w i l l be n o te d t h a t 12 A ctinom yce t e s w ere in o c u la t e d (some on m anure, some I n b r o t h and some i n b o th ) in t o s o i l o f a p l o t t h a t had n o t b een p la n t e d to p o ta to e s I n many y e a r s , i f e v e r. A lo n g w it h p o ta to e s w ere p la n t e d 5 - f o o t rows o f o th e r h o s ts : b e e ts , t u r n i p s , r u ta b a g a s , and r a d is h e s , b u t o n ly two o r t h r e e o f th e s e fo u r w ere te s t e d as p o te n ­ t i a l h o s ts f o r each o rg a n is m . I t was in te n d e d t h a t t h i s w o uld a ls o be a t e s t o f th e p a th o g e n i­ c ity o f th e s e A c tin o m y c e te s on p o ta to e s , b u t n o t a s in g le c le a n tu b e r was fo und i n any o f th e b lo c k s , t r e a t e d and c o n t r o l a l i k e , a t h a rv e s t. E v e ry t h i r d b lo c k was l e f t as a c o n t r o l ( s t e r i l e manure o f b e e f-p e p to n e b r o t h added to s o i l i n th e same q u a n t i t i e s as i n t r e a t e d b l o c k s ) . None o f th e b e e t s , t u r n ip s , r u ta b a g a s , or r a d is h e s scabbed e x c e p t: 5 o u t o f 9 o f th e b e e ts i n s o i l i n f e s t e d w it h A c t , # 5 ( i s o l a t e d fro m b e e t s c a b ), 2 o u t o f 6 ru ta b a g a s i n s o i l in f e s t e d w it h A c t . # 4 2 ( I s o l a t e d fro m scab on r o o t o f Am aranthus r e t ­ r o fle x u s ), and 3 o u t o f 11 r a d is h e s i n s o i l i n ­ -162- f e s t e d w it h A c t . # 4 3 ( i s o l a t e d fro m r a d is h s c a b ); T a b le X L V I w ould in d ic a t e t h a t t h i s s o il, a l­ th o u g h In f e s t e d w it h organism s c a p a b le o f scabb ing p o ta t o e s , was n o t in f e s t e d w it h or g an organism s c a p a b le o f i n f e c t i n g th e s e o th e r h o s ts o f A c t­ inom yces scab and t h a t o n ly c e r t a i n s p e c ie s a r e e a p a b le o f i n f e c t i n g th e s e o th e r h o s ts . As has b een n o te d b e f o r e , i n a p l o t a fe w ro d s d i s t a n t th e s o i l was n a t u r a l l y i n f e s t e d w it h A c tin o m y c e te s c a p a b le o f sc ab b in g a l l o f th e s e h o s ts , in d ic a ­ t i n g th e p re s e n c e i n t h a t s o i l o f p a r a s i t i c A c t­ in o m ycetes d i f f e r i n g i n h o s t r e l a t i o n s h ip s th o s e t h a t w ere so u n if o r m ly abundant i n th e s o i l o f th e p l o t t h a t had n o t r e c e n t l y ( i f prod uced a p o ta t o c r o p . fro m e v e r) 163TABLE XLV I P a t h o g e n ic it y o f A c tin o m y c e te -is o la t e s on H o s ts o t h e r th a n P o ta to e s u n d e r F i l e d C o n d itio n s , 1958 Act. Source # No . of roots scabbed beet turnip baga radish 2 potato 0 - - 0 3 soil 0 - 0 - 5 beet 5/9 - - 0 0 - - 0 6 eggplant 8 soil 0 0 - - 9 soil 0 - - 0 35 potato 0 0 - 0 39 potato 0 - - 0 0 - 2^6 0 0 3 /1 1 42 Amaranthus retroflexus 43 radish 0 57 soil 0 6 - - 64 potato 0 — — 0 S ym bols: O ajno i n f e s t i o n . - : n o t te s te d 5 /9 : 5 r o o ts o u t o f 9 showing scab i n f e c t i o n P la n te d May 1 2 ; p a r t i a l l y r e p la n t e d June 21 due to p a r t i a l drow ning o u t o f f i r s t p l a n t i n g . -164- Discussion of pathogenicity trials The problem of testing the pathogenicity of actinomycete-isolates was complicated by invariable contamination of the controls under greenhouse conditions. This was not avoided by covering the soil in the pots with rock wool, nor by planting detached potato sprouts rather than tubers. Afanasiev (2) who ex­ perienced similar difficulty obtained fairly satisfactory results by using a greenhouse with a concrete floor which he could disinfect. The writer had much less contamination when the greenhouse floor was covered with asphalt coated roofing paper than when it was left uncovered, although the results even then were inconclusive. Little contamination of the controls occurred in a trial conducted out of doors, and also there was little evidence of contamination by ''Michigan scab organisms” of soil infested with "Long Island scab organ­ isms11 in pots placed in sin orchard in 1938. It seems probable that further work could be done successfully out of doors. -165- The results of these experiments indicate that not all Actinomycetes that attack potatoes are capable of parasitizing the roots of other hosts of Actinomyces scab, but that some of the species of Actinomyces attacking potatoes are also capable of infecting other hosts seems evident since these other hosts commonly be­ come scabby on soil infested with potato scab,though they do not always do so# -166- OBSERVATIONS PROM ISOLATIONS OP ACTINOMYCETES AND DILUTION-PLATE COUNTS During the course of this study a great many media were employed for plating-out soil for samples for actinomycetal counts and for isolating Actinomycetes from plant materials. In general, synthetic media containing no pro­ teins were superior to other media. In one in­ stance two composite soil samples, one from heavily scab-infested woil and the other from lightly infested soil, were plated on various media for comparison. The results of the counts of the 1-100,000 dilution are given in Table XLVII and the formulae for the media in Table XLIX. The effect of reaction of the medium and temperature of incubation for a single sample from scab-infested soil is given in Table XLVIII. Other dilution-plate counts with beef- peptone agar (Tables XXIV, XXV, XXX), sodium asparaginate agar (Tables XXIII, XXXIII)* egg albumin agar (Tables XXII, XVXT1), and glucose agar (Tables XXVII*XXIX) are also recorded in this paper. -167- Beef-peptone and potato glucose agars were the poorest media tried for plating of both soil samples and plant material because of the high incidence of "spreader” bacterial colonies. As illustrated in Table XLVIII beef-peptone agar of neutral reaction is somewhat worse in this respect than in the same medium slightly acidified. Furthermore, a low temperature of incubation was less favorable for the spreading bacteria, although damage from them is not al­ ways avoided by incubation at temperatures as low as 16°C. Sodium asparaginate agar gives less trouble from spreaders than does beef-peptone, but where the prime consideration is the actinomycetal count, synthetic media with either glucose, glycerine, or sucrose as the source of carbon are much preferable. No spreaders have been observed on glucose or glycerine agars, and only occasionally a few on sucrose agar, although the latter medium is sometimes distorted by gasproducing organisms. These media have given approximately the same actinomycetal countsas have sodium asparaginate and beef-peptone when incubated at the same temperature. -168- It has been observed frequently that high temperatures of incubation of media (32°C. and 37°C.) generally give higher dilution-plate counts of actinomycetes than do lower temperatures (Tables XXIV, XXV, XLVII, and XLVII) with 32°C. probably about the optimum. On the other hand, bacteria frequently give higher counts at lofr temperatures of incubation (same tables). That highest counts of Actinomycetes should be ob­ tained at about 32°C. is not surprising since that is near the optimum temperature for growth of the majority of soil Actinomycetes on nutrient media. On the other hand, 16°C. is certainly be­ low the optimum for the majority of soil bac­ teria which appear on dilution plates, and the high counts obtained at that temperature may be due to a reduction in antagonistic action between the organisms at lower temperatures. This seems reasonable in view of the fact that the inhibitive action of toxins generally increases with rise in temperature. That the actinomycetal -169- count is not affected in this way may be an indication that the Actlnomycetes in general are less subject to antagonism by other organisms than are the bacteria# This too seems probable in view of the fact that the actinomycete count is often apparently not reduced by •'spreader” bacteria which eliminate fungi and greatly reduce the number of other bacteria appearing on the plates # Considering that Actinomycetes in general are re­ ported to have rather high thermal death points (80) whereas fungus spores are more sensitive to heat (149), it was thought that possibly fungi could be removed from soil dilutions without greatly reducing the actinomycetal counts by heating the soil dilution# This would make it possible to count plating at fairly low dilutions# Sterile water blanks in test tubes ( °1cc, each) were incubated in water baths held at constant temperatures of 60°, 70°, and 80°C# respectively# After the water in the test tubes had reached that of the baths, 1 cc# of 1-100 dilutions of a soil sample were added# At