ll 1 M ‘ \ W A # —— _— 1| _cn4>co COLE TOLERANCE STUNES WITH HY3R!D SEED CORN ‘Tt'hesis for the Degme of Ph. D. MICHIGAN STATE COLLEGE Gsrard Neptune 3953 This is to certify that the thesis entitled JI‘FT‘ :' l“”‘l* 15“," ‘33?“ e (1 T "' presented bg '7’ r“: " ' Y fiur : has been accepted towards fulfillment of the requirements for 'l», F u ,7. ,v l“ , ‘ ~. - degree 1n__’i_‘_._~ 1—; 'A 5 (if. m Major professor “, «my y. 1'; an": I A ‘ ‘ I " / , Date ’ l 7 4 t f l ‘ . , . -' , m1 COLD TOLERANCE STUDIES WITH HYBRID SEED CORN By Gerard Eggtyne A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science ' in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Farm Crops 1953 ACKNOWLEDGMENTS The writer wishes to express his sincere thanks to Dr. E. C. Rossman for his guidance and appreciable help throughout the experiments and preparation of this thesis. He also deeply appreciates the Alumni fellowship and the scholarship provided by Michigan State College which.made it possible for him to complete this investi- gation. . WWé’Ar *fi’rfl‘ Wfl' fi-W Vii? TIBLE 01“ CONTENT 3 INTRODUCTION................ oooooo oo-ooooooooooooooooooooo oooooo one 1 REVIEVIOB‘ LITMiTUREgooooo00.000000000000000...one. oooooo coco-00000 3 PART I - INFLUENCE OF TEVLPERATURE, STAGE OF GEMINATION 1ND LENGTH OF EXPOSURE ON COLD TEST (ELIMINATION FATWLS A‘IQD iiiEI‘IiODSOOOOe0.0.0.000...OOOOOOOOOOOOOO00.... 1]- EXPERIIWTAL RESULTS AND DISCUSSION...”.................. 13 PART II - INFLUENCE OF PREVIOUS CROPS AND SOIL TYPE ON COLD TEST GYRMINATION MATMALS AND MILTHODS................... ....... 22 EXPERDEENTEL RESULTS AND DISCUSSION....................... 21; PART III - EARLY GENERATION TESTING IN SELHITING COLD TOLERANT INDEED LINES MATEELIALS AND MEI‘HODSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO.0... 32 EXPEDIBNTIL RESULTS AND DISCUSSION....................... 33 wivflWYOOOO.00.00.000.000.000.000.000.000... oooooo o-ooooooooooooooo 38 LITmATUR-E CITEDQIOOOQOOO00.000000.000000.0000.0.0.0000000000000000 m lllllfl'll'llllr‘.ll[llli INTRODUCTION A good stand of corn is essential for successful production since grain yields are markedly affected by the number of plants per acre. Much emphasis is currently being placed on the production of maximum yields by adjusting planting rates to soil fertility. Cold wet weather frequently follows corn planting, particularly in northern areas, kernels and seedlings become susceptible to attack by various soil pathOgens, and stands are reduced. Injury varies from pre- emergence killing to blighting and root-rotting of the seedlings. Corn hybrids and the seed for these hybrids adapted to northern areas should produce satisfactory stands under adverse conditions, and retain high yielding potentialities. The ability of hybrid seed corn to produce good stands under cold wet conditions has been shown to be affected by genetic constitution of the seed, naturity of the seed when harvested, frost injury to the seed before harvest, care during processing, amount and type of seed coat injury, seed treatment, and age of the seed when planted. Corn seed.may be subjected to a cold test germination prior to planting in order to ascertain its probable germination and stand when subjected to field conditions. Such tests are run in flats in the green- house. Soil from a corn field is placed in flats, the seed planted and the soil thoroughly soaked before placing in a cold chamber at hS-SOO F for 8 to 12 days. {Ill-I‘ll Since little or no information is available on several phases of cold test germination, this study was designed to study: (a) the effects of temperature and length of exposure on seed in various stages of germination; (b) the effect of previous crap and soil type on cold test germination , and (c) the effectiveness of early generation testing in selecting cold tolerant inbred lines of corn. REVIEW OF LITERATURE Several Species of microorganisms are responsible for the diseases that affect seed corn at low soil temperatures. Dickson (3) and Valleau (26) showed that Gibberella saubinetii_may cause seedling blight. Johann, Holbert and Dickson (12) reported that gythium arrhenomanes caused rot of the embryo prior to emergence, seedling blight after emergence, and.root rot that reduced size, vigor, and yield of the matur- ing corn.plant. Hoppe (9) pointed out that Pythium injury varies from severe stunt- ing of the seedlings to complete pre-emergence killing. Of 138 Pythium isolates made from decayed corn kernels, Happe and Middleton (11) found that 58, falling into six species, were capable of causing pro-emergence killing of corn at low temperatures. Ho (7) studied the soil pathogens which attack the roots of maize and found that Pythium debaryanum and Eythium graminicola, tagether with Gibberella saubinetii, were the most destructive, when the soil tempera- ture was below 169 C and moisture was abundant. Diplodia.gggg has been reported as a seedling blight pathogen under adverse conditions (21). Ho (7) classified the organism as only moderate- ly destructive and according to Raleigh (21), Diplodia injury is more serious at high (200-2hOC) than at low (150-1900) temperatures. Various species of Fusarium, notably a. moniliforme and E. Succisee, have been isolated from rotting corn roots and decayed kernels. They caused little injury and appeared to be only secondary organisms (7). _HH o 1 \ .— O — .. ~m—-——o—-—v .A- ---._———.—. ... i.., Five Species of HelminthoSporium i.e., g, mgygigj fl, bicolor, g, carbonum, g, sativum, and g. turcicum, have been reported as seedling blight pathOgens (2, 22). In general, these pathogens caused blight at ZOO-28°C, though one of them, 5. carbonum race II was pathogenic at temperatures as low as 100 C. Several other organisms i.e., Penicillium oxalicum, Trichoderma li orum, Rhizoctonia solani, Rhizopus £22,, and Aspergillus piggg, have been.mentioned in relation to cold germination injury (7), but their pathogenicity has not been demonstrated. According to Dickson (3), soil temperature is the most important factor which determines the extent of seedling blight. Dickson pointed out that while temperatures of 12° - 180 C favor the blighting of wheat, maize seedlings are more subject to attack between 80 and 20° C. He attri— buted the difference in reaction between wheat and corn to the changes produced in their metabolism by the differences in temperatures. The influence of temperature upon seedling blight development appeared to be primarily a host reaponse. Dickson and Holbert (h) concluded that the differences in resistance among inbred lines of corn were due to different influences of temperature upon the metabolism of the young seedlings. Additional evidence that some relationship might exist between the metabolism of the corn seedling and the development of blight at low temperatures is found in the work of Smith (2h). He planted two con- trasting inbreds: RID; and Gng at high and at low temperatures. At high temperature (2h? C) both inbred were normally green. But at low temperatures (169 - 190 C) the seedlings of RYD4 were of a normal green -—-—_._ -....