OF THL UNIVERSITY OF ILLINOIS' 1 G30.7 cop - 2- AGRICULTURE NOTICE: Return or renew all Library Materials! The Minimum Fee for each Lost Book is $50.00. The person charging this material is responsible for its return to the library from which it was withdrawn on or before the Latest Date stamped below. Theft, mutilation, and underlining of booKs are reasons for discipli- nary action and may result in dismissal from the University. To renew call Telephone Center, 333-8400 UNIVERSITY OF ILLINOIS LIBRARY AT URBANA-CHAMPAIGN L161 O-I096 \ UNIVERSITY OF ILLINOIS Agricultural Experiment Station BULLETIN NO. 284 THE INFLUENCE OF PLANT INJURY AND THE ROOT ROT DISEASES UPON THE PHYSICAL AND CHEMICAL COMPOSITION OF CORN GRAIN BY GEORGE H. DUNCAN URBANA, ILLINOIS, DECEMBER, 1926 SUMMARY Constricting the ear-shanks and stalks of corn by breaking the supporting tissue in these structures without completely severing the vascular elements had essentially the same effect upon the yield and composition of corn grain as premature harvesting. Constriction of the shank was much more detrimental to ear development than the break- ing of the stalk. Breaking the shanks when the ears were in the soft-dough stage caused the greatest degree of chaffiness in the grain. Chemical analy- ses showed that there was no definite correlation between kernel "starchiness" and quantity of starch in the grain, as has been com- monly supposed. In fact the quantity of true starch was often less in the "starchy" than in the horny grain. In the light of these results it is suggested that the term "floury" be substituted for the word "starchy" in describing the corn containing more than a normal quan- tity of soft, dry, friable material. The percentage of total nitrogen, hemicellulose, and non-hydro- lyzable material was distinctly higher in grain from ears produced on broken shanks than in grain from ears produced on sound shanks. The mutilation of the shanks, on the other hand, resulted in a greatly re- duced proportion of ether extract and starch in the grain. Inoculation of the seed at planting time with corn root rot organ- isms resulted in the production of grain having a specific gravity .027 lower than that from the adjoining uninoculated plants. The samples of grain produced by plants that grew in the inocu- lated hills absorbed an average of 5.78 percent more water than the samples from plants growing in uninoculated hills, with odds .greater than 9999 to 1 that this difference was not due to chance. Chemical analyses did not show any significant differences be- tween the grain produced in the inoculated and uninoculated hills. There was slightly more nitrogen, on the average, in the corn from in- oculated seed, and a little less ether extract and total sugar. Even tho the average differences in total nitrogen, ether extract, and total sugar were not large, the odds indicating the significance of these re- sults were above 30 to 1. Analyses of two lots of horny and two lots of floury corn of the Learning variety showed that there was no significant distinction in chemical composition between these two types of corn. Upon germi- nation, however, the horny corn was found to contain a greater pro- portion of soluble starch and dextrins than the floury corn, and starch digestion in the horny corn was somewhat more rapid than in the floury corn. This phenomenon offers an explanation for the superior vigor of seedlings from horny corn, so frequently observed. THE INFLUENCE OF PLANT INJURY AND THE ROOT ROT DISEASES UPON THE PHYSICAL AND CHEMICAL COMPOSITION OF CORN GRAIN By GEORGE H. DUNCAN, Assistant Chief in Crop Production INTRODUCTION For a number of years the Illinois Agricultural Experiment Sta- tion has advised farmers to eliminate all extremely "starchy" ears from their seed stock. This recommendation has been based upon the re- sults of investigations into the causes of the corn rot diseases obtained by the Station and the Office of Cereal Investigations of the U. S. De- partment of Agriculture working cooperatively. In these investigations by Holbert and associates 13 the fact is established that a high degree of correlation exists between certain ear characters in seed corn and susceptibility of seedlings developed from such ears to infection by root rot organisms. They give the important diagnostic features of diseased seed corn as: (a) a lack of luster in the ear; (b) a discolored or shredded shank attachment; (c) a bleached ear tip; (d) an extremely deep indentation at the crown of the kernel ; (e) a relatively high proportion of soft to horny starch in the endosperm; and (f) a dull wrinkled kernel, especially in the region of the germ. It is evident that these characters are not en- tirely independent of one another, for an increased percentage of soft starch in the endosperm tends to decrease the luster of the ear, and a high content of soft starch is often accompanied also by deep kernel indentation. Thus it would seem that the composition of the grain, especially the proportion of soft to horny starch in the endosperm, is a very important factor, if not the most important single factor, in determining susceptibility or resistance to disease, or at least in indi- cating such susceptibility or resistance. The data from one experiment in which horny and "starchy" kernels were selected from the same ears and grown in comparative yield trials, showed a reduction of 12 bush- els an acre for the "starchy" seed. The results of investigations over a period of seven years showed a decrease of 7.9 bushels an acre for "starchy" seed, with odds greater than one million to one that this difference was not the result of chance. Trost 38 found that ears of the "starchy" class were character- ized by a larger percentage of infection on the germinator than horny 255 256 BULLETIN No. 284 [December, ears from the same seed sample. In experiments using Reid Yellow Dent corn as seed, the "starchy" ears produced a larger number of weak plants and 14 percent lower yield than did ears less "starchy" in composition. No data, however, have as yet been obtained as to why corn con- taining a large proportion of soft starch is more susceptible to fungus invasion than corn containing a smaller proportion, and very little di- rect knowledge is available as to the factors that influence the physical composition of the endosperm in corn grain. In general it may be said that the agencies which determine the type of endosperm in corn divide themselves into two classes; namely, hereditary factors, and those factors that may be included under the general term environ- ment. The present work has been confined to a study of certain fact- ors in the second group ; namely, the influence that stalk and shank in- jury may have at two different stages in the development of the ear, and the effect of the inoculation of the seed at planting time with four organisms, viz., Diplodia zeae (Schw.) Lev., Fusarium moniliforme Sheldon, Gibberella saubinetii (Mont.) Sacc., and Rhizopus spp., any one of which is capable of producing a corn root rot disease. Data are also presented on the comparative chemical composition of starchy and horny kernels at different stages during the process of germination. REVIEW OF LITERATURE The literature reporting the influence of ecological factors on the physical and chemical composition of cereal grains is voluminous, and this summary is not presented as in any sense exhausting that general subject. Only those papers are reviewed that show the influence of the stage of harvesting and plant injury from mechanical or parasitic causes on the composition of the grain. The monumental researches of Hornberger 15 in 1882 constitute one of the most intensive pieces of work on the corn plant up to that time. Later Schweitzer, 28 Jones and Huston, 18 and Smith, 32 engaged in the study of the same problem. These workers agreed with Horn- berger, 18 who found that as the grain developed beyond the milk stage the proportion of ether extract increased, while the percentage of total nitrogen and ash decreased. They also noted that the relative amount of carbohydrates increased with maturity, while that of the crude fiber decreased. The investigations of Ince 17 also showed a larger proportion of crude fat and nitrogen-free extract in mature grain than in immature grain. Bushey 3 reported that corn cut short in its devel- opment by frost contained higher percentages of ash and crube fiber than unfrosted corn. A comparatively high proportion of the nitrogen of the soft corn was in the amid-albumin and globulin forms. Hume, Champlin, and Loomis 16 noted that completely mature corn contained 1926} INFLUENCE OF PLANT INJURY ON COMPOSITION OF CORN 257 the largest percentage of oil. Appleman 1 found that the grain of sweet corn decreased markedly in content both of total sugar and of reducing sugar as it developed from the milk to the dough stage. Kent, Patrick, Eaton, and Heileman 20 and Curtiss and Patrick 6 found that after the ears had reached the dough stage there was but little change in the composition of the grain, altho there was a gain in yield per acre. Analyses reported by Saunders 27 showed that there was a rapid reduction in proportion of crude protein in the wheat grain during its early development, and that afterwards there was a gradual percent- age increase in this material. Similar results were also obtained by McDowell 25 in both winter and spring wheat, altho the percentage in- crease in protein following the early rapid decrease was fluctuating in character. LeClerc 21 working with wheat, and Failyer and Willard 10 work- ing with both oats and wheat, found that late cutting resulted in a slightly reduced proportion of nitrogen in the grain. Kedzie 19 made analyses of wheat grain at a great many stages, be- ginning when the heads were just past the "blossom" stage and con- tinuing until the grain was dead ripe and the straw fallen. His data showed a decrease in the percentage of ash and crude-fiber content as the grain matured. The relative amounts of albuminoids and amid nitrogen decreased rapidly up to the hard-dough stage, after which they increased slightly. The proportion of ether extract decreased un- til the grain was in the milk stage, after which it rose gradually. The percentage of nitrogen-free extract increased up to the hard-dough stage, then dropped off slightly. Data presented by Shutt 31 indicated that wheat grain injured by frost before it was completely mature contained a lower percentage of both total nitrogen and albuminoid nitrogen. Blish 2 reported that "frozen wheat contains larger