SB 608 G6 D6 1922 Copy 1 FACTORS INFLUENCING THE PATHOGENICITY OF HELMINTHOSPORIUM SATIVUM A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY LOUISE DOSDALL, M.A. IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY JUNE, 1922 FACTORS INFLUENCING THE PATHOGENICITY OF HELMINTHOSPORIUM SATIVUM A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY LOUISE DOSDALL, M.A. IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY JUNE, 1922 CONTENTS Page Introduction and historical review 3 Problem 4 Methods 5 Source of pr.thogene 5 Selection of host varieties 6 Check plants 6 Specific identity of the pathogene 7 Temperature relations 16 Growth of fungus on potato dextrose agar 16 Spore germination 18 Effect of hydrogen-ion concentration and temperature on spore germination 21 Infection 25 Influence of type of soil 27 Influence of soil moisture 31 Influence of soil fertility 39 Comparison of several root-rot causing organisms 40 Summrry and conclusions 44 Literature cited 47 ILLUSTRATIONS Fig. 1. Types of curves obtained from measuring length of snores of Helminthosporium sativum produced on potato dextrose 11 Fig. 2. Length of spores produced on potato dextrose agar at various temperatures 15 Fig. 3. Length of spores produced on different substrata at 24 C 16 Fig. 4. Growth of H. sativum on potato dextrose agar in Petri dishes 17 Fig. 5. Daily rate of growth of H. sativum on potato dextrose agar in Petri dishes 18 Fig. 6. Percentage germination of spores in phosphoric acid-potassium hydroxide solutions of various hydrogen-ion concentrations 23 Fig. 7. Percentage germination of spores in Czapek's solution minus the sugar at various hydrogen-ion concentrations 24 Plate I. Helminthosporium sativum P.K.B. grown on potato dextrose agar at different temperatures 49 Plate II. Helminthosporium sativum P.K.B. grown on potato dextrose agar at different temperatures 50 Plate III. Marquis wheat showing effect of Helminthosporium root-rot in different soils 51 Plate IV. Lion barley plants, 3 weeks old, growing on soils inoculated with various organisms 52 Plate V. Lion barley plants 3 weeks old showing effect of soil organisms on development of root systems 53 Plate VI. Lion barley pbnts, 2,Vz weeks old, showing effect of root infection by H. sativum P.K.B 54 f LIBRARY OF CONGRESS „, I KIOUV6D •"May 2 4 1924 Idocum ENTS DIVISION FACTORS INFLUENCING THE PATHOGENICITY OF HELMINTHOSPORIUM SATIVUM By Louise Dosdall! INTRODUCTION AND HISTORICAL REVIEW In 1 910 Pammel, King and Bakke (9) described a new Helmin- thosporium disease of barley which they called "late blight." The causal organism was named Helminthosporium sativum n. sp. Pammel, et al., had observed the disease in Iowa in 1907 and 1908. In 1909 it was very serious. In the same year, they report that it was also found in South Dakota, Minnesota, and Saskatchewan. These authors de- scribe the disease as follows : "Brown spots of irregular outline occur upon the leaves causing them to turn brown. The leaves are easily broken up, and in some cases completely destroyed. The disease also occurs upon the glumes, spikelets and seed. The straw at harvest is dull brown, and instead of standing erect becomes a tangled mass. The date of ripening of the grain corresponds with the time of full development of the late blight." They observed that there was con- siderable difference in varietal susceptibility, the degree of infection ranging from o to 100 per cent. Late blight was considered the most serious disease of barley in Iowa. In 1913 A. G. Johnson (7) differentiated clearly the three Helmin- thosporium diseases of barley in Wisconsin, and he designated the one caused by H. sativum P.K.B., the "American blotch disease." Louise Stakman (n), in 1920, showed that a Helminthosporium similar to the organism described as H. sativum by Pammel, King and Bakke, but isolated from various parts of diseased wheat and rye plants, was capable of causing a serious seedling blight of these hosts, and could also attack the older parts of the plants, namely, the leaves, nodes, culms, roots, glumes, and grains. In addition to wheat and rye, successful infections were obtained on barley and a number of grasses. In the spring and early summer of 1919, serious attacks of seedling blight caused by Helminthosporium occurred in practically all the wheat-growing regions of Minnesota. F. L. Stevens (12), also in 1920, reported that a species of Helminthosporium was constantly associated with foot rot disease of wheat in Madison County, 111. Inoculations with the organism gave positive results. He concluded that Helminthosporium was the cause of the disease. 