¢- color, while those of 0026 were almost devoid of chlorophyll. At both temperatures endOSpenm utilization was more rapid in inbred 0025 than in RYD4. Seedlings of 6025 were very susceptible to Gibberella.blight whereas those of RID4 were highly resistant. Johann, Holbert and Dickson (12) found that soil temperatures, 160 C or lower, together with high soil moistures were so favorable for disease infection that either germination of corn kernels was prevented or seedling blight developed. Data presented by Ho (7), Hoppe (9), and Livingston (16) support the findings of Johann.gt‘21. Flor (5) reported that Pythium injury to germination of corn de- creased with rise in temperature. At 35° C, Pythium did not injure corn. At 30° C, there was an appreciable amount of injury and as the toruperature was lowered to 110 C, injury became more severe. He also found that injury increased with the moisture content of the soil, but was less severe in warm wet soils than in cold wet soils. Haskell (6) planted five inbred lines of sweet corn in a soil known to contain pathogenic organisms and held the flats for various intervals up to 32 days at hOO F (hp C) and 500 F (10° C). Germination was re- duced as the duration of exposure to cold increased. The treatments at hc° F were not as injurious as the corresponding treatments at 50° F. Eight days of exposure to 50° F were necessary to reduce germination by 50 percent. Haskell concluded that cold injury to the seed was in part reaponsible for the reduction in germination at h0° F, since the activity of the pathogens was partially inhibited. He indicated that the ho° 1“ temperature gave a truer picture of cold hardiness of the inbreds whereas the 50° F temperature was more critical and constituted a better test for disease resistance. HOppe (10) also found that disease level is higher at 11° C than at to C. His results indicated that low temperature in itself was not injurious but served merely to prediSpose unprotected, slowly germinat- ing kernels to attack by soil fungi. Pericarp injury has been found to affect germination and yield of corn. In general, germination decreases as the amount of injury in- creases. Under field conditions, Meyers (18) obtained a significant reduction in both stand and vigor of seedlings when broken seeds were' planted. The difference in vigor became less evident as the season pro- grassed and finally disappeared. There was no reduction in yield, except in the plots planted with broken seed inoculated with spores of Penicillium epp. In these plots stand was reduced from a.possib1e 837 to 396 plants. ‘Alberts (1) pointed out that when the pericarp was injured, various soil pathogens readily penetrated the kernels and caused a rapid de- composition of the endosperm. Seedlings developing from such kernels grew slowly and their mesocotyl was subject to attack by soil pathogens. Koehler (13) studied the pathologic significance of seed coat injury in dent corn. He found that a slight puncture in the seed coat at the crown caused a 12 to 16 percent loss in yield of grain, while removal of the seed coat from the whole crown resulted in a loss of 18 to 23 percent. A cut through the seed coat on the side of the kernel caused no injury to stand or yield. Injury from Giberella and other soil pathogens was most pronounced in plots planted with seed injured at the crown. Tatum and Zuber (25) presented data indicating that a close re- lationship exists between inconspicuous pericarp injury over the germ and stand which in turn affects yield of corn in the field. Mechanical processing of the seed has been found to cause injury to the pericarp. Koehler and Dungan (1h) compared bin-dried and hanger- dried seed corn. There was more seed coat injury in the seed that had been bin-dried and machine processed than in the grain that had been hanger-dried and shelled by hand. In field tests, hanger-dried hybrid seed averaged 3.2 bu. better in yield over a three year period. This difference was statistically significant. Similar results on.mechanical injury were reported by Wbrtmann and Rinks (28) who found also that hybrids differ in their susceptibility to mechanical injury; Tatum and Zuber (25) reported that seed samples which were hand shelled and not subjected to any mechanical treatment were free from injury and genuinated approximately the same in a cold test as in a normal test. The amount of pericarp injury in commercial seed depended upon processing:methods and the care during processing. Pericarp injurwaas reflected in lower cold test germinations. Neptune and Rossman.