" ' '- GIFT OF Professor W*ASetchell BIOLOGY LIBRARY W1LUAM A. SETCHELL STATE OF ILLINOIS DEPARTMENT OF REGISTRATION AND EDUCATION DIVISION OF THE NATURAL HISTORY SURVEY STEPHEN A. FORBES, Chief Vol. XIV. BULLETIN Article V. The Helminthosporium Foot-rot of Wheat, with Observations on the Morphology of Helminthosporium and on the Occurrence of Saltation in the Genus BY F. L. STEVENS PRINTED BY AUTHORITY OF THE STATE OF ILLINOIS URBANA, ILLINOIS June, 1922 Original isolations of the foot-rot Helminthosporium made in May, 1919, from bits of tissue from wheat grown in Madison county, Illinois. STATE OF ILLINOIS DEPARTMENT OF REGISTRATION AND EDUCATION DIVISION OF NATURAL HISTORY SURVEY STEPHEN A. FORBES, Cheif Vol. XIV. BULLETIN Article V, The Helminthosporium Foot-rot of Wheat, with Observations on the Morphology of Helminthosporium and on the Occurrence of Saltation in the Genus BY F. L. STEVENS PRINTED BY AUTHORITY OF THE STATE OF ILLINOIS URBANA, ILLINOIS June, 1922 LIBRA*! a GIFT OF BIOLuuY STATE OF ILLINOIS DEPARTMENT OF REGISTRATION AND EDUCATION W. H. H. MILLER, Director BOARD OF NATURAL RESOURCES AND CONSERVATION W. H. H. MILLER, Chairman WILLIAM TRELEASE, Biology JOHN M. COULTER, Forestry ROLLIN D. SALISBURY, Geology WILLIAM A. NOYES, Chemistry JOHN W. ALVORD, Engineering KENDRIC C. BABCOCK, Representing the President of the University of Illi- nois THE NATURAL HISTORY SURVEY DIVISION STEPHEN A. FORBES, Chief (52929-1200-7-21) CONTENTS PAGE Introductory 77 I. A foot-rot of wheat and its causal fungus: Symptoms 78 Fungi present 78 Growth of the causal fungus on various media 79 Various agars as media 80 Summary concerning growth on agars 85 Rice and similar substances as media, with special note of color phenomena 86 Summary concerning growth on rice and similar substances 88 Miscellaneous vegetable media 88 Summary concerning the foregoing vegetable media 90 Cereal shoots grown from aseptic seeds as media 90 Environmental factors which induce variation 91 Quantity of nutriment 91 Inhibitory influences . . 93 Humidity of media 93 Humidity of air > 94 Temperature relations 98 Light 99 Carbohydrates 100 Nutrients as affecting conidial length, septation, and shape 101 Summary concerning environmental factors which induce variation . . . 102 Morphology of the foot-rot fungus 103 Mycelium 105 Senescence phenomena of aerial mycelium 107 Conidiophores 109 Conidia 110 Etiology of foot-rot: Evidences of etiological relation of H. No. 1 124 Constant presence of the pathogene 124 Absence of other constant parasites 124 Identity of pathogene proved by culture 124 Evidence of infectiousness 124 Conidia produced in moist-chamber culture 125 Evidence from inoculation 125 Recovery of organism 128 Infection phenomena on wheat 128 Susceptibility of various hosts to infection 137 Summary concerning etiology of foot-rot 139 IV II. Evidence and discussion of the occurrence of saltation within the genus Helmin- thosporium : PAGE Introductory 139 Characters of saltants as shown in transfers 141 Tendencies in saltation 145 Stability of the saltants 145 Stability of the saltants through the conidia 146 Apparent reversions 147 Supposititious causes of the variant sectors 147 Saltations from single conidia 149 Frequency of saltation 150 Saltations occurred on various media 152 Saltations and modifications occurring in test-tube cultures . . . . . . 152 Saltations in nature . 154 Notes concerning selected individual saltants 155 General discussion of saltation 157 Taxonomy 164 Conclusion ^ 168 Summary 168 Literature cited 171 Appendix: Methods 179 List of Helminthosporiums used for purposes of comparison 181 Discussion of foregoing list with several brief descriptions 184 Graphs: Figures A Y. Plates: VII XXXIV. ARTICLE V. The Helminthosporium Foot-rot of Wheat, with Observations on the Morphology of Helminthosporium and on the Occurrence of Saltation in the Genus. By F. L. STEVENS. INTRODUCTCXRIT / , -,^ The present study of wheat disease is^baseti upon Vfo6t-fot/6r rot of the basal portion of the stems, of wheat plants, as it occurred in Madison county, Illinois, in 1919 and subsequently. This disease was first reported in United States Government publications as " take-all" ( Ophiobolus gram- inis] ; later, merely as "take-all," no cause being assigned; and for some time past, in Government publications it has usually been designated as "so- called take-all." An annotated bibliography of nearly one hundred titles concerning foot-rot