intervals of 5, 10, and 20 minutes tubes were removed and plunged in ice water# When all had been cooled they were plated# The results of this test are given in Table L. A short period of heating reduced the bacterial count; a longer period caused an increase in bacterial count -170- at all three temperatures. This increase may have been due to breaking up of bacterial clumps since the colonies from heated tubes were definitely smaller than those of the control. The number of fungi was very markedly reduced by heating five minutes at 60 C., but since the reduction in actinomycetal count was almost equally as great, the method had no partical application in the way intended. It will be noted that five minutes at 70°C. reduced the actinomycetal population to such an extent that no colonies appeared even in the 1-1000 dilution, whereas the control gave 4,100^,000 Actinomycetes per gram. It is perhaps worthy of note that Actinomycetes in soil samples plated by Lutman _et al (80) were much less sensitive to heat. Actinomycetes isolated from plant hosts at East Lansing commonly produced little or no pigmentation of potato media. A few of them produced b]a ck, greenish- black, blue, purple, or red pigmentation in various liquid and solid media. Actinomycetes isolated from potatoes from Lake City mostly produced blue, red, or purple pigmentation on various media. In plating soil samples from New Jersey and from Long Island, New York, it was noted that Actinomycetes which produced brown to black pigmentation of protein media were much more abundant in these soils than in any scab-infested Michigan soils that had been plated. -171- Likewise, Actinomycetes which produced coiled aerial mycelium (both clockwise and counter clockwise spirals) were more common in eastern than in local soils. -172TABLE XLVII Comparison of Media for plating Soil Samples Plate counts from two soil samples (July 25), one from a plot heavily infested with scab, the other lightly infested. Incubated 7 days. Dilution: 1-100,000.________________ Medium pH Temp. C. Beef-peptone 7.0 25° Potatoglucose 7.0 25° HEAVILY INFESTED bact. act. fungi LIGHTLY INFESTED bact. £L0*b# fU3 8 12 - 8 0 - % - - 4 (8) (4) (lu) 8 9 (1) (3) 7 12 12 17 5 3 6 7 2 3 2 5 51 ■itt* ■» ■» 55 12 (4) 31 38 # a& — 1 1 - Sodium 7.0 25° asparaginate 52 54 55 42 10 10 8 18 2 1 2 6 65 43 54 46 9 10 10 9 0 3 3 1 Sodium 7.0 32° asparaginate 46 59 61 21 19 18 7 0 8 - 79 55 45 43 19 17 11 15 1 3 4 1 1 0 11 6 43 60 -K54 13 15 15 1 0 CD 1 0 2 0 0 20 32 27 37 9 13 5 8 0 0 0 0 9 10 2 6 7 12 12 11 5 5 5 7 mm Sodium 7.0 37° asparaginate Sodium 7.0 40° asparaginate Sodium 5.4 25° asparaginate Sodium asparaginate 5.0 25 W |t> U 1 © w 19 20 20 18 31 32 81 44 21 21 (21 )*«(4)«* 6 23 6 22 - 18 15 14 10 11 10 12 5 6 3 4 8 3 6 2 4 7 4 12 7 9 1 2 9 5 3 7 1 5 2 3 2 6 2 3 6 dcnit)tflll or wor thle .ess C k ^ ■sHfr Fungi overran the plate* +■>» -173TABLE XLVII Medium pH Temp, C. continued HEAVILY SCABBY LIGHTLY SCABBY bact, act. fungi bact. act, fungi Tyrosinate 7,0 250 32 37 30 44 12 9 13 20 2 2 3 0 6 0 8 8 10 12 12 13 1 3 2 0 Sucrose 6.6 25° 54 32 27 51 11 21 27 15 5 3 0 3 24 15 24 13 17 5 10 7 2 6 1 3 Glycerine 6,7 25° 53 58 53 •35-tt 11 10 8 (5) 11 6 5 10 80 65 137 160 9 12 13 5 7 10 9 5 Glucose 6.8 25° 17 42 29 31 16 11 17 15 8 2 3 7 2 13 37 14 13 9 11 12 6 2 7 4 Succinic ac id 7.0 25° 14 15 10 14 6 6 4 6 5 5 10 4 2 3 0 3 7 3 4 4 3 3 2 3 Tartaric acid 25° 3 4 2 3 0 0 2 0 3 5 5 2 0 0 0 3 0 0 0 0 1 2 0 2 Citric acid 25° 1 1 0 5 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Oxalic 25° 1 0 0 0 0 0 0 0 8 5 15 6 0 0 0 0 0 0 0 0 3 20 20 18 14 4 11 2 4 1 42 48 47 8 2 11 13 2 acid Sodium 7.0 asparaginate + 10 p.p.m. 25° 4 4 5 2 ■^"Spreader" bacteria made the count doubtful or worthless ** Fungi overran the plate. -174TABLE XLVIII Effect of Reaction of Medium and Temperature of In­ cubation on Plate Counts Plate counts from a single soil sample; count made on 6th day and checked on 10th, Dilution: 1-100,000 Temp 37° oCM to 25° 16° pH 6 bact, act. fungi pH 7 bact. act. fungi 44 23 1 (14)*(6)* 0 43 0 12 45 0 23 * * * X 36 #5 16.0 0.25 * * * * * - * * * * * - 71 18 1 44 24 0 * * * * * * * * * x 57.5 21.0 0.5 4 29 76 * * 36.3 14 16 20 * 76 9 86 7 56 7 * * * * x 72.7 7.7 1 1 1 * * 1.0 107 5 2 58 5 3 62 5 1 87 13 2 88 5 2 80,4 6,6 2.6 * * * * — pH 8 bact. act. fungi 14 5 9 14 10 10.4 3 9 8 1 7 5.6 0 0 0 0 0 0 13.3 0 4 * * 2.0 55 10 14 9 (11)* (2)* (27)* (1)* (25)*(11)* 26.4 6.6 1 1 (0)* (0)* (1>* 0.4 74 6 57 11 45 5 * * * * 5877 ‘T73“ 1 2 4 -if-if 2.3 43 1 48 11 56 1 42 2 44 5 46.0 3.0 3 0 0 1 0 0.8 2 1 0 3 0 172 52 38 33 55 40 43,6 0 1 2 2 1 1.2 101 89 64 67 70 7872 -IS- l 6 7 0 2 372 12 2 0 0 0 1 ©76 * count damaged or ruined by Bspreader1* bacteria# -175TABLE XL IX Media Referred to in this Paper* Medium Beef-peptone beef extract Bacto peptone NaCl agar grams per liter of dist. water 10 10 3 12 Potato-glucose glucose 20 extract (filtered through cotton) of 240 gms. pared pota­ toes boiled ■§- hr* agar 15 Sodium asparaginate sodium aspar* 1 10 glycerine 1.5 » w ° 4 0.1 CaClo 0.2 MgS04 «7H0H e.i KC1 trace PeCl3 12. agar Cook's #n peptone glucose MgS04.7H0H k h 2p 54 agar Medium Glucose Sucrose Glycerine carbohydrate KHpP04 3M03 MgS04.7G0H PeClg KC1 agar grams per liter of dist. water 20 1 2 0.5 0.01 0.5 15. Egg albumin (Waksman1s ) egg albumin 0.15 glucose 10. KHp POa 0.5 MgSo4.7H0H 0.2 Fe2(S04 )3 trace agar 15. (egg albumin disolved in fl/lO NaOH until neutral to phenolphthalein, then added to the warm medium) 10. 20 0.25 0.25 30 ^Various hydrogen-■ion concentrations of some of these media were employed, and consequently the reaction of the medium employed is recorded individually for each plating* -176TABLE L Effect of Heating the Soil Dilution on Plate Counts Temperature room Time in minutes 1000*s per gram fungi bacteria Actinomycetes - 833 600.0 4100 60 C. 5 10 20 650 687 870 57.7 10.3 6.3 600 370 220 70 C# 5 10 20 675 853 1413 4.0 2.0 1.3 0*0* 0* 80 C. 5 10 20 307 797 920 0.7 1.0 0.7 0* 0* 046- No Actinomycetes at any of the dilutions poured: 1-100,000, I--10,000, and 1-1,000,» # Beef-peptone agar, pH 7.0; plates incubated at room temperature; counts made on 6th day and checked on 10th# Each number is the average count of 3 plates# Bacteria and actinomycetes counted a dilution of 1-100,000; fungi at 1-1000 (3rd day)# -177- DISCUSSION Parasitic Actinomyce te s in Michigan attack potato tubers, and the roots of beets, turnips, rutabagas, radishes, and eggplants, and evidence is presented that they attack also the roots of Amaranthus retroflexus, A* graeclzans. golanum nigrum, and Brassica arvensis, and the nuts of peanut plants. It is probable that further study would disclose more hosts. The parasitism of Actinomycetes on the roots of various crop plants and weeds likely plays an important part in the main­ tenance of Infestation in scab-infested soils, and may also have some bearing on the occurrence of Actinomycetes pathogenic to potatoes in soils where potatoes were never previously grown. Frequent mention is made in the literature of the multiplicity of species or strains of Actinomyces attacking potatoes. These species or strains have been shown to differ widely in their physiological as well as in their morphological characteristics. It is only with this in mind that the literature on potato scab becomes comprehensible. As was pointed out in review­ ing the literature, various investigators have reported very divergent results concerning the effect on scabbing of potatoes of soil moisture, soil reaction, soil -178- aeration, and. soil treatments with manures aa d anti­ septics. In fact, scarcely anything that has been said about potato scab has been found to be true by all investigators. Not only are several species of Actlnomyces in­ volved in scabbing of potatoes in a given locality as has been shown to be the case in Germany (30,61,163,164), England (98), Italy (24) and in New York (81), aid Michigan, and which is probably true elsewhere also, but apparently most or all of the parasitic Actinomycetes in one locality may differ in some physiological characteristics from most or all of those in another locality. For example, the results from mercurial soil treatments in this investigation clearly indicate that such ngeographical differences” in the at.ctinomycetal soil flora exist in regard to the tolerance of Actinomy­ cetes for mercurials as antiseptics. Broad differences in the frequency of Actinomycetes with certain cultural characteristics were also noted from platings of scabinfested Michigan soils as compared with those from Long Island, New York, and from New Jersey. Similar, tho less marked, differences were evident between the Actinomycetal floras of scab-infested soils from East Lansing