amounts of non-protein nitrogen, reduc- ing sugars, and acid-reacting constituents than does sound wheat. The non-protein nitrogen of the frozen wheat carries a considerably higher percentage of a-amino nitrogen than that of sound wheat." Severe rust injury hastened the ripening of wheat, and according to Shutt 30 markedly increased the percentage of protein, crude fiber, and ash, and lowered the proportion of fat and nitrogen-free extract. Similar results as to the effect of rust on the ash and protein contents of the wheat grain were obtained by Stoa. 35 Headden, 12 however, stated that the shrunken berries resulting from rust infection were not high in protein. He pointed out that protein and starch were laid down simultaneously, and the effect of rust was to prevent the transfer of the filling material to the berries. Microchemical studies by Eckerson 9 indicated that starch was formed in the endosperm cells of the developing wheat grain soon after the formation of the cell walls, and that this process continued until 258 BULLETIN No. 284 [December, desiccation began. Storage proteins were not formed in the endosperm until the drying of the grain caused the amino acids present to con- dense into proteins. MATERIALS AND METHODS In the experiment to determine the effect of breaking the stalks and ear-shanks, yellow dent corn obtained from James R. Holbert of the U. S. Department of Agriculture, was used as seed. It was of F^ hybrid material between pure-line strains, and consequently was much more uniform in genetic constitution than average open-pollinated corn. Seed of this corn was planted on the University South Farm and received the usual cultivation under field conditions. On August 17, when the grain was in the "milk" stage, and also on August 31, after the grain had reached the "soft-dough" stage, a num- ber of the ears were broken down to the extent of markedly constrict- ing the supporting shank but not severing the ear from the stalk. Also, on these two dates, a number of plants were broken over, so that a definite constriction resulted between the nodes of the stalk about one foot from the surface of the ground. The grain from plants so treated was analyzed and the results compared with similar data from ears produced on plants which had not been mutilated. The seed used in the experiment to determine the effect of plant infection with corn root, stalk, and ear rot-producing organisms on the composition of the grain was obtained from the Plant Breeding Divi- sion of the University of Illinois. This seed had been inbred for two generations. Two ears possessed an extremely horny endosperm and two an endosperm containing a high proportion of soft starch. These were planted on May 16, in soil that had grown alfalfa for the twelve years prior to this corn crop. At the time of planting, the alternate hills were inoculated with a pure culture of some one of the following organisms: Diplodia zeae (Schw.) Lev., Fusarium monitiforme Shel- don, Gibberella saubinetii (Mont.) Sacc., and Rhizopus spp. The grains were laid, germs up, in the hill. Two horny kernels were placed about an inch apart on the north side of the hill and two "starchy" kernels were similarly placed on the south side of the hill. Two drops of a heavy spore suspension of one of the above-mentioned organisms were placed on the germ face of each kernel in the alternate hills. In order to make sure of getting an infection with Diplodia zeae a frag- ment of shredded cornstalk bearing a pure culture of this organism was placed between the kernels in the hill in addition to the spore sus- pension placed on the kernels. Two drops of distilled water were placed on the germ face of each kernel in the uninoculated hills. After the moisture in the drops of spore suspension had partially dried down, the corn was covered with moist soil which was pressed firmly down over the seed. 1926] INFLUENCE OF PLANT INJURY ON COMPOSITION OF CORN 259 The cultures of Fusarium moniliforme and Rhizopus spp. were iso- lated from germinating corn. The cultures of Diplodia zeae and Gib- berella saubinetii (the latter designated as strain 259) were obtained from Miss Helen Johann, of the U. S. Department of Agriculture. In the study of the progressive chemical changes occurring dur- ing germination in horny corn, as compared with those occuring in "starchy" corn, the Learning variety was used. This lot of corn con- sisted of twelve ears, six of which were smoothly indented and high in percentage of horny starch and six of which were rough in indenta- tion and relatively high in soft starch. All of these ears had been carefully field-selected early in November and were alike in that they had all been produced on apparently healthy plants. These ears were divided into four lots of three ears each. Lots A and C were made up of horny ears, and Lots B and D of "starchy" ears (Figs. 1 and 2). Five sets of germination tests were started in the temperature- control chamber, which was maintained at 16 C. in the plant pathol- ogy greenhouse at the University of Wisconsin a . Each test contained 20 kernels from each ear, the kernels in each case being removed from the same rows on the ear. The corn was germinated between muslin cloths over moist sand. At the end of the 2d, 4th, 6th, 8th, and 10th days a set of the tests was removed and the kernels or seedlings dried for analysis. Drying was effected by placing the samples in the oven at 100 C. for one hour to kill the enzyms, followed by 18 hours of drying with the oven door open (Link and Tottingham 22 ) . CHEMICAL METHODS All samples of corn grain analyzed were finely ground and passed thru a 100-mesh sieve. The total nitrogen determinations were made by the Official Gunning method modified to include the nitrogen of nitrates. 