(l9) found that cold test germination of the same hybrid varied depending on the seed producer. Age of the seed was found to be a factor in cold test genmination (19). New seed consistently gave higher cold test stands than old seed though all seed lots were capable of satisfactory germination in the standard warm tests. Seed treatment has been reported as capable of reducing cold germi- . nation injurY. Rush and Neal (23) obtained better stands with Arasan treated seed than with non-treated seed, but seed treatment did not give canplete protection in cold tests. Normal emergence was obtained by Livingston (16) under cold test conditions when the seed was treated with Arasan, irreSpective of the drying method or moisture content of the seed at harvest time. Wbrtman and.Rinke (28) found reductions in.stand from samples treated with.Arasan in a slurry treater when compared with seed treated by hand with dust fungicide. Tatum and Zuber (25) presented data indicating that dust treatment was not an adequate means of preventing poor stands in the field. The use of seed with an intact pericarp was more effective. .Maturity of the seed and frost injury prior to harvest have been reported as factors affecting cold germination of seed corn. Data.pre- sented by Johann, Holbert and Dickson (12) showed that different degrees of maturity of the seed influenced the reaction to Giberelle seedling blight and the stand and yield of the crOp. Resistance to Giberella in- creased with the naturity of the seed. ‘ Koehler, Dungan and Burlison (15) harvested seed corn at different stages of maturity. They found that field stand varied directly tdth the vigor of the seed and the acre yield varied directly with the stand; the more mature stages produced the best stands and yields. Rush and Neal (23) obtained large differences in gemination be- tween seed harvested at 10-day intervals when planted in soil at low temperatures (10° C) . In general, stands improved with maturity of the seed. Seed harvested after a light frost gave significantly lower cold germination than unfrosted seed while both types were equal in warm tests. Genetic differences in cold test performance have been reported. Dickson and Holbert (h) pointed out that resistance of inbred lines to Giberella seedling blight was an inhertiable characteristic. The expression of resistance was constant over a given temperature range. First generation hybrids between resistant lines and susceptible lines at temperatures of 120 to 320 C were susceptible at all temperatures, indicating that susceptibility was dominant. Hoppe (8) observed a wide range of relative resistance to Giberella among long-time inbred lines at 160 C. The behavior of these lines was very consistent, suggesting a high degree of homozygosity for the genetic factors involved in resistance. He found that first generation hybrids between resistant and susceptible lines were as resistant as the resistant parent while crosses between susceptible lines were as susceptible as either parent. Analysis of F3 generations gave evidence of transgressive segregation since families were isolated which were more resistant or more susceptible than the parent strains. One F, family, practically immune, maintained its resistance in the F, while results in other families indicated that the F3 families were very heterozygous. lO McIndoe (17) studied the inheritance of reaction to Giberella in 27 selfed lines, all the possible crosses between these lines, and some F, generations of the F1 hybrids. Crosses of susceptible and resistant lines gave conflicting results in the F1 generation. Resistance was neither dominant nor recessive to susceptibility. The results were best explained when inheritance of disease reaction was assumed to be quanti- tative in nature. Further evidence of a quantitative inheritance was obtained frmm the behavior of the F3 progeny. A definite segregation of the F, lines occurred in conformity with a Mendelian explanation of such inheritance. Where the parental lines differed widely in reaction, highly resistant and susceptible lines appeared in the F3 generation, in sufficient preportion to suggest that, perhaps relatively few factors were involved for resistance. Pinnel (20) found that, at low temperatures, double crosses germi- nated best, followed by single crosses and inbreds in that order. The higher stands for double crosses and single crosses over inbreds were best explained on the basis of complementary gene action. Among the in- breds, he obtained wide differences which appeared to be heritable in crosses. Differences among hybrids were largely determined by the maternal parent, in both single and double crosses. There was no relation between the performance of an inbred as a female in crosses and its performance as a male. A very high ear to ear variation was obtained in some long- time inbred lines. PART I Influence of Temperature, Stage of Germination, and Length of Exposure on Cold Test Germination 11 MATERIALS AND METHODS Five double-cross hybrids were used: Michigan LBO, Michigan 350, Michigan 250, Nosco N, and Gries ZGlL. In previous cold tests conducted during 1950 and 1951 at h5° F, these hybrids germinated on the average: 92.3, 92.0, 89.6, 58.3 and h6.0 percent reSpectively. On the basis of these results, the three Michigan hybrids were rated as tolerant and the other two as susceptible to injury during germination under adverse conditions. All seed, as it was obtained from commercial companies, germinated 95 percent or better in standard germination tests conducted under the ideal conditions of the laboratory. A11 seeds were treated with a seed protectant. The seed was planted in flats filled with a Hillsdale sandy loam obtained from a.corn field. The soil was watered to saturation and the flats subjected to various treatments. There were five different pre-cold treatments and four different exposures to cold. Pre-cold treatments consisted of keeping the flats in a 750 - 800 F greenhouse for 0, 1, 2, 3, or 5 days. At the end of the five-day treatment, the seedlings had emerged and reached a maximum height of 1/2-in. After>pre-cold treatment, the flats were moved to a walk-in cold? chamber for h, 8, 12, or 16 days. They were then placed in a warm green- house for 12 days and the strong seedlings were counted. 12 The design of the experiment was a double Split-plot in which the five pre-cold treatments constituted the main plots; the four lengths of exposure to cold were the sub-plots, and the five hybrids constituted the sub-subaplots. Two replications of 25 seeds each were used. The entire procedure was duplicated at 32° and U00 F. The temperature in the 320 chamber varied from 30° to 3h? F, and twice went up to 380 F for about four hours. This was due to the accumulation of ice on the pipes of the cooling system. The temperature variation in the ho° chamber was only plus or minus one degree. 