and Lake City, Michigan, although those soils reacted similarly to mercurial soil treatments. -179- The various Actinomycetes attacking potatoes have been considered as separate species by several European authors, whereas American Investigators have mostly considered them as ”strains” or physiologic races of a single species, Act, scabies. In no other group of plants would such a heterogeneous population be consid­ ered as a single species. Objection has been raised to the practice adopted by Millard and Burr of estab­ lishing species differences in Actinomyces on the basis of every constant difference, since by that means hund­ reds, perhaps thousands, of species could be named (62,144); but whether these Actinomycetes are called species, strains, or physiologic races, their differences must be taken into consideration in attacking the potato scab problem. In this connection the following quotation from Millard and Burr (98) is especially apt: wIn any consideration of the incidence of scab, the physiological characteristics of many species must now be included* Certain investigators assert that the primary factor in the occurrence of scab is soil re­ action, and have tried to correlate the latter -180- wlth the range of hydrogen-ion concentration with in which a few *strains* of A. scabies develop* Such a correlation can obviously hold only for the parti­ cular species of scabbing Actinomyces present in the soil under consideration, and not for those which thrive in other types of soil.” The effect of soil reaction on scabbing is most curious. Some investigators find no effect of soil reaction on scabbing over a wide pH range (68), others show a consistent trend of increasing scabbiness with rise in soil reaction, while one author reports the opposite trend (47), and many find an optimum reaction with scabbing decreasing as the pH deviates from the optimum in either direction (14,128,129,157). This optimum reaction is extremely variable from one locality to another as is also the limiting acid re­ action for scabbing. In interpreting these results several factors must be taken into consideration. A portion of these discrepancies must certainly be due to differences in the optimum and limiting soil reactions for various species of parasitic ActinQmyces which have different geographical distributions, for differences so great as that of optimum reaction for -181- scabbing of pH 4.5 in one soil (17) while in another the limiting reaction on the acid side for scabbing was pH 5.8 (55), can scarcely be attributed to differences in the physical or chemical properties of the soil or to different associations of soil microorganisms. However, soil differences may be, in part, responsible for these discrepancies, since Wingerberg (162) found that Actinomycete-isolates not only had different optimum reactions, but that the optimum reaction of a given isolate depended upon the medium and the temperature of incubation. Soil may possibly also have an indirect effect on scabbing. An acid soil condition with a high moisture content favor the growth of certain fungi such as Trichoderma lignorum, which has been shown to be anti­ biotic to ”A. scabies” on culture media (28); where­ as other soil reactions may favor other organisms which might antagonize certain of the parasitic Actinomycetes, or would at least compete with them for nutrients and limit their development in that manner. Observations on the effect of soil moisture on scabbing are as divergent as those for soil reaction. Scabbing is most severe in some localities when the -182- soil is wet (77,20), and especially after heavy, packing rains that tend to encrust the soil (20,48). In other places a moderate amount of soil moisture appears to he optimum for development of the disease (9); while in other localities drought conditions appear to favor scabbing (88,96,116,118); and under irrigation fluctuations in soil moisture may aggravate scabbing (84). m pott experiments severity of scabbing may be unrelated to soil moisture when under field conditions in the same soil scabbing is correlated with a dry soil condition (96). in some soils little or less scabbing occurs during wet years (118,96,90); in others there is less scabbing during dry seasons (48); while in others scabbing is likely to be severe in both wet and dry years (124). At East Lansing, Michigan, the disease is rather severe in infested soil every year regardless of rainfall. It appears to be worst in encrusted soil, but where no enerusting takes place, a moderate amount of soil moisture appears to be optimum. In 1938, when the fall was wet, both the dampest and the dries parts of the field plots produced less scab than did the remainder. -183- These discrepancies might he interpreted as an indication of different geographic distributions of parasitic Actinomycetes with different optimum soil moisture levels for growth or infection. Possibly some of the strongly aerobic Actinomycetes are in­ hibited in wet woils thru a lack of oxygen as suggested by Millard (96) and by Sanford (116-118). Perhaps some of the parasitic Actinomycetes are suppressed by certain bacteria which thrive in wet soils, as suggested by Dippenaar (32). Increased scabbing in dry soil may^due in part to the higher soil tempera­ ture that obtains in dry seasons as pointed out by Millard (97). Likely much of the increased scabbing associated with drying out of soils results from the ability of the Actinomycetes to produce spores and survive and cause infection of tubers which come in contact with them in soils too dry for activity of other organisma which might interfere with them in more moist soils. Furthermore, certain Actinomycetes may be capable of parasitizing potatoes only when the latter are injured by drought as indicated in the statement of Millard and Burr that Inoculation experiments with A. flavus were "entirely negative -f 84- under normal conditions of summer heat and soil moisture, whereas when the soil was allowed to dry out so much that the plants died of drought, the inoculation was successful" (98). A wet soil condi­ tion does not inhibit scabbing in some soils probably because of the presence in such soils of parasitic Actinomycetes which are compatible with the other microorganisms, or the absence in such soils of the organisms which antagonize them in some wet soils. Successes (59,95-97,100,160) and failures (32,112,119) in control of scab have been reported for green-manuring experiments, That antibiosis Is the factor involved when control obtains seems highly probable, for, if it were not for other organisms, one would expect an increase rather than a decrease in number of Actinomycetes (including facultatively parasitic species) and a consequent increase in scabbing ^occurred in the writer fs green-manuring ex­ periment with both alfalfa and blue grass. The importance of antibiosis in natural control of scab has been a matter of much speculation. Its possible role in causing variable reports on the effect of environmental factors and of green-manuring -185- on scabbing has been discussed in the proceeding paragraphs. Sterilization of soil before infestation with scab-organisms generally results In more severe scabbing than does similar infestation of unsteril­ ized soil. A similar increase in scabbing may be obtained in Michigan and western New York soils by applications of mercury compounds. In both cases the probable reason for aggravation of the disease is that sterilization or disinfection destroy soil microorganisms which interfere with the development of the scab organisms* However the writer*s attempts at biological control of scab using 71 organisms inoculated Into the soil on various media, were wholely unsuccessful. The failure to readily obtain biological control of scab artifically under Michigan conditions is probably primarily due to the presence of numerous parasitic species of Actinomyces which are not all antagonized by any one organism used as an antibiotic. For another thing, Actinomycetes are in general re­ latively tolerant to toxins while they are themselves commonly antibiotic to other organisms* -186- The writer»s attempts at biological control were by the "cut and try" method. Not knowing which Actinomycetes were primarily responsible for scabbing of potatoes in Michigan, it was considered rather useless to precede field trials with extensive lab­ oratory experiments to study the associate action of the organisms with Actinomycetes in culture media or in sterile soil. Furthermore, toxins produced in culture media might not be produced in the soil, or if produced might be absorbed on soil particles; and the associative action of an organism with Actinomycetes might be greatly modified by