disease of wheat, prepared by the writer, was presented before the Cereal Pathologists of America at St. Louis in June, 1919, and this, expanded to one hundred and eighty-eight titles, was published in October, 1919 (116). As early as May, 1919, cultural studies quite clearly pointed to Helminthosporium as the true cause of the disease, and at the December meeting of the American Phytopathological Society I announced this fungus as the probable cause. In May, 1920, in a note in Science (117), I published the statement that it had been conclusively established that this foot-rot of wheat is caused by Helminthosporium. One purpose of the present paper is to present the evidence on which the foregoing conclusion is based and certain facts concerning the mor- phology and parasitism of the fungus; but far transcending in interest the disease itself which now appears to be one of much less alarming nature than was at first feared is the fact that very striking phenomena of variability are found in this and related fungi. In the following pages, therefore, appear (I) an account of the Illinois foot-rot of wheat and its causal fungus; and (II) evidence and discussion of the occurrence*of salta- tion within the genus Helminthosporium. ACKNOWLEDGMENTS In this study I have been assisted financially by grants from the Illinois Natural History Survey and from the University of Illinois. I am indebted for specimens to persons mentioned in the list of species used for comparison (pages 181-184), and to W. P. Snyder for compu- tation of data embodied in several of the graphs. I wish also to express my thanks to Prof. J. A. Detlefsen, who kindly read the manuscript and offered valuable suggestions regarding genetic questions. 78 I. A Foot-rot of Wheat and its Causal Fungus SYMPTOMS As the name implies, the most obvious symptom is a rotting of the basal portion of the stem of the wheat plant, that is, the lowesr portion of the lowest internode. In earlier stages than that of actual rotting, minute yellow or brown lesions occur on the stem (PI. VII), while the roots, if diseased, are slightly yellowed and largely or quite devoid of functional root-hairs. No weft of superficial mycelium or black incrus- tation, such as is so frequently described in articles concerning take- all, was seen. The diseased tissues, however, were invariably ramified by an internal mycelium. Certain cases of diseased wheat came under observation in which the plants had attained a nearly normal growth and were eighteen inches high, when they suddenly died throughout. In such cases there was a slight darkening of the lower node and a mycelial invasion at this point. The opinion of those who observed this wheat in the field was that the death was due to frost injury. It is probable that the actual cause of death was foot-rot following the frost injury. FUNGI PRESENT Direct microscopic observation of the diseased tissues, in all cases of foot-rot examined, revealed the presence of an internal hyaline or faintly tinted mycelium in great abundance permeating the diseased tissues. Mycelium of different character was also occasionally found, but so inconstantly as apparently to have no actual relation to the disease. Isolations of the fungi present in the diseased tissues were made by two methods: 1. Direct planting of bits of diseased tissue on poured agar (corn- meal agar or wheat-straw agar). The diseased tissue was secured in as clean condition as possible by stripping back the enclosing sheaths, ex- cising the diseased part with sterile tools, and tearing it apart in a sterile Petri-dish. 2. Direct planting of similar bits of diseased tissue after surface- sterilization in mercuric chloride (1-1000, 10 min.). Dilution plating was unsatisfactory owing to the paucity of conidia and the presence of numerous soil bacteria, particularly "spreaders." As might be expected, the methods employed gave rise to colonies of many genera and species, including Phyllosticta, Septoria, Fusarium, Epicoccum, Alternaria, and Helminthosporium. A striking fact, however, 79 was that with the exception of the Helminthosporium, these fungi were very rarely present, and then only a single colony or part of a mixed colony on occasional plates. Alternaria occurred with remarkable rarity; only two or three colonies among several thousand. Fusarium was found in only a few colonies and so mixed that it was isolated with difficulty. Ep- icoccum occurred in two colonies; Phyllosticta also in two colonies (two species) . A Helminthosporium, however, appeared in every plate and from nearly every bit of tissue used, no matter how great the care in securing the inoculum. On many plates this Helminthosporium (which throughout this article I designate as H. No. 1) appeared in pure culture Thus it may be said that the Helminthosporium was universally present in the plates; that it was the only organism that was present with any constancy; and that all other fungi were obviously strays.