13 EXPERIMENTAL RESULTS AND DISCUSSlON Tables I and II present the average cold test germination of five corn hybrids following various periods of exposure to 320 and uo° F at different stages of germination. Analyses of variance for the data are given in Tables Ill and IV. Since the five day pre-cold treatment coupled with the 320 cold treatment resulted in an almost complete kill- ing it was not included in the analysis of variance. At both temperatures, there were differences among the pre-cold treatments in their effect upon germination. Exposing the seed to cold immediately or one day after planting resulted in nearly the same de- creases in stand. Germination was less as the time interval preceding exposure to cold increased beyond one day. Apparently in the more ad- vanced stages of germination the seed became more susceptible to attack by the soil pathogens responsible for cold germination injury; Three and five days of favorable conditions followed by 8, 12, or 16 days of cold treatment seriously reduced the stand of all hybrids. Figures 1 and 2 illustrate the effect of the cold treatments at 320 and uo° on germination. In general, increase in duration of exposure to cold resulted in lower survival. The interactions, temperature x pre- cold treatment, temperature x cold treatment, and temperature x pre-cold treatment x cold treatment, were significant (Table IV) indicating that the two temperatures failed to affect germination in the same manner in the various treatments. Pre-cold treatments of O and one day lb TABLE I UERMIMTION resonances FUR mam seam com WITH EITHER o, 1, 2, 3, 014. 5 DAYS or FEE-COLD Tammm FOLLOWED BY EITHER h, 8, 12, 0a 16 DAYS OF GOLD TRAATMENT AT 32° F Days of Days of Pre-cold Cold Nosco Gries Mich. Mich. Mich. Treatment Treatment N4, 201A 250 350 L80 Average h 66 86 9h 98 92 86.h O 8 112 68 88 96 96 78 .0 12 M4 70 86 82 98 76.0 16 36 38 76 78 98 66.0 Average *fifi6.8 65.6 86:0w 87.2 9fifl8 75:0 8 82 78 96 92 100 89.6 1 b‘ 62 Sb 9h 96 ‘ 96 80.1; 12 b2 20 68 78 86 58 .8 16 b2 32 72 70 8h 60.0 Average 56.8 H6f0 82.h 85.0 91.2 72.0 h 78 68 90 68 90 82.0 2 8 58 58 7o 92 88 72 .L. 12 b0 2h 20 88 52 36.8 16 10 6 10 _26 g 29 18.2; Average h6th 38.8 h7.2 62.D 6lifir 231.2 )4 6b MA 814 88 914 714.8 3 8 b h h 12 12 7 .2 l2 0 O 2 8 6 3.2 16 2 2 6 10 6 5.2 Average 17.6 12.3 2h.0 29.6' 29.6 22.3 )4 2b 20 to hh 52 36.0 S 8 O O 0 0 O 0 l2 0 0 O O 0 O 16 o o o o o o A Average Grand Average h2.0 h0.8 60.0 65.6 69.2 55.6 __ (TERMINATION PERCENTAGES FOR HYBRID SEED CORN WITH EITHER O, 1, 2, 3, OR 5 DAYS OF FEE-COLD TREATMENT FOLLWED TABLE II BY EITHER h, 8, 12, OR 16 ans or COLD TREATMENT AT 80° F. 15 Days of Days of Hybrid Pre-cold Cold Nosco Gries Mich . Mich . Mich . Treatment Treatment 81.. 201A 250 3 50 h80 Average 14 50 11b 88 86 90 71 . 6 O 8 20 2b 80 86 92 60.11 12 82 32 72 80 9h 68.0 16 18 10 72 72 90 52 .11 Average 32 .F 27 .2 78.5 80 .8 91 .2 62 .0 h 66 86 96 88 9h 86 .0 1 a 38 m. 81. 86 as 68 .0 12 32 lb 76 Bh 98 60.8 16 8 16 36 72 80 A2 .1; Average 360 ho .0 72 .8 83 .2 88 .8 61:1; 14 30 50 914 9b 98 73 .2 2 8 114 2b 86 86 9h 60 .8 l2 16 10 60 78 86 50 .0 16 20 6 38 52 66 25341: Average 20 .O 27.1; 69 .6 77 .It 86 .0 .2 h h8 60 96 86 92 76.1; 3 8 18 22 82 80 80 59.2 12 16 18 36 38 6h 3h.h 16 6 6' A36 38 36 2h.h Average 20 .8 26.5 62 .74— 60 .11 72 11. 118 J: b 58 58 92 8b 100 78.h S 8 38 30 60 514 88 514.0 12 2 2 26 30 32 18 .14 16 2 0 22 28 38 18 .0 Average 21; .8 28 .D ‘50:.0 “18 .8 6E1 I12 .0 Grand . Average 26.8 28.0 66.h 70.8 80.8 Sh.b I -.—-o o 6 .—.—~- "- o u I _._._.. I l V-.. I I i... __.-.-—'—'- - - -—- _. a. . - - . - . - ‘ ‘ ‘ - .— - - a-o—n __.~*' _..———~ — ow— .- .—~r - ’- _.-———— -. —- a _ .Mc .—-"_ -- "- ——.~.- v... “ #a .—- - ..-. u .— ‘—— -..'. “.- TABLE III 16 ANALYSES 01' VARIANCE FOR COLD TEST GERMINATION AT 32° F. AND 10° F. 32° F. Ao° F. _ agrees 0 Mean Degrees of Mean Source Freedom Square Freedom Square Total 159 199 Replications l 57.6 1 5.5 Pro-cold 3 1 ,505 .1.” I. 212 .6‘H Error (8) 3 149 .2 b 10.14 Cold 3 1,085.6M 3 1,061.8” Pre-cold x 0618 9 162.1” 12 56.2M Error (b) 12 8.2 15 1h.0 Hybrid b 360.0” A 1,598.2M Pre-cold x Hyb. 12 35.2” 16 19 .1" Cold 1 Hyb. 12 8.2 12 217.0” P-c x C x Hyb. 36 10.1* 148 11.1 Error (c) 68 h.1 80 8.0 H i Significant at the 5 percent level of probability. Significant at the 1 percent level of probability. ‘m‘; w: H . ‘H‘ .I an,“ __.._'~LLA~ ‘ '..'-;,_,_‘_____§__‘ ——-—————._" " " I- #M' --- ' _._.'—l_£__‘-' JLJ; ‘- " TABLE IV COMBINED ANALYSIS OF VARIANCE FOR COLD TEST GERMINATION AT 32° AND 80° F. Source Degrees of Freedom Mean Square Total 319 Tempeature 1 18.5 Error (a) 2 31.1** Pre-cold 3 13112.11,“E Temp. x Pre-cold 3 390.1 Error (b) 6 31.5 Cold 3 1,676.11? Temp. 1 Cold 3 118.0" Pre-cold x Cold 9 129.7“E T x.P-C x.C 9 67.7 Error (c) 28 12.2‘M Hybrids 1. 1,6145 .3, Temp. x Hyb. b 198.8 Pre-cold x Hyb. 12 20.0 Cold x Hyb. 12 214.2 T x P-C x Hyb. 12 26.3 T x C x Hyb. 12 9.5 P-C xCxHyb. 36 15.5 'r x P-C x c x Hyb. 36 6.5 Error 128 60.1 *'Significant at the 5 percent level of probability. ** Significant at the 1 percent level of probability. .( .n D :. :53. 10.: uxh ch 4.51:; an :N-nle P. .53. < .J: .J _: attic Iv... m5 5.— v.7:33m L Chute—1hr. .-.z followed by cold treatments at 320 F were less injurious than similar pre-cold treatments followed by 80° F cold treatments. It is likely that the soil pathogens were more active at 80° F than at 320 F. Pre- cold treatments of 2, 3, and 5 days followed by cold treatments at 320 F were generally more injurious to subsequent germination than equivalent treatments at 80° F. It appears that in the more advanced stages of germination the seed and seedling were more injured by direct effects of cold at 320 F than by soil path0gens. The hybrids differed in their tolerance to cold wet soil conditions. Nosco h‘ and Gries 201A which had been previously rated as susceptible to cold germination injury were found to be relatively more susceptible in all treatments than the three Michigan hybrids. Among the hybrids, Michigan 880 was the most tolerant, followed by Michigan 350 and then by Michigan 250. Cold periods of four and eight days are more likely to occur in the field than 12- and 16-day periods. Germination of the tolerant hybrids was not seriously injured by four days of cold at 32° F and h0° F after 0, 1, 2, or 3 days of pre-cold treatment. Eight days at either 32° F or 80° F did not seriously reduce germination of the tolerant hybrids until the seeds had been under favorable conditions for three days prior to cold