other organisms in ^sterile soil. Consequently, a large number of organisms, Including several well known in the literature on antibiosis, were inoculated into scab-infested soil with the hope of finding one that would lessen the incidence of the disease. In view of the evidence for biological control of scab in nature, it does not seem iihprobable that the disease could be controlled by modifying the soil flora. Since many strains or species of Actin­ omycetes are involved In scabbing, It may be difficult -187- to find an organism antibiotic to all of them. There may be greater promise in use of mixed cultures. One of the two reports of successful experimental control of scab by antibiosis,-that of Kiessling (70), was obtained by use of mixed cultures of bacteria, but even in this case there was, in the treated plots, some scab which Kiessling considered due to Actinomyoetes other than A. scabies. Mercury compounds and various other antiseptics and disinfectants have given no promise of control in Michigan. The theory of Daines and Martin (27) that mercurials are inneffective in controlling scab in some soils through "mercury-binding" capacity of the soil which is associated with the oxidationreduction potential of the soil was not substantiated for Michigan soils. Likewise, the statement of Stormer (133) that mercury compounds are efficacious only in an acid medium (in her case in superphoysate in soil of pH 5.0) is not in harmony with the results of field and pot experiments in Michigan. It was shown that calomel would control scab in Michigan soil that was infested with scab organisms from Long -188- Island, whereas increased scabbing similar to that occurring under field conditions in Michigan occurred in Long Island soil Infested with scab organisms from Michigan. Therefore the diametrically opposite re­ sults obtained from mercurial soil treatments in scab-infested soil in the two areas (Long Island and Michigan) appears to arise from differences in the species of Actinomyces infesting those soils. This is substantiated by the fact that Actinomycetes from plant hosts in Michigan were, in general, far more tolerant of mercuric chloride as an antiseptic than were Actinomycetes isolated from plant hosts in areas where mercurials are reported to control scab. Stormer's failure to control scab in any but acid soil may have been due to the occurrence of numerous species causing scab rather than to an immobilization of the fungicide at higher pH values. It is not common for scab to be of consequence in soil of pH 5.0 such as that in which she obtained control with mercuric chloride. It is possible that there were parasitic species of Actinomyces there which were tolerant of the mercurial and so were not -189- control led in neutral soils or when the mercurial was applied with an alkaline fertilizer. If these species could not tolerate an acid soil condition whereas other species that were sensitive to mer­ curials could cause scabbing in soil of pH 5.0, the results of mercurial soil treatments would beafr a relation to soil reaction such as she reported* The failure of mercury compounds to cause an increase in scabbing when applied with sulphur may be due to the sulphur having an inhibitory action on the particular Actinomycetes that are normally benefited by the mercurial treatment without controll­ ing other parasitic Actinomycetes in the soil* That sulphur does sometime have a depressing effect on scabbing without greatly modifying the soil react­ ion frequently has been shown (35,49,136). Control of scab by acidification of the soil has not met with much favor in Michigan. The trials that have been made with sulphur soil treatments have generally been of only one year duration on a given plot, and In most instances reduction in scabbing, -190- if any, was not sufficient to justify the cost of the treatment (134,150,158)* Since a single applica­ tion of sulphur has been shown to reduce scabbing in some instances (150,158), the question arises as to whether several successive sulphur treatments might not increase the acidity of the soil to a point where scabbing would no longer be of much consequence. The problem is complicated by the common prac­ tice of growing alfalfa In rotation with potatoes* Although potatoes are relatively tolerant of an acid soil condition 55,126,129,157) the optimum re­ action for alfalfa is near neutral. Liming the soil for the benefit of the alfalfa not only aggravates scabbing of potatoes, but buffers the soil with carbonates so that a large quantity of sulphur is requires to lower the soil reaction appreciably for the next potato crop# Thus it appears that where common scab is a liiiiiting factor in the production of marketable potatoes, alfalfa and potatoes are not especially compatible in the same crop rotation* Whether, in general, Michigan soils could be econom­ ically acidified to a reaction at which potato scab -191- would not be troublesome, and whether that soil reaction could be economically maintained using acid tolerant crops in rotation with potatoes, is a problem that appears worthy of further study. Crop rotations in which potatoes occur not oftener than every 4-6 years frequently have been reported to reduce scabbing (47 ,77,113), although they certainly do not always do so. Why the organisms that cause scab should die out or lose their virulence in some soils and not in others has not be determined. It might be explained by differences in species of Actinomyces causing scab, or differences in soil con­ ditions such as soil reaction, soil moisture, soil aeration, or the presence in some soils of organisms antagonistic to parasitic Actinomycetes. At any rate, it Is clear that with our present knowledge crop rotation cannot be depended upon to control scab under all conditions. In fact, reports of the occurrence of pathogenic Actinomycetes in virgin soils are not rare (11,36,64,79,110). In recent years much emphasis has been placed on breeding potatoes for resistance to scab in the United States, both by the United States Department -192- of Agriculture and by various state institutions. Aside from difficulties in the breeding of potatoes (such as bud abscission, pollen sterility and poly­ ploidy), th© problem is complicated by the fact that many species of Actinomyces, and it has already been shown by other investigators that a variety which is resistant to scab in one locality may not be so in another (132), and evidence has been presented that this is due to "physiologic races of A. scabies", or species as some authors would term them (73). Much can be done in the way of improving varieties as regards resistance to scab, but an early complete solution of the problem by plant breeding is not to be expected. In this discussion no attempt has been made at completeness in reviewing the literature but only sufficient citations have been made to show the great diversity of opinion regarding the influence of various factors on scabbing. More thorough re­ views on the biology and morphology of Actinomycetes (33,34,38,75,166) antibiosis (45,82,111,146,148,156y), and the effect of soil conditions on numbers of sold. Actinomycetes (/45) will be found by referring to the papers cited here* -193- SUMMARY 1* In field trials at East Lansing, Michigan, on scab-infested soil of pH 6.9-7.3, no control of po­ tato scab has been obtained from soil treatments with compounds of aluminum, arsenic, boron, cerium, chromium, copper, fluorine, lead, manganese, mercury, nickel, and zinc, nor with tetrachloroethane, various phenol derivaties, sulphur, and sulphuric, hydrochloric, and oxalic acids* In greenhouse trials wFormaciden, gentian violet, malachite green, wood creosote, and coal tar creosote were also ineffec­ tive, while nickel cyanide apparently gave control at 500 lbs. per acre,- a rate too high to be considered for practical control. 2. In field trials at Lake City, Michigan, in 1937 on soil of pH 5.2-5.8, sulphur, red copper oxide, and powdered zinc all were apparently of some value in reducing scabbing at all rates of application although none of the reductions in scabbing reached statistical significance by analysis of variance. The following year, after this field had been limed, none of these treatments showed any tendency to reduce scabbing. -194- 3* Lead acetate was of no value as a soil treatment at Lake City. Ammonium thiocyanate caused a marked reduction in yield without effecting the severity of scabbing. 4* Mercury compounds as soil treatments have gen­ erally caused a marked increase in scabbing of po­ tatoes at both East Lansing and Lake City at all rates of application from 6 to 100 lbs. per acre. Yellow oxide of mercury has been somewhat more effective in aggravating scabbing than have calomel, corrosive sublimate, and ethyl mercury iodide. 