7 The soluble nitrogen was extracted by soaking the ground grain for four hours in 50 cc. of distilled water to each gram of sample. The sugars were extracted from the ether-extract-free sample with 90 percent ethyl alcohol, by gently boiling the sample for one hour. After the alcohol was evaporated, the sirupy residue was taken up with water and the solution clarified with neutral lead acetate. The excess lead acetate in the filtrate was then removed by adding sodium sulfate and sodium carbonate in the proportion of nine parts of the former to one of the latter. Phenolphthalein was used to indicate when defeca- tion was complete. After filtering, the reducing sugar was determined, an aliquot of the clear filtrate being used for this purpose. Total su- gars were determined after hydrolysis in 2.5-percent hydrochloric acid, by boiling on a sand bath for one hour. "All the germinative tests and chemical analyses in this investigation were made at the University of Wisconsin. 260 BULLETIN No. 284 [December, FIG. 1. HORNY EARS OP LEAMING CORN USED IN STUDY OF CHEMICAL CHANGES OCCURRING DURING GERMINATION Kernels from these two lots of horny corn and from two lots of "starchy" corn (Fig. 2) were germinated, and the chemical changes taking place during germination were determined by analyzing samples taken at two-day intervals. The results are described on pages 269 to 275. Dextrins and soluble starch were extracted with cold water in which the sugar- free sample was allowed to soak overnight. The fil- trate was hydrolyzed by boiling in 2.5-percent hydrochloric acid for 2Y 2 hours. The solution containing the insoluble starch was boiled for three minutes to gelatinize the starch present. When cooled to 38 C., fresh saliva was added to hydrolyze the starch to maltose. The complete conversion of starch to maltose was indicated by failure of the mater- ial to give a blue color with iodin. The maltose extract was hydro- lyzed to glucose by adding hydrochloric acid until the concentration of the acid in the solution was 2.5 percent and by boiling over a sand bath for 2% hours. The residue was washed into a flask and submitted to hydrolysis in 2.5-percent hydrochloric acid for one hour. The filtrate was consid- ered to be hemicellulose. 1926} INFLUENCE OF PLANT INJURY ON COMPOSITION OF CORN 261 FIG. 2. "STARCHY" EARS OF LEAMING CORN USED IN THE GERMINATION STUDY The relative proportion of horny and soft starch in these ears is .indicated by the transverse and longitudinal sections of the grain. A preliminary analysis showed practically the same percentage composi- tion for both horny and "starchy" lots, but their specific gravity was markedly different. The quantity of sugars, soluble starch, and dextrins, insoluble starch, and hemicellulose was ascertained by determining the power of these substances to reduce the copper of Fehling's solution. The amount of copper reduced was measured by the Shaffer-Hartman 29 iodometric titration method. EXPERIMENTAL RESULTS EFFECT OF BROKEN STALK AND EAR SHANKS Weak and diseased corn plants are frequently not strong enough to support the ears to complete maturity ; the increasing weight of the ear may exceed the strength of the supporting tissue of the shank, or the stalk itself may be deficient in mechanical substance and break over under stress of wind. To simulate what so often happens in na- 262 BULLETIN No. 284 [December, 1926} INFLUENCE OF PLANT INJURY ON COMPOSITION OF CORN 263 ture, a number of ear shanks and cornstalks were artificially broken on August 17, when the grain was in the milk stage, and on August 31, when the grain was in the soft-dough stage. The breaking-over of the stalks and ear shanks did not in any case involve the complete severance of the ear from the shank, nor of the upper portion of the stalk from the stub. The vascular elements, or at least most of them, remained intact. There was, however, a dis- tinct constriction produced, which doubtless interfered greatly with the translocation of reserve and building materials to the ear. It would seem that the constriction of the shank, brought about by breaking down the ears, would be greater than that of the stalk, owing to the fact that the break caused a departure of approximately 120 from the normal position of the shank, whereas the break of the stalk re- sulted in a declination of only a little over 90. That the constriction of the shank and stalk produced a marked effect upon the size and character of the ears and kernels may be ob- served by reference to Fig. 3 and by a study of the data presented in Table 1. The breaking of the ear-shank produced a much greater hindrance to the proper filling of the grain than the constriction of the stalk. This is particularly pronounced in the weight of ears, the weight of TABLE I.