treatment. Germination of the susceptible hybrids was reduced by all treatments at all stages of germination. The relative ranking of the hybrids was generally consistent with all treatments. In most seed testing and corn breeding programs, cold tests are conducted with eight 21 to twelve days of cold treatment immediately after planting or one or two days after planting. On the basis of these results, this appears to be a satisfactory treatment for cold test evaluation. PART II Influence of Previous Cr0ps and Soil Type Upon Cold Test Germination 22 MATERIALS AND METHODS Michigan A80, Michigan 350, Michigan 250, Gries 201A, and Nosco N‘ were used. All seed was capable of 95 percent or better germination in standard tests. All seed was treated with a seed fungicide. Two types of soil: A Brookston silt-loam and a Hillsdale sandy loam, were used. Samples of the Brookston soil were taken in May from seven different plots which had been planted to wheat, corn, barley, beans, alfalfa, sweet clover, and sugar beets, the previous year. The Hillsdale soil samples were taken in early July from three plots on which corn, wheat, and sweet clover respectively had been grown the previous year. Seed of each hybrid was planted in flats containing the various soils. The soil was watered to saturation and the flats transferred to a walk-in cold chamber for 12 days and maintained at 80° F. Germination was completed in a warm greenhouse and strong seedlings were counted after 21 days. Experiment I - Three replications of 25 seeds each were planted in the Brookston soil samples. The experimental design was a Split-plot in which the previous crops grown on the soil constituted the main plots and the five hybrids made the subsplots. ggperiment II - In early July, the Brookston samples used for the previous experiment were mixed with the remainder of the original soil dug in May. Both samples had been kept in the greenhouse and were completely dry. 23 Half of the dry Brookston soil and half of the freshly dug Hillsdale soil was sterilized in the following manner. Two flats were filled with soil from each sample and placed cross-wise in a steam sterilizer for seven hours and thirty minutes. After sterilization, the flats were removed to a greenhouse for 2h hours. Two replications of 25 seeds each were then planted in both the sterilized and the nonsterilized soils. For each soil type, the design was a double Split-plot where the two treatments - sterilization and no sterilization - constituted the mainrplots; the previous creps made up the sub-plots, and the five hybrids constituted the sub-sub- plots. 2h EXPERIMENTAL RESULTS AND DISCUSSION Influence of Previous Crops Table V contains the cold test germinations for five double-cross hybrids in samples of a Brookston silt-loam on which different craps had been grown previously. Analysis of variance is given in Table VI. There were significant differences in the effects derived from the previous crops upon germination. The average stand on the soil which had previously grown sweet clover was 36.h percent as compared to 66.h percent on the soil which had grown wheat. The results for the other craps were intermediate between these two extremes. The legumes tended to be more detrimental than the cereals, corn excepted. The interaction, previous crap x hybrid, was not significant, indicating that the previous cr0ps affected the germination of the five hybrids in the same manner. 2 Reduction in genmination of seed corn.under cold wet soil conditions has been shown to be caused primarily by soil pathogens. Among these pathogens, Pythium spp. appear to be the most common and the most destructive (ll). Rythium can also cause a root rot of the corn.plant in the field (12). The same group of organisms has been found to be pathogenic to several other crops ander greenhouse conditions (22). Reaction to Pythium varied from normal growth of the plant in cats, rye and soybeans, to chlorosis of the leaf tips in wheat, severe 1"." I '1' I'll A|I TABLE V CERMINATION PERCENTAGES FOR HYBRID SEED CORN WHEN TESTED AT 80° F IN A BROOKSTON SILT-LOAN ON WHICH DIFFERENT CROPS HAD PREVIOUSLY GROWN Nosco 25 Cries Mich“ Mich. Mich. Average Previous Crop 201A N‘ 250 350 880 Hheat 28.0 88.0 81.2 88.0 90.8 66.8 Barley 37.2 32.0 58.8 76.0 76.0 57.6 Beans 18.8 37.? 50.8 77.2 92.0 58.8 Alfalfa 20.0 36.0 80.0 58.8 88.0 86.8 Sugar beets 13.2 25.2 85.2 58.8 78.8 88.8 Corn 12 .0 20 .0 82 .8 50 .8 78 . 80.0 Sweet clover 10.8 25.2 29.2 52.0 65.2 36.8 Average 19.2 32.0 51.2 68.8 80.0 89.6 TABLE VI ANALYSIS OF VARIANCE FOR COLD TEST GERMINATION IN A BROOKSTON SILT-LOAN Source Degrees of Freedom Mean Square Total 108 Replications 2 5.5g Previous creps 6 105.0 Error (a) 12 25.2 Hybrids 8 783.5“ CrOp x Hybrid 28 10.5 Error 56 6.9 'i Significant at the 5 percent level of probability. Significant at the 1 percent level of probability. damping off in alfalfa, sugar beets and timothy, and stunting of growth in corn. The results obtained in this experiment suggest that parasitism may have been involved in the detrimental effects derived from the vari- ous crops tested. The growth of sweet clover, corn, sugar beets and alfalfa may have favored the multiplication of the pathOgens responsible for cold injury, while the growth of wheat and barley may have altered the balance of’microorganisms in the soil and reduced the quantity of microorganisms pathogenic to seed corn. The previous craps may also have caused some change in the soil, favorable or unfavorable to the develOpment of the pathogens. The differences in the effects from the previous craps do not warrant any generalization of their significance under field conditions. It is probable that the influence of previous crepe upon cold germination will vary with the type of soil and its composition, as well as the climatic conditions, since these factors influence greatly the nature and abundance of the soil micro-biological papulation. Large differences were obtained among hybrids. The Michigan hybrids were more tolerant than.Nosco N4 and.Gries 201A. This is in agreement with the previous rating of the hybrids. Influence of Soil Type Table VII presents the average cold test germinations for the seed of five corn hybrids in sterilized andrunsterilized samples of a.Brookston 27 silt-loam and a Hillsdale sandy loam, on which three different craps had been previously grown. .