* Though conidia were never found in great numbers on plants brought in direct from infested fields, when the plants were placed in moist chamber for two or three days conidia developed in abundance. This was also the case with portions of wheat stems which had been placed in bichloride of mercury for ten minutes and then placed in moist chamber for several days. In passing it may be remarked that although great numbers of nematodes and amebae appeared in the plates there is no reason to believe that they had any relation to the disease under discussion or to any diseased condition. GROWTH OF THE CAUSAL FUNGUS ON VARIOUS MEDIA Since the characters exhibited by various Helminthosporiums when growing in artificial culture have been considered as of importance as a means of distinguishing one species, variety, race, or strain from another, many media were employed in the present study. This was done in part for the purpose of comparing the growth characters of the Helminthosporium with characters reported by others in connection with other forms; in part with the hope that some of the media tested might give emphasis to cer- tain characters and thus serve to differentiate between species or strains of the forms under observation. The following notes are, in the main, statements of the characters presented by the foot-rot Helminthosporium (H. No. 1), though for the purpose of comparison notes are added regarding the growth of several *A letter from Professor Hoffer written in May, 1919, tells me of a similar result from platings of wheat foot-rot from Indiana, and similar reports reach me from several other sources. 80 species or strains of Helminthosporium. These are throughout referred to by number rather than by name, partly for brevity and partly because the species of many of the races had not been determined, while in some cases the names were of more or less doubtful reliability. That the reader may formulate his own judgment of these forms, introduced for comparison, a complete list of them is given in the appendix (pages 181-184) together with certain notes regarding them. VARIOUS AGARS AS MEDIA Corn-meal agar in Petri dishes. This medium, prepared after the di- rections given by Shear and Stevens (104), was found to be admirably suited to Helminthosporium and was the medium chiefly used. The fungus grew rapidly, the colony being at first nearly hyaline both in the submerged and aerial parts, but when a diameter of about 2-3 cm. was attained the whole colony became much darker. Profusion of conidia was the chief factor in giving the dark hue to a colony, the slight darkening of the mycelium having little to do with it. The aerial mycelium varied largely with change of conditions, sometimes being very scant and at other times 5-6 mm. high, with windrow effects corresponding with the zones. After the colony was about 3 cm. in diameter zonation became quite pronounced, the zones corresponding approximately with the growth of each day. At room-temperature the colony attained a diameter of about 4.5 cm. in six days. Conidia-production was quite uniform over the surface of the colony unless checked by some growth- inhibiting cause, as drying, cold, or the antagonism of another colony near by, when it was much increased, as evidenced to the eye by black bands in such regions. By transmitted light the mycelium, and to some extent the conidia at certain ages, had a distinctly greenish tinge. H. No. 1 could be distinguished from H. Nos. 5-8, which were paler and produced fewer conidia. H. No. 6 approached nearer to H. No. 1 in these regards than did the others. H. Nos. 3, 4 (see PI. IX), 6, 15-17, and 18 typically developed more aerial white mycelium than did H. Nos. 1 and 14. H. No. 36 was of very distinct character owing to large development of aerial mycelium (see PI. X). Corn-meal agar in Freudenreich flasks. The flasks, of about 100 c.c. capacity, each received 50 c.c. of agar and were slanted. The large amount of nutriment available