1 The writer wishes to express her appreciation to Dr. E. C. Stakman, under whom the work was done, for advice and criticism, and to Mr. M. N. Levine for his helpful criticism in the presentation of the biometrical studies. 4 TECHNICAL BULLETIN 17 In January, 1922, Hamblin (5) reported a Helminthosporium foot- rot disease of wheat in New South Wales, Australia. The disease symptoms are very similar to those of the true take-all caused by Ophiobolus graminis Saccardo, but there are distinguishing characters. Hamblin's description of the foot-rot in Australia corresponds very closely with that of Mrs. Stakman and of F. L. Stevens. His descrip- tion of the poorly developed root system with an abnormal develop- ment of root hairs close to the culm, giving the dead or dying root a "fuzzy" appearance, and the frequent growth of secondary roots above the first node of the affected straws, applies equally well to conditions observed in Minnesota during 1921. In Hamblin's opinion, the Hel- minthosporium disease was responsible for far more damage in 1921 in Australia than was the better known take-all. In recent years, a foot-rot disease of cereals, particularly wheat, rye, and barley, has been destructive in certain localities in Minnesota. This was especially true on certain peat lands in Anoka and St. Louis counties and on some of the sandy soils in Anoka, Nicollet, and Mahnomen counties. A Helminthosporium of the sativum type has been consistently isolated from the diseased plants. This organism is very widely distributed throughout the cereal growing region. The severity of its attack apparently must be greatly influenced by eco- logical conditions. In order to obtain more detailed and accurate in- formation concerning these conditions, a study of the physiology of the fungus, to the extent of its possible correlation with the pathogenicity under given conditions, was undertaken. PROBLEM In this study attention was directed primarily to the root- and foot- rots caused by II sativum. Little attention was given to secondary in- fections on leaves and heads. The soil environment was, therefore, of chief concern. In analyzing the factors which might influence the development of a disease of this type, temperature, moisture, and acidity would affect the growth of both the pathogene and the host,< and possibly also the reaction between the two. The vigor of the host conceivably might greatly influence the development of a disease caused by a facultative parasite, such as H. sativum. The type of soil in which they grew and the available nutriment might, therefore, change the balance between host and pathogene. It is difficult to separate and analyze the individual factors, because certain combinations intro- duce various complexities which are difficult to interpret. The following phases of the problem were investigated especially : 1. Relation of temperature to the growth of the fungus, to spore germination, to infection, and to the development of the disease. PATHOGENICITY OF H. SATIVUM 5 2. Relation of hydrogen-ion concentration and temperature to spore germination. 3. Development of the disease in various types of soil. 4. Influence of soil moisture on the development of the disease. 5. Influence of soil fertilization on the development of the disease. 6. Comparison of several root-rotting organisms. 7. Morphological variation in the fungus with regard to its specific identity. METHODS SOURCE OF PATHOGENE During the spring and summer of 1920, tissue cultures were made from lesions caused by Helminthosporium on cereals and grasses. Twenty-two strains (isolations from various parts of different hosts or from different localities) of the sativum type were obtained from the roots, stems, nodes, leaves, and kernels of barley ; from the roots, stems, leaves, and kernels of wheat ; and from leaf spots of various grasses. Material was obtained from Anoka, Clay, Mahnomen, Nicol- let, Ramsey, and St. Louis counties in Minnesota ; from Tennessee, and from Spruce Grove and Edmonton, Alberta. Seven of these strains were selected for preliminary inoculation experiments. As a virulent root-rotting organism was desired, only soil inoculations were made. Four-inch pots filled with soil were treated with live steam for two hours on each of three successive days. Six pots of such soil were inoculated with each of the various strains of H clminthosporum. For this purpose, spores were scraped from the surface of potato dextrose agar cultures and mixed with water. The suspension of spores was poured over the soil, and