-'~nalyses of variance are given in Tables VIII and IX. Considering both sterilized and unsterilized soils, soil type did not have any influence on cold germination. On the average, 86.2 per- cent of the seed planted in the Hillsdale soil produced strong seedlings compared to 82.0 percent in the Brookston soil. The difference, h.2 percent, was not significant (Table IX). Perfect stands were not obtained in the sterilized soils, suggest- ing that either sterilization of the soils was not complete or the cold temperature was in itself injurious to germination. Haskell (6) has presented evidence of cold injury to seed corn at ho° F. The interaction soil 1 treatment was highly significant, indicating that the two treatments (sterilization and no sterilization) failed to affect genmination in the same manner in the two types of soil. Soil sterilization improved germination in the Brookston soil, but had no beneficial effect in the Hillsdale soil (Table VII).. Heppe and Middleton (ll) pointed out that Pythium app. which are among the most common pathogens reaponsible for cold germination injury, were associated with soil type. Possibly, the Hillsdale soil contained fewer pathogens capable of causing injury to seed corn at low temperatures. It is also possible that the drought period which preceded the collection of the soil in July had adversely influenced the pathogens in the Hillsdale soil, since climatic conditions can affect the nature and abundance of the soil microbial pepulation. 28 «.mm m.gm w.~m o.mw N.~m 2.0m N.~w 4.m© wwwnm>a N.mm we ooH 00a 0.4m mm mm mm om; .aoaz o.mm on em 4m 4.0m mm om 4m 0mm anoaz «.mm om om em «.mm om mm mm 0mm .noaa mamemaaam N.mo we pm we 0.4m om om om «z oomoz o.oe we no :m 4.45 em on 4% «How «mane o.- o.mp m.ma o.am . e.mm m.mm o.mm m.om mwmnm>< N.am om mm mm m.~m om mm mm on: .aoaz 0.0m mm mm no 4.0m om mm om 0mm .aoaz «.mm so we as «.mm om mm mm omw .eoaz copmxoonm m.mm on om No 0.0m om an no «2 oomoz 4.4m so On we 4.4a we we so «How ”mane me hmbfi hm>OHU nhoo 9w 02.3 mmWh0>< hm>OHU CHOU am mag ammsm . pmmzm sateen sexy Haom done nsowaoam mono meowbopm eoapaaaaanwpm oz eoaamuaaanmam iHr meEpmmne m 004 84 Z¢OQ anqm quawqu: 4 92¢ z wqmda TABLE VIII ANALYSIS OF VARIANCE FOR COLD TEST GERMINATION IN A BROOKSTON SOIL AND A HILLSDALE SOIL 29 Degrees Brookston Hillsdale Frezgom Mean Square Mean Square Total 59 Heplic ations l 2 6 .6 3 .3 Treatment 1 72 .6* h .3 Error (:1) 1 0.3 h.2 CrOp 2 1.8 3.5 Treat. 3: Crop 2 2h.0* 1.3 Error (b) h 3.3 h.6 Hybrids b 17 6 .3“ 82 .1“ Treat. x Hyb. h 35.8M 16.3“ CrOp x Crap 8 hit“ 7.1“ Treat. x Crap x Hyb. 8 11:5“ 3.0fl Error (c) 2h 3.6 2.2 4.:Significant at the 5 percent level of probability. Significant at the 1 percent level of probability. Ililillill .II{ 111'. TABLE IX 30 COMBINED ANALYSIS OF VARIANCE FOR COLD TEST GEWMINATION IN A BROOKSTON SOIL AND A HILLSDALE SOIL Source Degrees of Freedom Mean Square Total 119 Replication l 9.7 Soil type 1 3h.l Error (a) 1 17.6% Soil treatment 1 56.1. Soil szreat. 1 20.8** Error (b) 2 0.2 Previous Crops 2 1.6 Soil x Crap 2 3.8 Treat. x Cr0p 2 17.8 Soil x Treat. x CrOp 2 7.5 Error (c) 8 h.8 , Hybrid h 2148.8:it Soil x Hyb. h , 9.2 Treat. x Hyb. 1. My“ Crop x Hyb. 8 10.6" Soil 3: Treat. x Hyb. h 8.8‘ Soil x.Cr0p x Hyb. 8 1‘25 Treat. 1 Crop x Hyb. 8 10.2 Soil x Treat. x.Cr0p x Hyb. 8 6.8 Error h8 3.0 a» {-19 Significant at the 5 percent level of probability. Significant at the 1 percent level of probability. 31 In this connection, the average germination of all hybrids in the unsterilized Brookston soil, though lower than in the sterilized soil, was considerably higher than in the preceding experiment (Table V) con- ducted in May immediately after taking the soil from the field. The large differences in germination level between the two emeriments sug- gest that the pathogens may have been adversely affected by the drying- out of the soil in the greenhouse after the first experiment. Additional indication of the detrimental effect of drying-out of the soil on the pathogens is found in the fact that the previous craps had no influence upon germination. In the preceding experiment (Table V), significant differences were obtained anong the effects of the same previous craps on cold germination. It appears that the soil to be used in cold test studies should not be allowed to dry out. Large differences were found among the germinations of the hybrids. The three Michigan hybrids were again more tolerant to cold injury than Gries 201}; and Nosco N‘, in both soil types, sterilized and unsterilized. The consistency of the relative ranking of these hybrids under so widely different cold test conditions indicates that, in corn breeding programs, the cold test method can be effectively used to determine the relative tolerance of inbreds and hybrids to cold germination injury. PART III Early Generation Testing in Selecting Cold Tolerant Inbred Lines 32 MATERIALS AND METHODS 80, 31, and S; seed for hl families from a cold tolerant double- cross hybrid, Pioneer 373, was cold tested at the sane time in one experiment. The So seed was produced in 1950, the 81 seed in 1951, and the S; seed in 1952. Random samples of the seed indicated good germination under standard laboratory conditions. No seed fungicide was used. Each fanily was composed of seven lines developed from a single 80 plant selected on the basis of desirable agronomic characteristics. These lines represented three generations of inbreeding. There were: one Saline, two 51 lines, and four 52 lines in each family. Seed for each line was obtained from a single ear. Three replications of 25 seeds each were planted in flats of Hillsdale sandy loam soil obtained from a corn field. The soil was watered to saturation and the flats transferred to a walk-in cold chamber at ho° F for 12 days. Germination was completed in a warm greenhouse and strong seedlings counted 21 days later. 33 EXPERIMENTAL RESULTS AND DISCUSSION Table 1 presents the average cold test germination.percentages for the hl families. Analysis of variance is given in Table XI. The mean square for replications was highly significant indicating that there was great variability within the lines even though the seed for each line was obtained from a single ear. There were significant differences in average germination for the three generations.