and the sustained moisture gave noteworthy characters. At 7 days, with H. No. 1, the surface of the slant was com- 81 pletely overgrown and of an even black color, largely curtained by an abundant, even overgrowth of white aerial mycelium. At contact with the glass a sharp, black line gave clear evidence of the black surface-coat. No clumps or balls of mycelium were present. At 22 days a few clumps developed, though not so many as on H. Nos. 9, 13-16. Cultures of H. No. 1 on corn-meal agar in large flasks, as those of Kolle or of Piorkowski (PL XI-XIII) gave colonies very different from those on the ordinary Petri dish, due presumably to the larger quantity of nutrient available and to different humidity relations. These flasks gave increased density of colony and conidia-formation, more aerial mycelium, and some clumping of the mycelium. Though colonies of H. No. 3 and H. No. 1 differed in these characters in these flasks (PI. XII, XIII), portions of the colonies of these strains were indistinguishable. Corn-meal agar made at various temperatures. Corn-meal agar was made in the usual manner excepting that the temperature in three cases was held at 43, 85, and 100 respectively, instead of at 60, before filtering. Duplicate plates were made. The four resulting agars are designated according to the temperatures held, and colony data for each are presented in the following table. CORN-MEAL AGAR MADE AT VARIOUS TEMPERATURES Temperature Growth in 8 days Zonation Density Colors 43 43 7.5 7.2 distinct thin pale 60 60 5.5 6 sharp thick dark 85 6.5 In above characters, ranks between 43 and 60 agars 100 100 8 7.8 none very thin very pale The 100 agar is most favorable to linear growth, 43 agar stands next; 43 and 85 agars give growth of poorer color than 60 agar, but 100 agar ranks lowest in this regard. Color is directly due to quantity of conidia, and it is uniform in the mycelium on the four agars. Nutrition in 100 agar was very little better than in plain agar. In general, it 82 appears that their order of nutritive value for this fungus, from poorest to best, is 100, 43, 85, 60. Evidently a temperature of 43 is in- sufficient to extract the nutrient proteids sufficiently, while 100 pre- cipitates too many of them. While leucosin, a prominent proteid of the embryo, is largely precipitated at 52 and a second coaguIuriTgOes down at 82, no more is precipitated even by boiling (Osborne, 89). Graphs 1-4 (Fig. A), indicating conidial length on these four agars, show that although the quantity of conidia produced varied materially, the length and general variability are not greatly influenced by varying the composition of the agar done in this case by change of temperature. The conidial length of all these agars is, however, considerably less than that on wheat shoots (cf. graphs in Fig. A and Fig. K). Graphs of conidial breadth and septation on 43 and 60 agars given in Fig. B also show but little influence of these agars on these two characters. A "Difco" corn- meal agar, prepared according to my directions by the Digestive Ferments Company, gave growth-characters almost identical with those of my own 60 agar. On "Difco" beef-agar the conidia were short, and were frequently deformed (M, 17.44.22, line cd was tangential to the surface of the conidium at the point of maxi- mum diameter, and was parallel to the longitudinal axis of the conidium, the line ef being 3.4 /* from the line cd and parallel to it. Then the points x and y are where the surface-line of the conidium cuts the line ef. It is obvious that as the line xy increases in proportion to the length of the conid- ium, gh, the conidium more nearly approaches the form of a cylinder; and as the line xy becomes proportionately shorter the conidium becomes less xy like a cylinder. The ratio may therefore be termed the coefficient 120 of cylindricity. For these determinations only conidia of approximately modal length were used, and to obviate unconscious selection, measure- ments were made of only the left side of the conidium, the basal end being toward the observer. Determination from 11 conidia of H. No. 1 of sub- cylindrical shape gave a coefficient of .74, while that frorrPST elliptical conidia was .67. The above findings for H. No.