5. Combinations of oxidizing and reducing agents with mercurials and other combinations of chemicals as soil treatments were of no value in controlling scab. At East Lansing in 1937 singnificant increases in scabbing were obtained with combinations of calomel with zinc, with sodium nitrite, and with potassium permanganate* Sulphur alone and combinations of sulphur with calomel and with sodium nitrite, and a combination of copper sulphate with sulphuric acid had no significant effect on scabbing. 6. At Lake City in 1937, on unlimed soil, where sul­ phur tended to reduce scabbing, the mixture of mercury -195- eompounds with sulphur in seed and soil treatments was in no case better than sulphur alone. Combina­ tions of zinc with yellow oxide of mercury and with calomel, and combinations of both calomel and corro­ sive sublimate with red copper oxide, and corrosive sublimate with oxalic acid had no significant effect on scabbing* 7. In 1937 scab-infested soil was obtained from Long Island, New York, and from New Jersey from fields were calomel had been effective in controlling potato scab. Both soils had been fumigated with carbon disulphide before shipment* In a pot experiment calomel as a soil treatment caused an increase in scabbing in proportion to the amount applied in Long Island soil that had been artificially infested with scab organisms from Michigan potatoes. Calomel had little, if any, effect on scabbing in similar soil that had not been artificially infested. The results with Hew Jersey soil were about the same as those for Long Island soil. Evidence is presented that the soils that had not been artifically infested were nevertheless contaminated with local parasitic Actinomyoetes, this explaining the failure of calomel -196- to control scab under those conditions. Calomel did not cause an increase in scabbing of potatoes in local soil in a parallel experiment probably only because the controls were excessively scabby. 8. In 1938 scabby potatoes as well as scab-infested soil were obtained from Long Island, New York, for further pot experiments. In these trials, calomel mixed thoroughly through the soil controlled scab in both Long Island and Michigan soils that had been steam-sterilized and infested with strains of Actinomyces from Long Island. Conversely, calomel aggravated scabbing in both Long Island and Michigan soils, in­ fested with Michigan strains of Actinomyces, at the same time controlling Rhizoctonia scurf. 9. In a placement test at Lake City In 1938, yellow oxide of mercury caused a highly significant increase in scabbing when (a) mixed thoroughly threw.gh the soil along the planting row, (b) applied on the sur­ face of the soil in a band 6 inches wide after the tubers were planted, (c) banded two inches from the seed pieces and on a level with them, (d) applied in the planting furrow, and (b) applied in a band two -197- inches directly below the seed pieces. However, when the mercurial was placed four inches below the seed pieces, the increase in scabbing was small ani not significant. 10* In general, in soil treatment experiments, results obtained from pot experiments were comparable to those obtained in field trials. noted. A few exceptions are Calomel even at 50 p.p.m. gave complete con­ trol of Rhizoctonia scurf in East Lansing soil in a pot experiment; whereas calomel and yellow oxide of mercury applied in the planting furrow showed no ten­ dency to control scurf under field conditions at Lake City in 1957. In pots calomel caused a significant increase in yield of potatoes in Michigan soil that had been fumigated to make it more comparable to the Long Island soil. No increase in yield occurred in soil that had not been fumigated with carbon disulphide. Mercurial soil treatments have shown no tendency to increase yield under field conditions In Michigan; at rates up to 50 lbs. per acre they have had no effect on yield. It is noteworthy in this connection that signif­ icant increases in yield from mercurial soil treatments -198- have been obtained in western New York. 11. Combinations of calomel with zinc tended to re­ duce scabbing in both Long Island and Michigan soils in pots. Apparently this was due to the zinc rather than to the combination. reduction in scabbing. Zinc alone also caused a Zinc, but not combinations of zinc with mercurials, has shown a tendency to re­ duce scabbing under field conditions in some instances. 12. In pots there was much more Rhizoctonia scurf in local soil that had been fumigated with carbon disulphide than in similar soil which had not been fumigated. 13. In field trials in 1937 calomel did not greatly affect either the incidence or the severity of scabbing on the roots of radishes, turnips, and rutabagas, but greatly aggravated scabbing on the roots of beets and eggplants. On this approximately neutral soil lim­ ing caused no appreciable increase in scabbing of these hosts except in the case of beets. A combina­ tion of calomel with lime aggravated scabbing of all of these hosts except one of the three varieties of radishes (White Icicle), and caused a greater increase in scabbing than did either calomel or lime applied -199- separately except In the case of White Icicle radishes and eggplants* 14* In 1938 yellow oxide of mercury caused a signif­ icant increase in scabbing of beets. 15* Several attempts were made to test the hypo­ thesis that increased scabbing of potatoes as a re­ sult of mercurial soil treatment is due to predis­ position of the host plant to the disease* Although the results of these trials were rather unsatisfactory, no evidence was obtained that would support this hypothesis* 16. Dilution plate counts of soil microorganisms from field, pot, and glassware experiments showed that calomel even at rates up to 1 part per 100 parts of soil did not eliminate Actinomycetes from local soil, and that at lower rates of application there was in some instances a marked increase in the total number of Actinomycetes as compared with the control. That an increase in number of parasitic Actinomycetes usually occurs as a result of mercurial soil treat­ ments might be inferred from the effect of such treat­ ment on scabbing of susceptible hosts. 17. Mercurial soil treatments, even at low rates of application, greatly affect the bacterial soil flora causing a decrease of some types of soil bac­ teria and an increase of others. In several instances -200- the total bacterial count of treated soil was con­ siderably higher than that for untreated soil. 18. Media containing proteins or peptones or as- paragin have been unsatisfactory for soil plating studies. Synthetic media with glucose or glycerine as a source of carbon have been most satisfactory for soil plating in which the desired count is that of Actinomycetes, and also for isolation of Actinomycetes from scab lesions on plant tubers and roots* 19. In a pot experiment sodium thiocarbonate re­ duced scabbing of potatoes in proportion to the amount applied, but only at rates at which the yield was materially reduced. In this instance the number of Actinomycetes in the soil, as determined by dilutionplate counts, closely paralleled the severity of scabbing as measured by number of lesions per tuber. However, as a general rule, there appears to be no marked correlation between the total Actinomycetal count of a soil and the severity of soil infestation with parasitic strains of Actinomyces. 20. A study was made of the tolerance of 31 Actinom­ ycetes to mercuric chloride in nutrient solutions. These included two from England (Act. Setonli M. & B. and Act, viridis M* & B.), two from Maine, three from -201- Long Island, New York, and 24 others isolated from various plant hosts and from soil in Michigan. The results, for the most part, bear out the theory that mercurials control scab in England and on Long Island because the parasitic species (or strains) of Actinomyces in the soils there are relatively sus­ ceptible to mercury as an antiseptic, whereas mer­ curials do not control scab in Michigan soils because the parasitic Actinomycetes are highly tolerant of mercury. 