-W-EFFECT OF BREAKING THE SHANKS AND STALKS OF CORN ON WEIGHT OF EARS (GRAIN AND COB), WEIGHT OF TEN KERNELS FROM EACH EAR, AND GERMINATIVE VIGOR OF THE KERNELS Average Average length of Group Treatment Average weight of ears Average weight of 10 kernels Average germi- nation in soil number of secon- dary roots per seed- plumule at end of 8-day germi- nation ling test in soil grams grams perct. mm. A Parent ears 2.96 98.0 2.88 120 B Shank broken when grain was in milk stage 78.4 .69 52.3 1.60 74 C Shank broken when grain was in soft-dough stage . . 191.8 1.59 97.1 1.65 75 D Stalk broken when grain was in milk stage 272.4 2.37 88.1 2.67 87 E Stalk broken when grain was in soft-dough stage . . 261.0 2.38 88.3 2.96 100 F Ears from erect plants and from unbroken shanks . . . 331.1 2.86 98.1 3.43 117 264 BULLETIN No. 284 [December, ten kernels, the number of secondary roots, and the length of plumules. Even tho the delay in breaking the shanks until the soft-dough stage resulted in a much greater weight of ear and kernel, and also increased the percentage of germination, the vigor of the seedlings produced by this corn, as measured by number of secondary roots and length of plumule, was only very slightly better than that of the corn the shanks of which were broken when the grain was in the milk stage. The chaffy character of the ears in Group C, Fig. 3, is striking. All the ears in this experiment that had their shanks broken when the grain was in the soft-dough stage contained a very high proportion of soft starch. A comparison of the various lots of ears in respect to the relative quantity of soft and horny starch in the endosperm of the kernels is afforded in Table 2. The parent ears and those that matured on erect plants and on unbroken shanks contained the highest percentage of horny and the TABLE 2. EFFECT OF BREAKING THE SHANKS AND STALKS OF CORN AT Two DIFFERENT STAGES OF GROWTH, ON THE KERNEL CHARACTER OF THE EARS PRODUCED Group Treatment Number of ears repre- sented Kernel character: percentage of ears that were Horny Medium Starchy A B C D E F Parent ears 5 13 7 10 12 11 80 20 8 27 30 10 18 20 70 100 70 92 55 Shank broken when grain was in milk stage Shank broken when grain was in soft-dough stage ... ... Stalk broken when grain was in milk stage Stalk broken when grain was in soft- dough stage Ears from erect plants and from unbroken shanks lowest percentage of "starchy" individuals. However, as may be seen from Table 3, the chemical composition of the kernels in respect to the quantity of true starch which they contained, did not harmonize with their physical appearance. The early constriction of the shanks resulted in grain high in total nitrogen, hemicellulose, and crude fibrous material that was non-hy- drolyzable in 2.5-percent hydrochloric acid and low in ether extract and insoluble starch. When the breaking of the shanks was delayed until the grain was in the soft-dough stage, the proportion of total ni- 1926} INFLUENCE OF PLANT INJURY ON COMPOSITION OF CORN 265 trogen, hemicellulose, and non-hydrolyzable material was not so high as when the breaking was done earlier, but it still was considerably higher than in the normally matured ears. There was also a signifi- cant increase in the relative amount of ether extract and insoluble starch as the grain developed from the milk to the soft-dough stage. The breaking of the stalk did not affect the composition of the grain so much as did the constriction of the shank, altho it did result in a slight increase in total nitrogen and non-hydrolyzable material, as well as in a small decrease in ether extract and insoluble starch. The time of breaking the stalks had very little effect on the relative quan- tity of these materials in the grain. It appears that either the plant above the constriction contained a sufficient amount of previously elaborated food materials to fill out the grain to an almost normal de- gree, or the bending of the vascular elements in the stalk was not acute enough to prevent the passage of solutions for synthetic pur- TABLE 3. EFFECT OF THE BREAKING OF THE SHANKS AND STALKS OF CORN ON THE CHEMICAL COMPOSITION OF THE CORN GRAIN (Amounts expressed in percentages) Material Dextrins non-hy- 3 Total Ether Total and Insolu- Hemi- drolyz- Treatment nitro- ex- sugar solu- ble cellu- able in O gen tract ble starch lose 2. 5- per- starch cent HC1 A Parent ears 2.07 4.83 1.32 2 01 53.17 6.62 6.16 B Shank broken when grain was in milk stage 3.40 3. 16 1. 17 1.76 39.76 9.55 9.90 C Shank broken when grain was in soft-dough stage 2.27 3.92 1.54 1.44 44.71 9.04 7.68 D Stalk broken when grain was in milk stage 1.90 4. 