the pots were in- cubated for several days. Three pots which had been inoculated with each strain were then sowed with Marquis wheat and three with Manchuria barley (Minn. 105). Some infection was obtained in each case, on both the leaves and the roots. (The check plants were slightly infected, as the seed had not been treated.) The plants inoculated with strain 82a, however, were decidedly more heavily attacked than the others. This was especially true of the barley plants. A Helmin- thosporium of the sativum type was re-isolated from lesions on both the barley and the wheat. A single spore culture was then made from the original 82a culture, and all subsequent work was done with this single spore strain. Culture 82a was originally isolated from the darkened base of badly stunted barley plants sent to the laboratory from the peat plots on the Fens experimental field, St. Louis County, Minn., in the summer 6 TECHNICAL BULLETIN 17 of 1920. A similar Helminthosporium was isolated from the nodes, sheaths, and blades of the same plants. In addition to H. sativum, Alternaria was frequently obtained from blackened kernels and nodes; a pink Fusarium was sometimes found on the base of the stem and roots; and Helminthosporium teres Sace. was occasionally isolated from the leaves and stems. SELECTION OF HOST VARIETIES In all experiments, the effect of the fungus on wheat and barley only was studied. In most cases where barley was tested, both Man- churia (Minn. 105) and Lion (Selection) were used. Manchuria is the barley most commonly grown in Minnesota. It is somewhat re- sistant to H. sativum, as shown by the work of Pammel, King and Bakke (9), of Hayes and Stakman (6), and of Christensen (3). For this reason it was used in the breeding work of Hayes and Stakman. It was crossed with the smooth-awned Lion, which is very susceptible to Helminthosporium, in an attempt to obtain a high yielding, smooth- awned, resistant variety. Marquis (Minn. 1239) was used in most of the experiments with wheat. In some cases Haynes Bluestem (Minn. 169) also was used. CHECK PLANTS Since it is difficult to obtain seed entirely free from Helmintho- sporium, especially in susceptible varieties, it was necessary to treat the seed in order to reduce infection in the check plants to a minimum. Silver nitrate was found to be the most useful disinfectant because the seed coats of both barley and wheat are impermeable to it (10), and the seed can be soaked for a long time in the solution without being injured. It also is more effective, especially against Helmintho- sporium, than mercuric bichloride. For experimental purposes, the method of seed treatment followed was essentially that recommended by Schroeder (10). The seed was dipped in 50 per cent alcohol to remove the air from the surface, soaked over night in X 100 silver nitrate solution, dipped in a dilute sodium chloride solution to precipi- tate as insoluble silver chloride the silver nitrate remaining on the surface of the seed, washed thoroly in running tap water, and dried. Such treatment reduced the germination of Lion barley from 90 per cent to 78 per cent, and of Marquis wheat from 99 per cent to 97 per cent. PATHOGENICITY OF H. SATIVUM 7 SPECIFIC IDENTITY OF THE PATHOGENE Three species of Helminthosporium are known to occur on barley in the United States. These are readily distinguished on the host by the symptoms. II. gramineum Rabh. causes the systemic stripe disease characterized by long, narrow, yellowish to brownish spots on the leaves and sheaths. Many spots often coalesce to form parallel stria- tions which run more or less the entire length of the blade and often down the sheath. Eventually the leaves may be reduced to shreds. II. teres. Sacc. and IT. sativum P.K.B. both cause local lesions which are characterized by peculiar blotches on the leaves. H. teres causes the European blotch or net blotch disease. The spots are yellowish brown in color, irregular in shape, and are scattered on the leaves. When held to the light, a characteristic net work is apparent. H. sativum causes the spot blotch disease characterized by irregular red- dish brown spots on the leaves. The spots are usually longer than they are broad, and, when abundant, may tend to form stripes. These three species also may be distinguished readily by their growth on potato dextrose agar. H. gramineum grows slowly, forms a fluffy, aerial mycelium which does not sporulate (at least not readily), and usually gives the medium a reddish or purplish tinge. H . teres also grows rather slowly. The mycelium grows very close to the