21. The Actinomycetes from England and from Long Is­ land were all less tolerant of mercuric chloride as an antiseptic than 12 out of the 13 Actinomycetes isolated from plant hosts in Michigan. Some of the actinomycete-isolates from Michigan tolerated more than 100 times the concentration of mercuric chloride than did some of the isolates from regions where mercurial soil treatments control scab. 22. The evidence for natural biological control of potato scab is discussed, 23. Pield soil was infested with various organisms which were added to the soil in broth, on agar, on sterilized manure, and on sterilized oat sprouts. -202- In trials over a three year period no indication of biological control was obtained# 24# In 1936 all organisms added to the soil on agar or on oat sprouts resulted in a striking increase in scabb­ ing whereas those added in broth had no effect on scabb­ ing# A slight tendency was noted also in later trials for organic matter alone to cause an inorease in scabb­ ing, but, because the controls were severely scabbed in later trials, no such great differences were to be observed, 25# In all, 21 bacteria, 41 Actinomycetes, seven fungi, and two yeasts were employed in attempts at biological control of scab under field conditions. Many of these organisms were not identified, and a few may be dupli­ cates except in the case of the fungi, each of which was a representative of a different genus. Among the organisms employed were Act# praecox, Triohoderma lignorum, Pseudomonas fluorescens, a^nd Bacillus me­ gatherium,- all of which are well-known in the liter­ ature on the associative action of microorganisms. 26# In plating out media containing proteins it has been constantly observed that Aotinomycetes in general are less subject to the inhibitive effects of wspreaderw bacteria than are other organisms# 27. In liquid media containing sugars some bacteria inhibited Actinomycetes by changing the reaction of the medium# -203- 28# In broth to which no sugars were added the majority of bacteria tested were not antibiotic to various Actinomycetes when the bacteria and Actinomycetes were seeded simultaneously. The Actinomycetes that were in­ hibited were in most cases those that made slow growth* Frequently the bacteria were inhibited. In many cases the bacteria and Actinomycetes were compatible. When bacteria were seeded one or more days in advance of the Actinomycetes the latter were generally inhibited, prob­ ably due to competition for nutrients. 29. A bacterium which is antibiotic to one Actinomycete- isolate may itself be inhibited by another. 30. Five bacteria, three of which had been shown by Clark to be strongly antibiotic to Phymatotrichum and other fungi on agar media, failed to inhibit scabbing of potatoes even when inoculated heavily into sterilized soil that was not artifically infested with Actinomycetes, the latter presumably contaminating the soil from dust in the air of a dusty greenhouse in which the soil was infested with scab organisms. 31* Green manures of both blue grass and alfalfa applied at the rate of 20 tons per acre caused a signif­ icant increase in scabbing. 32. Evidence is presented that Solanum melongena, -204— S. nigrum, Amaranthus retroflexus, and possibly also A* graecigans, Brassioa arvensis, and Arachis hypogaea are hosts of phytopathogenio Actinomycetes. 33* The results of pathogenicity tests with pure cul­ tures of Actinomyces were not very satisfactory due to contamination of sterilized soil with parasitic Act­ inomycetes under greenhouse conditions. This dificulty was less conspicuous in pots placed out of doors. 34. Evidence is presented that several Actinomycetes were pathogenic to potatoes. These include Act, viridis and several Actinomycete-isolates from plant hosts and soil* 35. Actinomycetes isolated from plant hosts at East Lansing commonly produced little or no pigmentation of potato media. A few of them produced intense black, greenish black, blue, purple, or red pigmentation in various liquid and solid media. 36. Actinomycetes isolated from potatoes from Lake City mostly produced blue, red, or purple pigmentation in various media. 37. Differences were noted in the abundance of certain types of Actinomycetes in eastern soils as compared with local soils. -205- Literature Cited 1* Adams, J. F. An actinomycete the cause of soil rot or pox in sweet potatoes. topath. 2. Afanasiev, M. M. 19j 179-190. Phy­ 1929. Comparative physiology of Actinomyces in relation to potato scab. Nebraska Agr. Exp. Sta. Res. Bui. 92. 1937. 3. Alexopoulos, C. J., R. Arnett, and A. V. Mclntoch. Studies in antibiosis between bacteria and fungi. 4. 234. 1938. 350. 1939). Ohio Jour. Sci. 38: 221(Exp. Sta. Rec. 80j Allen, M. C. and C. M. Haensler. Antagonistic action of Trichoderma on Rhizoctonia and other soil-fungi. 25: 244-252. 5. Phytopath. 1935. Arthur, J. C. and K. Golden. Diseases of the sugar beet root. Sta. Bui. 39. Indiana Agr. Exp. 1892. (Cited by Lut- man and Cunningham, 1914)« 6. Asthana, R. P. Antagonism in fungi as a measure of control in ’red-leg’ disease of lettuce. Proc. 4: 201-207. 16: 150. Indian Acad. Sci. 1936. 1937). (Rev. Appl. Myc. -206- 7* Banga, 0. Over ziekteverschijnselen van de aardbei en over voorkomen van een Actinomyceet in de weefsels van dese plant. Meded. v. de Landbouw Hoogeschool, Deel 35, Vehr. 5(15): 35» 1931 (Wageningen). (Rev. Appl. Myc. 11: 250-251. 8. Baten, W, D. 1932). Formulas for finding estimates for two and three missing plots in random­ ized block layouts. Sta. Tech. Bui. 165, 9* Beckwith, M. H. Michigan Agr. Exp. 1939. Report of assistant horticulturist. N. Y. (Geneva) Agr. Exp. Sta. Rpt. 6: 307315* 1888. (Cited by Lutman & Cunningham, 1914). 10. Beijerinck, M. W. Sur la production de quinone par le Streptothrlx chromongea et la biologie de ce microbe. Central, f. Bakt. Abt. 2, Orig., Bd. 6: 661. 11. Bisby, G-. R. 1900. Potato seed treatment in Manitoba. Phytopath. 14: 58. 1924 (Abstr.). 12. _____ , N. James, and M. Timonin. Fungi isolated from Manitoba soils by the plate method. Canad. Jour. Res. 8: 253-275* 13. Bliss, D. E. 1933* Soil disinfection as a means of combating decline disease in date palms. Rev. Appl. Myc. 15: 15-16. 1936. -207- 14# Blodgett, F. M. and F, B# Howe. Factors influen­ cing the occurrence of potato scab in New York. 581. 15. Cornell Agr. Exp. sta. Bui. 1934. Bonde, Reiner. Effect of applying chemicals to the soil on the control of Rhizoctonia. Maine Agr. Exp. Sta. Bui. 391: 296297. 16. 1938. Botjes, J. G. 0. Zwaartbeenigheid van de aardapp&lplant• Tidschr. over Plantenziekten 34: 91-105. 17. Boysen, H. 1928. Flatschurv - Actinomyces-schurv - og jordreaksjon. Noen iakttagelser fra hvam forsksgars Tidsskr. Norske Landbruk 1932 (10), 6 pp. 1932. * 9 18. Brunchorst, J. TTber einige Wurzelanschwellungen, besonders derjenigen von Alnus undder Elaeagnaceen. TJnters. Bot. Inst. Tubingen 2: 151. 1886/1888. (Cited by Rob erg, 1938). 19. Butler, Karl D. The cotton root rot fungus, Phymatotrichum omnivorum, parasitic on watermelon, Citrullus vulgaris. Phy­ topath. 25: 559-577. 20. Carpenter, C. W. Potato diseases in Hawaii and their control. Bui. 45. 1935. 1920. Hawaii Agr. Exp. Sta. -208- 21. Caspari. Ueber Spaltoffnungen (Stomata) der Kartoffel und Entstehung der Pocken (des gchorfes) beitfenselben. Sitz. d. mederrheinischen Gesillsch. fur Natur —und Heilkunde. Jan., 1857. (Bot. Zeitung 15: 116-117. 1857.) 22. Cholodny, N. TJeber eine neue Methode zur Unter- suchung der Boderunikroflora. Mikrobiol. 1:620-652. 23. Christensen, J. J. Arch. f. 1930. Associations of microorgan&ims in relation to seedling injury arising from infected seed. 1091-1105. 24. Gocchi, F. Phytopath. 26: 1936. Ricerche sulla 'scabbia1 della patate. Boll. R. Staz. Pat. Veg. N. S. 13: 74139. 25. Conn, H. J. 1933. A microscopic study of certain changes in the microflora of the soil. New York (Geneva) Agr* Exp. Sta. Tech, Bui. 204, 26* 1932. Cunningham, H. 8. The addition of mercury com­ pounds to the fertilizer mixture as a control for common scab of potato under Long Island conditions. Jour. 13: 100-103. 1936. Amer. Potato -209 27. Dairies, R. H. and W. H. Martin* Further studies with mercury as a seed and soil disin­ fectant. Paper presented before the American Potato Assoc., Atlantic City. Dec. 31, 1936. 28* Daines, R. H. Antagonistic action of Trichoderma on Actinomyces scabies and Rhizootonia solani. Amer. Potato Jour. 93. 29. 14: 85- 1937* Darling, H. M. A study of scab resistance in the Potato. Jour. Agr. Res. 54: 305-317. 1937. 30. De Buijn, Helen L. G. Het schurftvraagstuk van mycologische zijde beteken. Tijdschr. Landbouwk. Wageningen 47* 579. (Rev. Appl. Myc. 15: 250-251. 31. De Haan, K. 1935. 1936). Beschouwingen over de parctische Suiker-bietenteelt. V. Ziekten en vijanden van de Bieten en hun bestrijdigswijze. Meded. Inst. Suikerbiet., Bergen - o. - Z. 6: 151-166. (Rev. Appl. Myc. 15: 190. 32. Dippenaar, B. J. 1935. 193&) Environmental sx d control studies of the common scab disease of potatoes caused by Actinomyces scabies (Thax.) Gus. Dept. Agr. Union South Africa Sci* Bui. 136. 1933. -21033# Drechsler, Charles# Morphology of the genus Actinomyces, I and II. 65-83, 147-168. 34. Duche, Jacques, Bot. Gas. 67; 1919. Les Actinomyces du groupe albus. / Encyclopedie Mycologique 6; Paul Lechevalier & Pils, Paris. 