18 1.05 1.78 51.85 6.65 6.19 E Stalk broken when grain was in soft-dough stage 1.85 4.38 1.53 1.77 51.61 7.25 6.81 F Ears from erect plants and from unbroken shanks 1.66 4.56 1.61 2.67 54.35 6.66 5.95 poses into the lodged plant. It is very likely that both the transloca- tion of storage material already in the stalk and the deposition of re- serves synthesized after the stalk was broken were contributing factors in producing grain of a practically normal composition. Even tho the relative amounts of materials in the grain produced on broken stalks closely approached that of the checks, the total weights of ears and grains were considerably below those of the checks (Table 1). A comparison of the data presented in Table 2 with that in Table 3 brings out the fact that the condition of the corn grain which has so often been described by the term "starchy" is not necessarily associ- ated with a higher percentage of starch in the grain, but that, on the 266 BULLETIN No. 284 [December, contrary, there is a slight suggestion that the ears which are more "starchy" in appearance may actually contain less starch hydrolyzable with saliva than horny ears of the same variety. EFFECT OF PLANT INFECTION WITH CORN ROOT ROT ORGANISMS Not all the plants that were inoculated with the four corn root parasites gave evidence of being injured appreciably by these organ- isms. In order to obtain data on the effectiveness of the inoculation, both shelled grain weights and root anchorage figures were taken. The TABLE 4. SPECIFIC GRAVITY OF THE GRAIN PRODUCED FROM INOCULATED HILLS COMPARED WITH THAT OF GRAIN FROM PLANTS IN ADJACENT, UNINOCULATED HILLS Number by which Specific g grain f ravity of rom Difference Organism with which seed was inoculated inoculated plant was designated Inoculated hill Adjoining uninocu- lated hill uninocu- lated hill Diplodia zeae . ... 2b.. 1.111 1.116 .005 9b 1.096 1.042 054 27b 1.085 1.126 .041 37b 1.132 1.161 .029 55b 1.053 1.188 .135 Average. . 1.095 1.127 .032 Fusarium moniliforme 3a. . 1.222 1.302 .080 lOa 1.217 1.243 .026 28a. . . 1.214 1.245 .031 56a 1.312 1.302 -.010 3b 1.090 1.100 .010 lOb 1.127 1.117 -.010 : f^J 17b 1.066 1.040 - .026 28b. .. 1.167 1.196 .029 38b 1.193 1.298 .105 Average. . 1.178 1.205 .027 Gibber eUa saubinetii 54a. . 1.281 1.293 .012 12b. . 1.047 1.069 .022 19b 1.142 1.132 -.010 40b 1.188 1.239 .051 54b 1.233 1.320 .087 Average. . 1.178 1.211 .033 Rhizopus spp 18a. . 1.243 1.243 .000 25a 1.173 1.226 .053 4b 1.063 1.066 .003 25b 1.040 1.040 .000 32b 1.219 1.239 .020 Average. . 1.148 1.163 .015 Grand average 1.154 1.181 .027 . Odds that the average difference is not due to chance = 302:1 1926] INFLUENCE OF PLANT INJURY ON COMPOSITION OF CORN 267 root anchorage was determined by measuring the pulling resistance of the hills at harvest time. Grain from only those plants that yielded less, or that had a root anchorage below that of the adjoining uninocu- lated check, was selected for analysis. The yield, however, was given primary consideration in making this selection. No ear was taken for this study unless its weight was less than its check. It was possible in many instances by visual comparison to detect a slightly greater proportion of soft starch in the corn from the inoc- ulated hills. This was not possible, however, in every case. In order to determine more accurately the influence that infection had upon the density of the grain, specific gravity determinations by displace- ment in 95-percent alcohol were made. The results of this experiment are given in Table 4. In only five cases out of the twenty- four sets tested did the grain TABLE 5. PERCENTAGE OF WATER ABSORBED BY GRAIN PRODUCED ON CORN PLANTS GROWN FROM INOCULATED SEED, COMPARED WITH THAT ABSORBED BY GRAIN PRODUCED ON PLANTS FROM UNINOCULATED SEED Organism- with which seed was inoculated Number by which inoculated plant was designated Amount of water absorbed in 24 hours by grain produced on plants from Difference in favor of corn from inoculated seed Inoculated seed Adjoining uninocu- lated seed Diplodia zeae 27b. . perct. 72.53 64.27 67.82 68.21 perct. 64.16 63.09 62.28 63.18 perct. 8.37 1.18 5.54 5.03 37b 55b Average. . Fusarium moniliforme 3a 53.09 51.40 55.41 65.26 61.32 73.06 54.87 59.20 49.26 44.89 49.42 58.97 62,57 54.53 47.86 52.50 3.83 6.51 5.99 6.29 -1.25 18.53 7.01 6.70 28a 56a 3b lOb. . 28b 38b Average. . Gibberella saubinetii 54a.. 12b 47.58 72.03 67.57 62.91 56.62 61.34 48.34 63.12 64.97 54.66 48.59 55.93 - .76 8.91 2.60 8.25 8.03 5.41 19b 40b 54b Average. . Rhizopus spp 4b. 72.04 56.99 64.52 65.04 54.66 59.85 7.00 2.33 4.67 32b Average. . Grand average 62.04 56.26 5.78 Odds that this difference is due to inoculation > 9999:1 268 BULLETIN No. 284 [December, from the inoculated plant exceed that of the check in specific gravity. This occurred once with Diplodia, three times with Fusarium, and once with Gibberella. In every case, however, the average specific gravity was higher for the corn from uninoculated plants, and when the results from all the organisms were averaged the grain from uninoculated plants was found to be .207 higher than the specific grav- ity of the infected plants, with odds of 302 to 1 that this difference was not due to chance. In some previous work by the author 8 it was shown that corn pos- sessing a high proportion of soft starch in the endosperm is capable of TABLE 6. RESULTS OF CHEMICAL ANALYSES OF CORN GRAIN PRODUCED BY PLANTS GROWN FROM INOCULATED AND FROM UNINOCULATED SEED (Results expressed in percentages) Kind of corn Plant No. Total nitro- gen Ether extract Total sugar Dextrins and solu- ble starch Insol- uble starch Hemi- cellu- lose Non-hy- drolyza- ble residue Diplodia-inoculated. . . . Adjacent check 2b.. . 6b.... 2.77 2.22 4.26 4.70 1.32 1.68 2.58 3.15 48.22 50.06 7.72 7.91 5.94 5.32 Diplodia-inoculated. . . . Adjacent check 9b... 13b 2.26 1.81 4.67 4.61 1.13 1.39 1.89 1.75 51.23 52.92 7.67 7.36 5.87 6.49 Fusarium-inoculated. . . Adjacent check lOa. . . 