surface of the agar. The color of the reverse side of the colony is greenish black. Grayish white tufts of mycelium are formed irregu- larly on the surface of the colony. Cylindrical, thin-walled spores are formed, but usually they are not abundant. In contrast to both these species, H . sativum grows very readily and sporulates abundantly, forming a flat, black or greenish black colony on agar. The abundance of conidia gives the surface a powdery appearance. Organisms similar to the one isolated from typical barley spot blotch have been isolated hundreds of times by workers in this laboratory from various parts of barley, wheat, and rye plants, and from numerous grasses. Pammel, King and Bakke (9) described the spores as cvlindric in shape, straight or curved, slender, widest at the middle, from 105 to 130 microns in length by 15 to 20 microns in width, pale greenish gray to dark brown in color, with 7 to 14 cells. Later workers have found much shorter spores, altho observations on shape agree fairly well. Johnson (j) states that the spores are narrowly spindle-shaped, usually more or less curved. Mrs. Stakman ( 1 1 ) describes the spores of the organism with which she worked as either straight or curved, dark blue-green to brown in color, averaging 41 by 20 microns in size, and containing from 3 to 8 septa. Two types were isolated from dis- eased wheat : one a fuscous type measuring 35 by 22 microns and containing from 3 to 4 septa ; the other straw-colored to fuscous, 8 TECHNICAL BULLETIN 17 measuring 60 by 20 microns, and containing from 4 to 7 septa. Both of the latter are described as elliptical in shape. Stevens (12) makes the following statement regarding the form causing the foot-rot of wheat : "The spores, observed as grown on autoclaved wheat leaves or stems in humid air, are from 24 to 122 microns long, the majority of them falling within the limits 80 to 90 microns, with septa or pseudo-septa varying from o to 13, usually 5 to 10. The spores are usually typically thickest in the region about midway between the base and the middle point of the spore, approach- ing a narrow or broadly elliptical shape, tapering somewhat toward each end. They possess an outer dark wall that is thin and extremely fragile and an inner, colorless, thick wall that is frequently soft and gelatinous . . . The spores usually, perhaps always, germinate either from one or both ends, not laterally, and are functionally only one-celled." After making a large number of isolations from H clminthosporimn lesions on barley, wheat, and rye, great variations were found in the size of the spores of the various cultures, altho they resembled each other more or less in shape and color. In order to find out just what variations might be expected in one strain, as a guide to the interpreta- tion of the species, a single spore was again isolated from culture 82a and a biometric study was made of the spores produced under various conditions. The single spore was planted on a potato dextrose agar slant and incubated at 24 C. for ten days. Transfers were then made to potato dextrose agar and to ripe autoclaved barley heads. Agar cultures were incubated at the following temperatures: 5 , 14 , 18 , 24 , 28 , 32 , and 36 C. The cultures grown at 5 and 36 did not produce spores. The barley head cultures were incubated at 24 C. Fresh barley leaves were taken from the greenhouse, placed in moist chambers, inoculated with spores of the same culture, and incubated at 24 . The length of time required for the cultures to sporulate at the different temperatures varied considerably; those at 24 , 28 , and 32 were ready for measurement in 16 days, while those at 14 required 37 days. \o 00 «* 1". o\ M .-. u-, r^ CO in n to O M m o -f O -t- o 11 +1 " +i " +1 W +1 3 O G\ o -t M n vo *£> -1- CO <«i en N 1 o -t O -+ O it O *+l " +1 ~+l " +1 3 in •+ r^ VD in r^ * •5 CO -f m *o -+ O ro 1 o t-« o 0\ O G\ O J +1 1/1 +1 w +| "■ +1 O oj d E £ 3 S * 1 1 K L-i_ 1 i / \ 7 7 / \\l i J f KlCi T}e(ni\ i Irlrritj ._ i ""4^., 7 / V *"\ >\i 4j / / ^ ^ j Vi V t -j t J ^ JUi J — *- ^ / \Il4 1 L > L4lX io tt ^ J / \aX It \ I ... i nfn tt y^ — j |i — m ^ jri ~\ih ~u It ti J It aT- II / I T'"Z t ! \\ " H \ f || j 1 V - t X — - -- -- .