35. 1934. Duff, G. H. and Catherine G. Welch. Sulphur as a control agent for common scab of pota­ toes. 36. Eastham, J. W. Phytopath. 17: 297-314. Some potato disease problems in British Columbia. 89-94. 37. Scient. Agric. 4: 1923. . Plant-disease survey in central British Columbia. Agr. Jour. Brit. Columbia 8: 224-225, 233. Appl. Myc. 3: 195* 38. Erikson, Dagny. (Rev. The pathogenic aerobic organisms Council Spec. Palck, R* 1923. 1924). of the Actinomyces group. 39. 1927. Medical Res. Rpt. Series No. 203. 1935. Ueber den Einfluss des Flossens auf die Widerstandsfahigkeit des Bauholzes gegen Trockenfaule und uber den Holzchutz durch Schimmelbefall und Diffusionstrankung. Mitt. Forstwirtsch. und Forstwissensch. 1931: 480-485. -211- 1931* (Rev* Appl* Myc. 11; 414-415* 1932). 40. Ploess, (The present status of the potato scab question). 1933. 41. Phosphorsaure 3: 726-734. (Chem. Abst. 1457(6).’ 1934.) prank, A. B. Die Krankheiten der Pflanzen. (Breslau): 141-142. 1880. (Cited by Dippenaar., 19 33). 42. Frutchey, C. W. Studies on potato scab caused by Actinomyces scabies. Mich. State College M. S. Thesis. 43. 1932. ____________ and J. H. Muncie. Soil treatments with mercurials for the control of potato scab. Mich. Agr. Exp. Sta. Quart. Bui* 16: 259-263. 44. Garbowski, L. i Mme. H. Juraszkowna. 1934. choroby roslin uzytkpwych W okresie 1926-1930. Zestawienie notowafi zakladc>n Ochrony Roslin. dgoszcz. Rocznik Ochrony Roslin, By­ Sect. A, 1: 97-235. (Rev. Appl. Myc. 13: 288. 45. Garrard, E. H. and A. G. Lochhead. 1933. 1934). Relationships between soil micro-organisms and soilborne pathogens. A review. Scien. Agric. 18: 719-737. 1938. -212- 46. Gordon, Ruth and W. A. Hagan. A study of some acid-fast Actinomycetes isolated from soil. 47. Goss, R. w. Jour, Bact. 31s 89. 1936. (Abst.) A survey of potato scab and Fusarium wilt in western Nebraska. 24: 517-527. Phytopath. 1934. 48.______ ______ The influence of various soil factors upon potato scab caused by Actinomyces scabies. Nebraska Agr. Exp. Sta. Res. Bui. 93. 49. Graham, J. C. 1937. Some experiments with sulphur as a control for potato scab. 50* Gram, E. Proc. Potato Assoc. Amer. 10(1923): 161-165. 1924. Plantesygdorame i Danmark 1930. Tidsskr. f. Planteavl. 37; 458-508. (Cited by Palm, 1934). 51. Greig-Smith, R. The action of certain organisms upon the numbers of bacteria in the soil. Proc. Linn. Soc. New South Wales 42: 162-166. 52. Halsted, B. D. 1917. Field experiments with potatoes. New Jersey Agr. Exp. Sta. Bui. 112, 1895. 53#_____________ Field experiments with potatoes in 1896. Ibid. 120. 1897. -213- 54. __________ _____ Experiments with potatoes. New Jersey Agr. Exp. Sta. Rpt. 18 (1897): 276-284. 55. Hardenburg, E. V. program. 195. 56. Hiltner, L. 1898. Potato rotations and fertilizer Amer. Potato Jour. 8: 192- 1931. Tiber die Bedeutung der Wurtzelknoll- chen von Alnus glutinosa fur die U Stickstoffernahrung dieser pflanze. Landwirtsch* 153. 57. Hino, I. Versuchsstationen Bd. 46: 1896. Antagonistic action of soil microbes with special reference to plant hygiene. Trans. Third Int. Cong. Soil Sci. 1: 173-174. 395. 58. 1935. (Rev. Appl. Myc. 15: 1936). Hohne, E. und G. Che'lard. Hat die Dungung einen Einfluss auf die Schorfbildung bei Kartoffeln. Die Phosphorsaure 4; 161- 167. (Rev. Appl. Myc. 13: 651. 1934. 1934). 59. Huisman. T. J. knol. De gewone Schurft van de AardappelTidschr. over Plantenziekten. 39: 173-188. 1933. -214- 60. Humphrey, J. E. The potato scab. Mass. Agr. Exp. Sta. Rpt. 7: 214-223. 1890. (Cited by Dippenaar, 1933). 61. Janehen, E* Der Kartoffelschorf. Oesterr. Zeitschr. fur Kartoffelbau 1: nos. 34. 62. Jensen, H. L. 1921. Contributions to our knowledge of the Actinomycetales. I & II. Proc. Linn. Soc. New South Wales 56: 79-98, 345-370. 63. Johnson, L. R. 1931. Trials of mercuric chloride for the prevention of potato sickness. Appl. Biol. 23: 155-165. 64. Jones, L. R. and A. W. Edson. ments of 1901. Ann. 1936. Potato scab experi­ Vermont Agr. Exp. Sta. Rpt. 14 (1900): 231-235. 1901. (Cited by Morse, 1912). 65. H. H. McKinney, aid H. Fellows. The Influence of soil temperature on potato scab. Bui. 53. 66. Katser, Annie. Wis. Agr. Exp. Sta. Res. 1922. Ein Beitrag zur Anwendung des Antagonismus als biologische Bek&npfIf* ungsmethode unter besonderer Berucksiehtigung der Gattungen Trichoderma und -215- Phytophthera. Bol. della R. Staz. di Pat. Veg. (N. S.) 18: 221-330. 67. Katser, Annie. 1939. Weiters Studien zur Awendung des Antagonisraus als praktische Bek&mpfungsmethode des Keimlingsstrebens der Tomaten. 68. KenKnight, G. Ibid. 18: 367-382. Boil treatments for the control of potato scab. Thesis. 69. 1939. Mich. State College M. S. 1937. and J. H. Muncie. The relation of compactness of soil samples to the actinomycetal plate-counts. 70. Kies sling, L. E. (Manuscript). Biologische Massnahmen zur Unterdruckung des Kartoffelschorfes. Kuhn-Arch. 38: 189-201. Abst. 9: 1003 entry 8940. 71. Krebber, 0. 588-608. 312. Kroger, F. (Biol 1935). TJtersuchungen uber die Wurzelknoll- chen der Erie. 72. 1933. 1932. Arch. f. Mikrobiol. 3: (Rev. Appl. Myc. 12: 1933). TJntersuchungen uber den Gurtelschorf der Zuckerruben. Arb. a. d. Biol. Anst. f. Land- und Forstwirtschaft 4: 254: 318. 1905. (Cited by Wollenweber in Sorauer, 1932). -216- 73. Leach, J. G., Phares Decker, and Hannah B§cker* Pathogenic race of Actinomyces scabies in relation to scab resistance. topath. 29: 204-209. 74. Lewis, J. M. Phy- 1939. Bacterial antagonism with special reference to the effect of Pseudomanas fluorescens on spore forming bacteria in soils. 75. Lieske, Rudolf. Jour. Bact. 17: 89-103. Morphologie und Biologie der Strahlenpilze. Leipzig. 76. Lode, A. Gebruder Borntraeger, 1921. Experiment ale TJnter suchungen tiber Bacteriensantagonismus. Gentbl. Abt. 1, Orig., Bd. 33: 196-208. 77. 1929. Lutman, B. F. and G. C. Cunningham. Bakt. 1903. Potato scab. Vermont Agr. Exp. Sta. Bui. 184. 1914. 78. ____________ Resistance of potato tubers to scab. Ibid. 215. 1919. — 79*_____________ Potato scab in new Land. 13: 214-244. 80. Phytopath. 1923. , R. J. Livingstcn , and Alice M. Schmidt. scab. 1936. Soil Actinomyces and jbotato Vermont Agr. Exp. Sta. Bui. 401. -217- 81. McCormick, R. B. Effect of certain soil micro­ organisms on the growth, development and pathogenicity of Actinomyces spp. caus­ ing potato scab; an attempt to deter­ mine the soil conditions and reactions governing the occurrence of scab. Ann. Rpt. Cornell Agr. Exp. Sta. (1934), 1935. 82. . The associative action of some species of Actinomyces. Ph. D. Thesis. 83. Cornell Univ. 1935. MacLeod, D. J. and J. L. Howatt. Soil treatments in the control of soil-borne diseases of potatoes. 60-61. 84. MacMillan, H. G. Amer. Potato Jour. 11: 1934. Potato seed treatments in western states. Phytopath. 12: 39. 1922 (Abst.). 85. MacMillan, H. G. and 0. A. Plunkett. Plant diseases observed in southern California in 1936. Plant Dis. Rptr. 21: 76-79. 86. Mader, E. 0. and F. M. Blodgett. and potato scab. 12: 137-142. 87. Mar., 1937. Potato spraying Amer. Potato Jour. 1935. ____________ , and Mary T. Mader. Effect of Bordeaux mixture on three varieties of potatoes with respect to yields, com­ position of tubers, and control of scab. Phytopath. 27; 1032-1045. Martin, W. H. 1937. Influence of soil moisture and acidity on the development of potato scab. Soil Sci. 16; 69-73. 1923. The value of organic mercury com­ pounds in the control of seed and soil borne scab. Proc. Potato Assoc. Amer. 14 (1927): 102-108. 1928. . The relation of soil conditions to the development of potato scab. 17 (1930): 62-73. Ibid. 1931. ____________ . The use of disinfectants in fertilizers for the control of potato scab and Rhizoctonia. 104. Phytopath. 21: 1931 (Abst.). . Fertilizer-mercury studies. New Jersey Agr. Exp. Sta. Rpt. 55 (1934): 58-59. 1935; 56 (1935): 65. 1936. ____________ . some recent developments in the control of potato diseases. Ann. Rpt. Veg. Growers* Assoc. Amer. 1937; 190205 . Martin, W. J. and L. H. Person. Pathog®. icity of -219- Actinomycete isolates on sweet potato. Phytopath. 29; 17. 95. Meyer, P. G. Landbouwk. 1939. Tidschr. 47: 643. by Wieringa and Wiebols. 96. Millard, w. A. (Cited 1936). Common scab of potatoes. Univ. Leeds and Yorkshire Council for Agr. Educ, Bui. 118. 1921. Also: in Ann. Appl. Biol. 9: 156-164. 97. 1922. _____ . Common scab of potatoes. Ann. Appl. Biology 10: 70-88. 98. ______ _____and Sidney Burr. Part II. 1923. A study of twenty- four strains of Actinomyces and their relation to types of common potato scab. Ibid. 13: 580-644. 99. __________ , and P. Beeley. 1926. Mangel scab - its cause and histogeny. 311. 100 Ibid. 14: 296- 1927. . ____________ , and G. B. Taylor. Antagonism of microorganisms as the controlling factor in inhibition of scab by green-manuring. Ibid. 14: 202-216. 