14a 2.31 1.99 4.07 4.42 1.23 1.53 2.48 2.67 48.93 51.30 6.13 6 46 4.52 4.35 Fusarium-inoculated. . . Adjacent check 17b... 21b 1.64 1.84 4.45 4.74 1 32 1.34 1.73 1.82 53.32 53.21 8.29 7.53 6.33 5.95 Gibberella-inoculated . . Adjacent check la.. 5a 1.55 1.68 3.73 4.12 .95 .95 3.07 2.43 61.96 55.76 5.85 5.91 5.33 5.56 Gibberella-inoculated . . Adjacent check Ib... 5b.. .. 2.92 1.79 4.44 5.16 1.03 1.24 2.71 2.76 45.65 56.97 8.16 7.45 7.76 6.25 Rhizopus-inoculated . . . Adjacent check ISa 14a 2.02 1.99 5.01 4.42 1.70 1.53 3.92 2.67 52.94 51.30 5.74 6.46 4.81 4.35 Rhizopus-inoculated. . . Adjacent check 25a 21a.... 2.37 .2.09 4.02 4.38 1.08 1.13 2.30 2.43 52.58 55.28 6.57 6.79 7.29 4.70 Rhizopus-inoculated. . . Adjacent check 25b... 21b.... 1.87 1.84 4.65 4.74 1.29 1.34 2.01 1.82 53.92 53.21 6.94 7.53 5.14 5.95 Average difference in favor of inoculated plant + 31 22 12 + 13 -1 14 - .14 + .45 Odds that above dif- ference is not due to chance 44:1 57:1 57:1 4:1 4:1 5:1 18:1 more rapid water absorption than corn containing a less amount of this material. Accordingly, the water-absorptive capacity of the corn in the present experiment was determined with a view to applying the results as a measure of soft-starch content. The data included in Table 5 were obtained by soaking samples of corn produced by inoculated and check plants, in distilled water, under the same conditions, for a period of twenty-four hours. The percentages are based on the water- free weight of the sample. 1926} INFLUENCE OF PLANT INJURY ON COMPOSITION OF CORN 269 In every instance except two one with Fusarium and one with Gibberella the corn from infected plants absorbed more water than that from the adjoining checks. When all the determinations with corn from plants inoculated with the four organisms were averaged, the difference between the percentage of water absorbed by the grain from inoculated plants and that by grain from the checks was 5.78, and the odds were greater than 9999 to 1 against a difference as great as this being due merely to chance. Notwithstanding this conclusive evidence, the results of the chem- ical analyses of representative samples, as presented in Table 6, do not show any marked differences between corn grain produced by plants from inoculated seed and those from uninoculated seed. When the differences between the two types of grain in all the nine pairs of determinations are averaged, it is found that the corn from the inoculated plants contained .31 percent more nitrogen, .13 percent more soluble starch and dextrins, and .45 percent more non-hydroly- zable substance than the accompanying checks. The odds that these average differences were not due to chance were 44 to 1, 4 to 1, and 18 to 1, respectively. The corn from the uninoculated plants exceeded that from the inoculated plants in ether extract by .22 percent, with odds of 57 to 1. The average difference in total sugar was .12 percent in favor of the uninoculated checks, and this result also carried odds of 57 to 1. Insoluble starch and hemicellulose averaged slightly higher for the check corn, but the significance of these results as measured by odds is very low. CHEMICAL CHANGES OCCURRING IN "STARCHY" AND HORNY CORN DURING THE GERMINATIVE PROCESS A preliminary analysis of the four lots of Learning corn repre- sented in Figs. 1 and 2 showed that the "starchy" lots, B and D, had practically the same percentage composition as the horny lots, A and C. That there was a marked difference in these lots of corn may be seen from their specific gravity, a record of which is given in Table 7. TABLE 7. SPECIFIC GRAVITY OF THE GRAIN OF LEAMING CORN, Two LOTS OF WHICH WERE HORNY AND Two "STARCHY" Average specific Type of corn Lot Weight of 20 kernels Specific gravity gravity of the two lots of the same type (grams) Horny A.. 7.85 1.312 1.292 C 7.87 1.271 "Starchy".. . B.. 6.54 1.208 1.204 D 6.40 1.199 270 BULLETIN No. 284 [December, In order to obtain data on the difference in quality of the mater- ials contained in these two types of seed, a germination test of both was made at 16 C., and the kernels and the seedlings with grains attached were sampled for analysis every two days. The degree of development is illustrated in Fig. 4. The results obtained are presented in Table 8. The data from Lots A and C and from Lots B and D are averaged and set forth in this table under the descriptive heads, horny and "starchy," respectively. TABLE 8. CHEMICAL COMPOSITION OF THE CoRfr, GRAIN, AND SEEDLINGS OF HORNY AND "STARCHY" CORN DURING THE PROCESS OF GERMINATION AT A CONSTANT TEMPERATURE OF 16 C. Material Type of corn Percentage composition of seedlings after being on the germinator for different numbers of days days 2 days 4 days 6 days 8 days 10 days Ether extract Horny. . . . "Starchy" . 4.81 4.96 4.81 4.95 4.74 4.87 4.58 4.64 4.21 4.31 4.03 4.00 Total nitrogen .... Horny. . . . "Starchy" . 1.94 1.89 1.94 1.85 1.94 1.88 1.93 1.85 1.90 1.79 1.91 1.85 Soluble nitrogen. . . Total sugar Horny. . . . "Starchy" . Horny. . . . "Starchy" . .24 .25 1.27 1.26 .19 .20 1.34 1.26 .21 .24 ' 1.66 1.27 .33 .35 2.91 3.00 .41 .42 3.87 3.42 .53 .53 5.66 6.10 Reducing sugar. . . Horny. . . . "Starchy" . .15 .02 .68 .57 1.47 1.04 1.84 1.92 2.53 2.40 3.71 3.98 Dextrins and solu- ble starch Horny. . . . "Starchy". Horny. . . . "Starchy" . 