- j ill ' i 5ixe p. 10 20 ^o 4o 5o Co 70 So Qo 100 Fig. 1. Types of Curves Obtained from Measuring the Length of ioo, 300, 500, and 1000 Spores of Helminthospormm sativum Produced on Potato Dextrose Agar at 32° C. 12 TECHNICAL BULLETIN 17 a normal one. In general contour the 300-spore curve and the 500- spore curve approach the 1000-spore curve, altho the first is somewhat more irregular. The slight rise at the lower extreme indicates that the short spores tend to group themselves about a mode of their own. It is possible that improvement in the method of sampling might in- crease the accuracy of the results obtained from a smaller population. In the present study about 100 spores were measured from one mount. The spores were distributed as evenly as possible in the drop of water and each spore was measured in passing systematically over the slide from the upper left to the lower right hand corner. An attempt was made to make the mount so that two or three spores would come into the field at once. For all other conditions, 500 spores were measured. Results obtained in the study of the morphology of spores de- veloped on potato dextrose agar at various temperatures are interesting. Table III shows very little difference in the means of spores de- veloped at 18 and 24 . From the comparisons in Table IV it is seen that these differences are insignificant. If, however, we examine the coefficients of variability, we find that there is a significant difference in the amount of relative variation in the length of spores. This fact is very clearly brought out in the curves in Figure 2. The degree of variation is not increased by a temperature 4 degrees lower (14 C), but the mean length of the spores is slightly increased. This may be due to the fact that at a lower temperature the black outer wall on the spores and mycelium is laid down much more slowly, so that the spores have a longer time in which to form. This is further sub- stantiated by the fact that at 32 ° the spores are very much shorter. The amount of relative variation is practically the same as at the lower extreme. These differences in length of spores produced at various temperatures are graphically represented by the curves in Figure 2. The most striking difference in spore morphology was obtained by comparing the spores produced on different media. As the fresh leaf and the autoclaved head cultures were incubated at 24 C, we may compare these results with those obtained from the agar culture at 24 C. Comparing first the spores from the head and from the agar, we find that the former are slightly longer. The amount of relative variation in the two is practically the same. On the fresh leaves, however, the spores are very much longer and decidedly more uniform. 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E 2 ° s « i; ° r T 2 S * 2 ^ < 6 > a u > 8 36 TECHNICAL BULLETIN 17 An attempt was made to check up the moisture relations of the disease under field conditions. In a series of six square-rod plots, three were planted to barley and three to wheat. The north half of the barley plots was planted with Manchuria and the south half with Lion; the north half of the wheat plots was planted with Marquis, the south with Bluestem. A field drill was used for planting. Several days before planting, the soil was inoculated by applying //. sativum grown on sterilized wheat seed. One half gallon of the culture was applied to each square rod. After the seed was planted, one of the barley plots and one of the wheat plots was sprinkled for ten minutes each morning and evening, a second of each was sprinkled for five minutes each morn- ing and evening, and to the third no water was added. During the first part of the season, very little infection appeared on any of the plants except Lion barley. The weather was very cold during the first two weeks after seeding and scarcely any infection occurred. There was no seedling blight on the unwatered plot, very little on the moder- ately watered plot, and only a moderate amount on the heavily watered plot. Infection occurred mainly on the above-ground parts, resulting in leaf spots and lesions on the sheaths. Less than 1 per cent of the plants were killed. After the first two weeks, the amount of infection increased very rapidly on the heavily watered plot, so that at the end of six weeks 100 per cent of the plants were infected, and most of them were severely attacked. On the moderately watered plot about 80 per cent of the plants were infected, the severity of the infection ranging from light to moderate plus. On the unwatered plot, about 50 per cent of the plants were infected, the severity of the infection ranging from light to moderate. The method of applying the water tended to keep a film of moisture on the lower, shaded leaves, forming almost a moist chamber near the surface of the soil. The sprinkling also offered a good opportunity for the spores to be splashed from the soil onto the leaves. During