- The average stand for the So seed was 39.5 percent compared to hh.h percent for the 81 seed and 29.3 percent for the 8; seed. The lower germination of the So seed as compared to the 31 seed may have been'due to the fact that the So seed was one year older than the S1 seed at the time of testing. Cold test germination decreases as the age of the seed increases (19). The lower average for the 82 seed was probably a result of inbreeding and consequent reduction in general vigor. The frequency distribution of the families in each generation (Table III) shows that in the So and 51 generations, l6 and 18 families, reapectively, had an average germination greater than 50 percent compared to only five families in the 32. Large differences were found among the families. Their average stands ranged from 80.h percent to 5.1 percent. Germination in the SO varied from 0.0 percent to 92.0 percent. Variability within the lines increaSed with inbreeding as indicated by the larger mean square for "within lines in 82" than for "within lines in 51" (Table XI). However, % TABLE I we a r m S A. E l u _ .... S M 1. mm a F0 . I #2 NWT 25 GM F a... ... am a 2 a: s .S S P e D n 1 NN .1 . Obn L 1 I . ml) 2 A... S .nuS u, m% G 2 E . TH 1 ET 5 Tm m w. .1 O 1. C S a m o v is Ir V. n a F fififi35755flfiflfifi3161391fifiSJJJfiflfiflfiflfifiJ2flfi2JJ m6w9h3385 h22 9887666h3399666hh399765h205 6 SSSShh hhh 33333333332222222211111111 JfiflfidfififldfiflJJfiJflfifiJflflfififiJflJfiflfifififlbflfiflflfififl 76 halo/6 9h879858h812h861fl76122h66h02608670 7a..“ 56 9 h3|u125iu233|u1 2 511 11122 11 22 1 atJennnettnbaatatnttsaaaaabattsen3ttnebtn 9 S90 lulu IO 2 9 96614639011992 had 9 2 58 fififljfififlfiJfifiJDJJDJJDflJfiADfiJDfiflfiJflflfifiJfiJflJfi 88830 0&7283 9187.7 h1h28616 207|u0h2529 50 958353” 3217.5m2 213m8 162nm» 3w131 12 O 1 JDJQDJJfiJfiJfifiJDfiJfiJDJfiJDfiJJDfiJDQ336336Dflfl 921W63121672xu106389m161221901u1681383520OO 67 - 7.9 623321 N231 no 16 221 3 111 Jeannettenosesennntnatsnttnensnteeecnsaep 51171308280389326280309222h969863138 .5970, 866567865h27h275575h7565 82 «(Jinn/.23 12 221 0 30006539363606630 3066.60.66.6O33O6.6 3....“in 336:0 ooooooooooooooooooooooooooo o o o e 368660525 1282147. 7. 2 688‘u6 1366 57.385969 w7763h5h56®682h13M5Mh922356M2332mh132 22 .Dflfiflfifidflfifidflfififififlfiflfifi 0 h60868872h290983626226 27 35 117-52 h2h31121 1 8895l42/0 890123h567890123h56789 u 1122222222223333333333m 29.3 Mt Average 39 .5 TABLE XI ANALYSIS OF VARIANCE Fon COLD TEST GEEMINATION OF bl INBnED FAMILIES IN THE so, 5,, 1ND 52 GENERATIONS 35 Source Degrees of Freedom Mean Square Total 860 ‘ as Replications 2 10,877.2 ‘ a* Lines 286 1,931.8 ‘ as Families b0 5,539.6 as Generations 2 l9,999.1 Families x Generations 80 1,215.9** as Between lines in So hO 1,77h.2 “ Between lines in S; b0 1,878.9 as Between lines in 5; LG h,318.b Within lines in s1 bl 876.1;M as ‘Within lines in S; 123 1,282.2 Error 572 261.2 ** Significant at the 1 percent level of probability. 36 TABLE XII FREQUENCY DISTRIBUTION OF THE hl FAMILIES IN 50, 51, and 5; GENERATIONS Percent Germination So S]. 82 0-10 h l 6 10-20 7 2 10 33-30 7 8 7 30-10 3 h 7 140-50 14 8 6 50-60 8 9 3 60-70 3 8 1 70-80 2 l 0 83-90 2 O 1 90-100 1 0 0 TABLE XIII INTERGENERATION CORRELATION COEFFICIENTS I Degrees of r Freedom 50 "' S]. .. ' 0 J42” 110 53 "' 52 0 J42” ’40 51 " S; O .57“ 110 ** Significant at the 1 percent level of probability. 37 variability between lines increased more with continued inbreeding. In S; and 8; generations there was considerably more variation between lines than within lines indicating that possibilities for selection were greater between lines than within lines. Intergeneration correlation coefficients for germination were highly significant (Table XIII), indicating that cold test germination in the So and 5; generations was a fair indication of the relative tolerance in the 5;. The correlation of So with S; was O.h2** and.the correlation of So with S; was 0.h22*. Therefore only 17.6 percent of the variation in cold tests in So could be accounted for by inheritance in S; and 8;. Correlation of S; with S; was 0.57NE and 32.5 percent of the variability in 8; cold tests could be accounted for by inheritance in 3;. There is an indication here that the S; generation would be a better place to start selection for cold tolerance than the So. In the SO material, three lines germinated 80 percent or better. imong the 12 5; lines from these three tolerant SO selections, there were six iines (50 percent of these 12 8; lines) that germinated 70 percent or better and three lines (25 percent of.the 12 5; lines) that germinated 80 percent or above. From the 38 50 lines that germinated below 80 per- cent, there were only ten out of the 152 5; lines (6.5 percent) that germinated 70 percent or above. It appears that early generation testing can be effectively used in breeding corn for cold tolerance. 38 SUMMARY Commercial seeds for two susceptible and three tolerant doubler cross corn hybrids were cold tested in wet soil at 32° F and hfio F. All samples were treated with a seed disinfectant and were capable of highly satisfactory germination under standard laboratory conditions. After pre-cold treatments of O, 1, 2, 3, or 5 days of warm temperature, seeds of each of the five hybrids were given cold treatments of h, 8, 12, or 16 days at 32° F or ho° F. At both temperatures, exposing the seed to cold immediately or one day after planting resulted in nearly the same decreases in stand. Germination decreased as the pre-cold treatments increased beyond one day. Apparently in the more advanced stages of germination the seed became more susceptible to attack by the soil pathOgens responsible for cold germination injury; Three days of favorable conditions prior to cold treatment reduced the stand of all hybrids by 50 percent or more. In general, increase in duration of exposure to cold resulted in lower germination. Pre-cold treatments of O and one day followed by cold treatments at 320 F were less injurious than stmilar pre-cold ' treatments followed byhOO F cold treatments. It is likely that the soil pathogens were more active at uo° F than at 32° F. In the more advanced stages of.germination, cold treatments at 320 F were more ine jurious than.similar treatments at ho° F and apparently the seed was more injured by cold itself at 32° F than by soil pathogens. 