2.73 2.90 51.52 50.91 2.67 1.92 50.16 50.45 2.92 2.52 50.54 50.82 3.22 2.41 49.49 50.06 3.76 2.64 47.23 47.78 3.20 2.45 45.57 46.48 Insoluble starch . . . Hemicellulose Horny. . . . "Starchy" . 6.96 7.15 7.12 7.34 7.13 7.51 7.10 7.48 7.26 7.34 7.35 7.82 From the second day to the end of the period there was a grad- ual but distinct reduction in the percentage of ether extract in both the horny and the "starchy" groups. This decrease is illustrated graphically in Fig. 5. These data harmonize with the results obtained by Toole, 37 who found that the fat in the maize embryo was rapidly oxidized during the germinative process. The fact that the "starchy" corn possessed a slightly greater per- centage of ether extract than the horny ears should not be taken as of any particular significance, for the "starchy" Lot B contained some- what less fat than either Lots A or C. Lot D, however, was abnor- mally high in fat, and this contributed to the slightly superior average INFLUENCE OF PLANT INJURY ON COMPOSITION OF CORN 271 of the "starchy" group over the horny group. It is also probable that the more rapid exhaustion of the fat of the "starchy" kernels is of doubtful import, since the differences between these percentages are well within the error common to these determinations. There was a very slight, tho fluctuating, reduction of total nitro- gen during germination. The proportion of soluble nitrogen, however, FIG. 4. APPROXIMATE AVERAGE GROWTH OF THE SEEDLINGS TAKEN FROM THE GERMINATOR AT TWO-DAY INTERVALS 5.00 4.75 4.50 4.25 4.00 - *>Sf-arch y" corn -Horny corn Days on cferminator at /6"C FIG. 5. PERCENTAGES OF ETHER EXTRACT IN HORNY AND IN "STARCHY" CORN AT DIFFERENT STAGES OF GERMINATION 272 BULLETIN No. 284 [December, after a small decrease at the end of the second day, increased appre- ciably thereafter. It is worthy of note that the soluble nitrogen was consistently higher in the "starchy" than in the horny corn. When the soluble nitrogen was calculated as a percentage of the total nitrogen, the greater solubility of the nitrogen of the "starchy" type of corn is more strikingly emphasized. Fig. 6 was prepared from such a calcula- tion. Percentage of nitrogen that was water so/ub/e * fe <5 5 % *< /' // ~ IS 1 Tarchy" corn >rny corn > / / f ''A f / * / t / H / / / , / / / / / / / / / / / / / / / / / ^ON ^.x ^** / ) 2 4 C 6 /< Days on cjerminator at /6"C FIG. 6. PROPORTIONS OF TOTAL NITROGEN THAT WERE WATER- SOLUBLE IN HORNY AND IN "STARCHY" CORN AT DIF- FERENT STAGES OF GERMINATION A larger quantity of the nitrogen contained in the "starchy" corn was in a soluble form than that in the horny corn. In this respect the "starchy" corn resembles grain lacking in maturity. There is the suggestion that the nitro- gen of the "starchy" corn is more readily converted to the soluble form during the process of germination than is the case with horny corn. The amount of total sugar in the horny corn increased from the very beginning of germination. The rise was more gradual, however, during the early than during the later stages of germination. The in- crease of sugar in the "starchy" corn was initiated somewhat more slowly than that in the horny corn, altho at the end of the sixth day the amount of total sugar in the "starchy" corn slightly exceeded that INFLUENCE OF PLANT INJURY ON COMPOSITION OF CORN 273 in the horny corn. A comparison of these two types of corn in respect to total sugar content may be made from Fig. 7. The increase in reducing sugar was much more marked from the very beginning of germination than was the case with total sugar. At the end of the second day, when the total sugar was increased only .07 I c: "t! 6 5 4 3 2 ( Joto ' \ Horni. / corn hy corn i [ Horntj i corn -hy corn / /..- i , i ^. / k ^^ ^/^ ir / ^ '"' 924 6 8 /(. Days on germ/nator af /6C FIG. 7. PERCENTAGES OF TOTAL AND REDUCING SUGARS IN HORNY AND IN "STARCHY" CORN AT DIFFERENT STAGES OF GERMINATION There was no uniform difference in the sugar content of the horny and starchy corn during the process of ger- mination. On the 6th and 10th days of germination the percentages both of total and of reducing sugars were higher in the "starchy" corn than in the horny corn. The reverse was true at the beginning of the test and at the end of the 2d, 4th, and 8th days of germination. of one percent in the horny corn, and not at all in the "starchy" corn, the amount of reducing sugar was multiplied 4% times in the horny 274 BULLETIN No. 284 [December, corn and 28 times in the "starchy" corn. Fig. 7 shows that the rela- tion of the quantity of reducing sugar in the two types of corn was roughly the same as that of the total sugar. It is evident from this chart that the proportion of reducing sugar to total sugar increased markedly until the fourth day of germination, after which time there was a reduction in this proportion. The ratio of reducing sugar to total sugar approached the 65-percent mark on the sixth day of germination, and remained there with minor variations to the end of the period. It is significant that the soluble starch and dextrins were consist- ently higher in the horny group than in the "starchy" lot. The quan- tity of these materials was somewhat greater in the "starchy" lots, So/ub/e starch and dextrins in percent ^ I\J Co -t HA rny corn jrchy'corn ^ \ ._"