June, the weather was very hot. As a result of this combination of circum- stances, the plants on the heavily watered plot were literally covered with 77. sativum lesions. In many cases the plants were so badly in- fected at the base that they rotted off. This was not true to such a marked extent on the moderately watered plot owing, probably, to the fact that the surface of the soil was not kept wet enough to maintain a more or less constant layer of moisture just above the surface of the ground. The Manchuria barley was moderately affected, but the wheats only slightly. PATHOGENICITY OF H. SATIVUM 37 Final data were taken just before the heads ripened. An attempt was made to obtain a quantitative estimate of the percentage of plants. infected for the whole plot and an average of the degree of infection on the roots, foot, and node from individual plants. For this purpose, approximately equally large groups of plants were dug from the center, and also from each corner of the plots, two feet from the margins. Ten plants were taken from each group and for these fifty plants, the following data were recorded : the number of culms which headed ; the number of tillers which did not head ; the degree of infection — indicated as heavy, moderate, or light on the roots, foot, and nodes. Finally, the seed from each plot was weighed after threshing and the yield per acre was calculated from this. These data are summarized in Table XI. In order to obtain a simple mathematical expression for making com- parisons, the percentage of heavy infections was multiplied by 10, the moderate by 5, and the light by I, and the sum was taken as the index of the total infection. In order more easily to make comparisons, these sums were reduced to unity. Finally, these expressions for root and foot infections were totaled to obtain a means of comparing the com- bined foot- and root-rot with the relative amount of soil moisture and with the yield. For the sake of comparison these were also reduced to unity. In some cases the Helminthosporium infections were so com- plicated by Fusarium infections that it is quite impossible to say how much of the damage was due to each organism. This was especially true on the Manchuria barley. In general, the amount of injur}- was small. Altho the root-rot and the basal infection as measured by the degree of browning was sometimes heavy on a large number of plants, the plants were not noticeably stunted or immature as is often the case in severe cases of foot-rot. On the whole, there was more foot- and root-rot on the barley than on the wheat. The Lion barley alone shows an increase in the amount of foot-rot as the relative amount of soil moisture is increased. The differences are so small, however, that they can not have much significance. There were no indications of a correla- tion in yield with either the relative amount of soil moisture or the amount of foot- and root-rot. For both varieties of wheat, foot- and root-rot was slightly worse on the driest plot. In no case did the rela- tive amount of soil moisture or infection influence the tillering of the plants. tf I I I '" fa .5 3 o >> 01 ^D ++ O M •H- co in M in r^ Ov O " M " M S3 *_ H 2 s CO 00 CO t 4 3 H 10 6?J ° ° <£ 2 n * 00 c : -- * 4 -- <5> 5 s a s * ^ •2 2 t^ffi CN s § M 3 £ 10 is - 1 M 1 30 1 O 1 O H H N " " O fa I"" 1 O CO 1 ° 1 1 >o 1 s 1 5- 1 il \n 12 IR O 3- S ! 5. '--- IN 1% 1 ^ 1 3. 1 S 1 ■*■ 1 r ' 1° IS 1* * 1* E^E | CO 1 n I 1 ! 1 1° 1° 1° 1° 1 ° v-\ I" I 2 Immature ey CO VO * * •? X) vo Oi •> 00 V 3 00 ?; 00 ■* CO ■ M + IT + + + + + + + + + 2 y >> . "3 "S ^ n, ° CU jii ^ a s>, h +3 ? • - " - « g £ g U 3 r- • - fl -S3 ■300. E "0 o ■- a £ £ 2 ■^ 33 -•-• rt PATHOGENICITY OF H. SATIVUM 39 Many factors that are difficult to control enter into field experiments and complicate the results in such a way as to make them indicative rather than conclusive. In these experiments foot- and root-rot de- veloped slightly more vigorously on Lion barley in the wettest soil and on the two varieties of wheat in the driest soil. This may be only a confirmation of the earlier greenhouse experience that root infection tends to be worse under either extremely dry or extremely wet conditions. INFLUENCE OF SOIL FERTILITY The effect of soil fertilization on the development of foot- and root- rot caused by H. sativum was studied in field plots on Lion and Man- churia barley, and on Marquis and Bluestem wheat. Potassium and nitrogen in the form of muriate of potash and nitrate of soda were added to square-rod plots at the rate of 600 and 300 pounds of fertilizer to the