39 The hybrids differed in their tolerance to cold wet soil conditions. Seed of the tolerant hybrids consistently gave higher germinations than seed of the susceptible hybrids. Germination of the tolerant hybrids was not seriously injured by four days of cold at 32° F and LOO F regard- less of the stage of germination. Eight days at either 320 F or bo° F did not seriously reduce germination of the tolerant hybrids until the seeds had been under favorable conditions for three days prior to cold treatment. Germination of the susceptible hybrids was reduced by all cold treatments at all stages of germination. Exposing seed corn to cold temperatures for eight to twelve days, immediately or one or two days after planting appears to be a satisfactory method for evaluating cold tolerance. , Seeds for the same five hybrids were cold tested at hOO F in samples of a.Brookston silt-loam soil and a.Hillsdale sandy loam soil from crap rotation experiments. Cold test germination percentages differed depend- ing on the soil type and the previous crap grown in the soil. In the Brookston soil, germination was best in soil that had previously been in wheat and decreased progressively in soils that had previously been planted to barley, beans, alfalfa, sugar beets, corn, and sweet clover. In the Hillsdale soil there were no significant differences in genmination in the soil samples that had previously grown corn, wheat, and sweet clover. Soil sterilization decreased injury to germination but did not re- sult in perfect stands. Part of the reduction in germination may have been caused by cold injury at hc° F. Drying out of the soil before its ho use for cold test germination seemed to have an adverse effect on the soil pathogens responsible for cold germination injury; SO, 5;, and S; seeds for hl families from a cold tolerant double- cross hybrid were cold tested at the same time in one experiment. Cold test germination decreased with inbreeding. Variability was greater between lines than within lines, indicating that selection would be relatively more effective between lines than within lines. Intergenera- tion correlations for germination indicated that early generation testing may be effectively used in selecting cold tolerant inbred lines. 12. LITERATURE CITED Alberta, H. W} Effect of pericarp injury on moisture absorption, fungus attack, and vitality of corn. Jour. Amer. Soc. jgron., 19: 1021-1030, 1927. . Davidson, R. S. Helminthcsporium seedling blights of corn. PhytOpath., to; 6, 1950. Dickson, J. G. Influence of soil temperatures and moisture on the development of the seedling blight of wheat and corn caused bngibberella.saubinetii. Jour. Agr. Res., 23: 837-8W3, 1923. , and J..R. Holbert. The influence of temperature upon the metabolism and expression of disease resistance in selfed lines of corn. Jour. Amer. Soc. Agr., 18: 3lh-322, 1926. Flor, H. H. Relation of environmental factors to growth and patho- genicity of Pythium isolated from roots of sugar cane. PhytOpath., 20: 319-328, 1930. Haskell, G. Effect of low temperatures on the germination of inbred lines of sweet corn. Science: 107: 150, l9h8. Ho, Wen-Chum. Soil inhabiting fungi attacking the roots of maize. Iowa Res. Bul. 332. 19th. Happe, P. E. Inheritance of resistance to seedling blight of corn caused by Gibberella saubinetii. PhytOpath. 19: 79-80, 1929. Heppe, P. E. Differences in Pythium injury to corn seedlings at high and low soil temperatures. PhytOpath., 39: 77-8h, l9h9. . A new technique for incubating seed corn in cold soil for disease tests. Phytopath., bl: 18, 1951. , and J. T. Middleton. Pathogenicity and occurrence in Wisconsin soils of Bythium Species which cause seedling disease in corn. Phytopath., 0: 13, 1950. Johann, Helen, J. R. Holbert and J. G. Dickson. A Eythium seedling blight and root rot of dent corn. Jour. Agr. Res., 37: th- héh. 1928. h2 13. Koehler, B. Pathologic significance of seed coat injury in dent corn. Abstract in.Phyt0path., 25: 2h. 1935. 111. , and G. H. Dungan. Disease infection and field performance of bin-dried and hanger-dried seed corn. Jour. Amer. Soc. Agron., 32: 768-781. 19h0. 15. , and W. L. Burlison. Maturity of seed corn in relation to yielding ability and disease infection. Jour. Amer. Soc. Agron., 26: 262-2714. 19311. 16. Livingston, J. E. Effect of controlled low temperature on the emergence of seedlings from articifially dried sweet corn. Phytopath., 111: 21;. 1951. 17. McIndoe, K. G. The inheritance of the reaction of maize to Gibberella saubinetii. Phytopath., 21: 615-639. 1931. 18. Meyers, M. T. The influence of broken pericarp on the germination and yield of corn. Jour. Amer. Soc. Agron., 16: 510-550. 19214. 19. Neptune, G. and E. C. Rossman. Cold test germination of hybrid seed corn. Mich. Agr. Exp. Sta. Quat. Bu1., 35: 3113-350. 1953. 20. Pinnel, E. L. Genetic and environmental factors affecting corn seed germination at low temperatures. Jour. Amer. Soc. Agron., 141: 562-568. 19119. 21. Raleigh, w. P. Infection studies of Diplodia zeae (Schw.) Lev. and control of seedling blights of corn. Iowa Agr. Res. Sta. Bul. 12h: 96-121. 1930. 22. Richardson, J. K. Studies on root rot of corn in Ontario. Can. Jour. Res. C., 20: 2141-256. 191:2. 23. Rush, G. E., and'N. P. Neal. The effect of maturity and other factors on stands of corn at low temperatures. Jour. Amer. Soc. Agron., 143: 112-116. 1951. 21;. Smith, 0. F. The influence of low temperature on seedling develop- ment in two inbred lines of corn. Jour. Amer. Soc. Agron., 27 8 h67-h79 . 1935 . 25. Tatum, L. A., and M. S. Zuber. Germination of maize under adverse conditions. Jour. Amer. Soc. Agron., 35: 118-59. l9h3. LB 26. Valleau, W, D., P. E. Karraker and E. M. Johnson. Corn root rot-- a.soil borne disease. Jour. Agr. Res. 33: h53-h76. 1926. 27. waksman, S. 1. Soil Microbiology. p. 32. John Wiley & Sons, Inc., New York. 1952. 28. Wbrtman, L. 3., and E. H. Rinke. Seed corn injury at various stages of processing and its effect upon cold test performance. Jour. Amer. Soc. .Agron., h3: 299-3011. 1951. W‘W‘w”