101. Moore, G. C. 1927. The effect of certain methods of potato cultivation on growth and yield and accompanying soil conditions. Potato Jour. 14: 175-184. 1937. Amer. -220- 102. Niethammer, Anneliese. pilze. 1937. Die mikroskopichen Boden- Tabul• Biol. Berl. 6: 279-284. (Rev. Appl. Myc. 16: 557-558. 1937). 103. Palm, B. T . Notiser om sydsvenska actinomycoser. Bot. Notiser. 104. Parris, G. K. 1934: 449-456. Potatoes. Hawaiian Agr. Exp. Sta. Rpt. (1937): 39-40. 105. Peklo, J, 1934, 1938. Die pflanzlichen Aktinomykosen. Centralbl. f. Bakter. Abt. II, Bd. 27: 451-579. 106. Pierstorff, A. L. 1933, 1910. Potato scab demonstrations in proc. Ohio Veg. Growers1 Assoc. 19; 154-156. 107. Poole, R. p. 1934. Recent Investigations on the control of three important field diseases of sweet potatoes. Sta. Bui. 365. 108. New Jersey Agr. Exp. 1922. ___________ . Some results obtained in the in­ vestigation of pox disease control of sweet potatoes. New Jersey Agr. Exp, Sta. Rpt. 44 (1923): 109. 394-398. 1924. . The relation of soil moisture to the pox or ground rot disease of sweet potatoes. 110. Pratt, 0. A. Phytopath. 15: 287-293. 1925. Experiments with clean seed potatoes -221- on new land In southern Idaho. Agr. Res. 6: 573-575. 111. 1916. Porter, C. 1. and J* C. Carter. among fungi. Jour. Competition Bot. Rev. 4: 165-182. 1938. 112. Riha, J. Obranne prostredky proti obecune stru- povistosti Branboru. 113* Rode,A. 6: 73-80. 1926. 246-247. 1927). Ochrana Rostlin (Rev. Appl. Myc. 6: Zur Frage des Kartoffelschorfes. Dtsch. landw. Pr. 63 (1): 4. Myc. 15: 394. 1936. (Rev. Appl. 1936). *4 114. Roberg, M. Tiber den Erreger der Wurzelknollchen europaischer Erlen. Bot. 86; 344-349. 115. Jahrb. f. wiss. 1938. Rohde, G-. Kali im Stoffwechsel der pflanzen unter besonderer Berucksichtigung der Kaliman% gelerscheinungen an Kartoffeln* Ernahr. Pfl. 51 (13-14): 237-243, 1935. (Rev. Appl. Myc. 15: 46. 1936). 116. Sanford, G.B • The relation of soil moisture to the development of common scab of potatoes, phytopath. 13: 231-236. 117. 1923. ____________ • Some factors influencing the devel­ opment of potato scab. 58. 1924. (Abst.) Phytopath. 14: -222- 118, Sanford, G. B. Some factors affecting the patho­ genicity of Actinomyces scabies. Phy­ topath. 119 » . 16: 525-547. 1926. > The relation of some soil factors to the development of scab of potatoes. Proc. Potato Assoc. 113-120. 120 . ______ ___• 1926. Some soil microbiological aspects of plant pathology. 638-641. 121 . Savulescu, Amer. 12 (1925): Sci. Agric. 13: 1933. T., C* Sandu-Vill©', T. Rayss, et V. Alexandri. L ’etat phytosanitaire en / Roumanie au cours de l fannee 1932-1933. Inst. Cere. Agron. 193#-. Romaniei 12: 1-93. (Rev. Appl. Myc. 14; 214-215. 1935). 122 . ____________ , ____________ ,A.Aronescu, Alexandri. et V. Letat phytosanitaire en / Ronmanie au cours de l*annee 1934-1935. Ibid. 25: 1-97. Myc. 16: 19. 123. Schacht, H. (Rev. Appl. 1937). Kartoffelpflanze und deren Krankheiten. p. 24. 124. 1936. Stewart, J. G. Yorks, 1854. (Cited by Dippenaar, 1933). Report 70. Univ. of Leeds and council for Agric. Cited by Millard, 1921). Educ. 22. -223- 125. Serbinov, I. L. Ein neuer epidemischer Ausbruch von Actinomyces an stisspfeffer (Capsicum). (russisch). La Defense des Plantes, Leningrad 2: 537-546. 1926. (Cited by Wollenweber in Sorauer, 1932). 126. van der Slikke, C. M. -fa- Verslag van Rijks^finbouw- proefvelden over grondontsmetting tegen de Rhizoctoniaaiekten en de schurft op Aardappelen. Tidjschr. enziekten 41: 65-73. 127. Smith, Ora. over Plant- 1935. Effect on soil reaction on the growth of the potato. 118-121. Amer. Potato Jour. 10: 1933. 128._____________ . Effect of soil reaction on growth, yield, and market quality of potatoes. Cornell Agr. Exp. Sta. Bui. 664. 129. Sorauer, P. 1937. Handbuch der Pflanzenkrankheiten. Berlin (1886): 227. (Cited by Dippenaar, 1933). 130. Spencer, E. R. Decay of Brasil nuts. 72: 265-292. Bot. Gas. 1921. ✓ 131. Stehlik, V. Einfluss des Bodens auf die Anfangs- entwicklung der Rube mit besonderer Rucksicht auf die Rubenkrankheiten. Zeitschr. fur Zuckerind. 58: 437-455. 1934. (Rev. Appl. Myc. 14: 73. 1935)). -224- 132. Stevenson, P. J., and C. P. Clark. varieties. 92. 133. New potato Amer. Potato Jour. 11: 85- 1934. «\ Stormer, Inge. Versuche zur BekAmpfung von Schorf und Rhizoctonia bei der Kartoffel durch Quicksilberhaltige Dunge - und Beizmittle, Nachr. SchadlBekampf., Leverkusen 13 (2): 45-54. 134. Taft, L. R. 1938. (Potato scab). Bui. 57: 23-25. 135. Taubenhaus, J. J. 1890. Pox, or pit (soil rot) of the sweet potato. 437-450. 136. Mich. Agr. Exp. Sta. Jour. Agr, Res. 13; 1918. ____________ • Studies in potato-scab control. Phytopath. 24: 836. 137. Taylor, C. P. Potato scab and Rhizoctonia and their control. 66-67. 138. 1934 (Abst.). Amer. Potato Jour. 10: 1933. ____________ . Field experiments on potato scab control in western New York. potato Jour. 11: 40-45. 139. , and P. M. Blodgett. Amer. 1934. Further field experiments on potato scab control in western New York. 1936. Ibid. 13: 145-150. -225- 140. Tims, E. C. An actinomycete antagonistic to a Pythium root parasite of sugar cane. Phytopath. 22: 27. 141. Tucker, C. M. 1932. (Abat.). Investigations of di seases of white potatoes. Florida Agr. Exp. Sta. Rpt. 45 (1931): 118-119. 142. Voelkel, H. und Klemm. 1932. Die hauptsachlichsten starken Sch&den an Hackfruchten im Jahre 1932. Nachrichtenbl. Deutsch. Pflan- zenschutzdienst 12 (12): 101-103. (Rev. Appl. Myc. 12: 266. 143. Waksman, S. A. soil? 144. Waksman, S. A. 1933). Is there any fungus flora of the Soil Sci. 3: 565-589. 1917. Cultural studies of species of Actinomyces. 145. 1932. Ibid. 8: 71-207. 1919. ____________ • Principles of soil microbiology. 2d ed. Williams & Wilkins, Baltimore. 1932. 146. ____________ . Associative and antagonistic effects of microorganisms. I. Histori­ cal review of antagonistic relation­ ships. 147. Soil Sci. 43: 51-68. ____________ , and J. W. Foster. 1937. Associative and antagonistic effects of microorganisms. 2. Antagonistic effects of microorganisms -226- grown on artificial substrates. 43: 69-76. 148. Ibid. 1937. ______ and I. J. Hutchings. Associative and antagonistic effects of microor­ ganisms. 3. Associative and antagonis­ tic relationships in the decomposition of plant residues. Ibid. 43*:77-92. 1937. 149. Wallace, G. I. and F. W. Tanner. on mold spores. Proc. Soc. Exp. Biol. Med. 28: 970-972. 150, Wedgworth, H. H. Effect of heat 1931. Investigations on the relation of sulphur to the Actinomyces scab of potato. Mich. State College M. S. Thesis. 1924. 151. Weindling, R. Trichoderma lignorum as a parasite of other soil fungi. 837-845. 152. Phytopath. 22* 1932. ____________ , Studies on a lethal principal effective in the parasitic action of Trichoderma lignorum on Rhizoctonia solani and other soil fungi. 24: 1153-1179. 153. Phytopath. 1934. __________ and H. S. Fawcett. Experiments in the biological control of Rhizoctonia -227- damping-off of Citrus seedlings. gardia 10: 1-16. 154. _______ Hil- 1936. t and 0. H. Emerson. The isolation of a toxio substance from the culture filtrate of Trichoderma. Phytopath. 26: 1068-1070. 155. _____________ • 1936. Isolation of a toxic substance from the culture filtrate of Trichoderma and Gliocladium. Ibid. 27: 1175-1177. 1937. 156. __________ Associative effective of fungi. Bot. Rev. 4: 475-496. 157. Wessels, P. H. 1938. Soil acidity studies with potatoes, cauliflower, and other vegetables on Long Island. 536. 158. Cornell Agr. Exp. Sta. Bui. 1932. Wheeler, E. J. and H. C. Moore. treatment tests, Bui. 246. 159. Potato seed Mich. Agr. Exp. Sta. 1933. Wheeler, H. J. and G. E. Adams. On the use of flowers of sulfur and sulfate of ammonia as preventives of potato scab in con­ taminated soils. Rhode Island Agr. Exp. Sta. Rpt. 10 (1897): 254-268. 1898. -228- 160* White, R. p. Potato experiments for the control of Rhizoctonia, scab, and blackleg, 1922 to 1927* Tech. Bui. 24. 161. Kansas Agr. Exp. Sta. 1928. Wieringa, K. T. ety G. L. W. Wiebols. De Aardappel- schurft en de Heterolyse der Schurftparasiet. Tijdschrift over Planten- ziekten 42: 235-240. 162. 1S36. Wingerberg, p. Studien uber den gewohnlichen »♦ Kartoffelshorf und seiner Erreger. Archiv 33: 259-295. 163. Wollenweber, H. W. Kuhn- 1932. Der Kartoffelschorf. Arbeiten des Forschungsinstitutes fur Kartoffelbau. 164. Heft 2: 1-102. 1920. ____________ . Die Gattung Actinomyces Harz. In Sorauer: Handbuch der PTlanzenkrankheiten. Funfte Auf. Bd. 3, Tl. 2: 830- 165. Zach. 843. 1932. ?> Tiber den in den Wurzelknollchen von Eleagnus augusjkifLoLia und Alus giutOQjQsj, lebenden Padenpilz. Sitzungsber. d. 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