acre. Treble superphosphate was added at the rate of 200 and 100 pounds. These fertilizers were so applied that there were plots with a heavy and a light application of each alone and in combination with a heavy and light application of each of the others, except that there were no combinations of nitrogen and potassium. In addition to these, complete fertilizer was applied at the rate of 600 and 300 pounds and manure at the rate of 20 tons and 10 tons per acre. Unfertilized plots were left as checks. All the plots were run in duplicate, one series planted with wheat and one with barley. The north half of the wheat plots was planted with Marquis, the south half with Bluestem, the north half of the barley plots with Lion, the south half with Manchuria. Several days before planting, H. sativum grown on sterilized wheat seed was applied on the surface of the soil at the rate of one half gallon of the culture to the square rod. The plots were seeded with a field drill, wheat at the rate of 90 pounds to the acre, and barley at the rate of 86 pounds. This is the normal rate of seeding for this section of the country. Practically no seedling blight developed on any of the plots. Leaf lesions and foot-rot first appeared on the barley during the second and third weeks, and soon after lesions developed also on the wheat. There was considerable difference in the vigor and height of plants on the different plots in response to the different fertilizers. During the latter part of the season, there were differences in the amount of lodging on the various plots. Final data on the amount of foot- and root-rot were taken just previous to the ripening of the grain. In order to obtain an approximately quantitative expression for the amount of infection in each plot. 50 plants were selected from each half square rod, 10 from each corner, two feet in 'from the margins, and 10 from the center of the plot. For each of these plants the following data 40 TECHNICAL BULLETIN 17 were recorded : the number of culms which developed heads ; the num- ber of tillers which did not mature; the degree of infection (designated as heavy, moderate, or light) on the roots, the foot, and the nodes. After harvesting, the weight of the straw and of the threshed grain was recorded, and from this the yield per acre was calculated. The infection of Lion barley was slightly more pronounced than on the other hosts. These data for Lion barley are summarized in Table XII. In order to arrive at a simple factor which would express the total infection for the roots, the foot, and the nodes for a single plot and would also take into account the severity of infection as well as the percentage of plants infected, the number of heavy infections was multiplied by 10, the number of moderate infections by 5, and the num- ber of light infections by 1, and the three products were summed. This was taken as an arbitrary index of the infection. In order to make comparison more simple, these summations were reduced to unity by dividing each by the lowest sum. This is the factor designated as total infection in Table XII. In order to compare the combined effect of root- and foot-rot, the summation for each was added and these sums in turn were reduced to unity. In order to bring out the relation of infection to fertilizers and yields, the arbitrary indices of infection were grouped into three classes and the yields per acre into three classes, and the fertilizer plots were arranged according to their infection and yield in the various classes as shown in Table XIII. From this summary table it is quite clear that the amount of foot- and root-rot is not correlated with any particular fertilizer. The disease did not appear in its severest form on any of the plots. Under the conditions of this experiment, there was no evidence of severe stunting of the plants or of excessive tillering. COMPARISON OF SEVERAL ROOT-ROT CAUSING ORGANISMS In order to obtain comparative results on the pathogenic effect of different soil organisms on Marquis wheat and Lion barley, a culture of Helminthosporium isolated from the foot-rot of wheat in Illinois by F. L. Stevens and a culture of Fusarium culmorwm (W. Sm.) Saccardo isolated from scabby wheat, were compared with the Helmin- thosporium sativum isolated from barley foot-rot in Minnesota. £ 0tOTt- >n ^t" m CO SO "1 H N t n a o vUh-| I JO 00 -cf so 00 -cj- 00 O PI -tOO Tt-PISO O t SO OsOsO Si '"^ -~ l/~, CO N — — — -I CI (SI m M lv IM N O t-v SO CO ,3j 1 00 00 B- ! CO co P) O 00 P4 PI O W O SO so -*■ CO SO SO ~ V. 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