*l are as follows: Coefficient of longitudinal eccentricity All conidia 43 Elliptical conidia 42 Subcylindrical conidia 45 Coefficient of cylindricity All conidia 70 Elliptical conidia 67 Subcylindrical conidia 74 Determinations of the coefficient of cylindricity made from drawings of Dr. Ravn (91) gave for H. gramineum and H. avenae respectively .86 and .95, showing a much higher coefficient than is given by any of the forms in my collection. A convenient method of measuring conidia for coefficients is given on page 179 of the appendix. Conidial length. From five separate plates, a, b, c, d, and e, inoculated with H. No. 1 under standard conditions, Graphs 36-40 (Fig. K) of conidial length were made. Two additional graphs were made from plate e, one of which is designated as e 1 '. The data pertaining to these graphs are given with the others (Fig. K). The differences between the means of conidial length on plates a to e and e' are as follows: Plates Differences between means Plates Differences between means a-b +0.70 .21 a-c +0.62 .24 a-d -0.78 .23 a-e +2.03 .24 a-e' +0.50 .16 b-c -0.07 .22 b-d +0.08 .23 b-e' -0.19 .16 c-d +0.16 .23 c-e + 1.40 == .24 c-e' +0.11 .17 d-e +1.24 .25 d-e' -0.28 .18 e-e' -1.52 .19 b-e +1.32 .24 Since the various plantings on these plates were all from the same in- oculum, made at the same time, and under as nearly identical conditions as possible, and so kept, the rather large difference in means seen, particu- 121 larly in a e, b e, c e, d e, and e e' is significant. If plate e be left out of consideration, the others agree reasonably well, with differences greater than the probable error in six out of ten cases, the difference being but slightly above the probable error in two cases, about twice the probable error in two cases; and about thrice that, in two cases, the largest excess, in plates a b, being 0. 70 .21. Plate e deviates widely, with a difference in case of a e of 2.03 24, the difference being more than eight times the probable error. The great difference in plate e must indicate variability of the fungus on this plate (cf.with page 152), modification due to influence of some unknown factor of environment, or error in sampling. But since such a variation did occur in a series of plates made with the greatest care and with the same organ- ism, it is clear that the occurrence of such a difference can not properly be interpreted as meaning specific difference. Data from the combined rec- ords of a, b, c, d, and e' (omitting e as questionable) give the most reliable data I have on length of conidia of H. No. 1 under standard conditions (cf. with Graph 42, Fig. K). To determine how wide a variability occurs in specimens collected in the open, on the natural host, H. ravenelii (PI. XX) a well-marked, easily recog- nized species of wide geographic distribution, growing on Sporobolus, was stud- ied in conidial-length graphs made from specimens listed in connection with the graphs (Fig. L) . The tabulated results of this study of H. ravenelii follow : Nos* 4346 4347 4349 4348 4350 4351 4352 4353 44-^8 4449 4450 4451 4452 4453 4550 4551 4552 4553 Differences between means Nos.* Differences between means 0.48 0.59 0.62 0.62 0.98 1.18 1.94 2.92 0.45 0.46 0.81 1.01 1.77 2.75 0.80 1.00 1.76 2.74 .26 .24 .28 .29 .27 .27 .27 .26 .31 .31 .30 .30 .30 .29 .24 .25 .25 .24 4650 4651 4652 4653 4751 4752 4753 4851 4852 4853 4951 4952 4953 5052 5152 5153 5253 0.50 0.70 1.46 2.43 0.59 1.35 2.33 0.56 1.32 2.30 0.55 1.31 2.29 0.96 .28 .28 .28 .27 .26 .26 .25 .30 .30 .29 .29 .29 .29 .29 0.76 =t .29 1.73 .28 0.97 .28 *For significance of numbers, see Figure L. 122 A difference of 0.97^.28 between the means of two samples from the same specimen (Nos. 52 and 53) a difference more than three times the probable error shows clearly the difficulties of sampling, and that such differences between samples of the same species grown under the same con- ditions may be expected. The differences in several instances, notably between Nos. 44 and 49, 44 and 48, and 46 and 51, are no greater than those between two samples of the same specimen and may well be due to sampling, and to this extent show the fungus, over a wide geographic range, to be remarkably uniform. In several other instances, however, there is a wide difference of means, above the probable error notably in all cases involv- ing sample No. 53. These differences are often four, five, or six times the probable error, and occasionally run as high as eleven or twelve times the probable error even with this remarkably uniform fungus. While these differences may in part be attributed to sampling they probably represent also morphological changes due to environmental differences, and differ- ences of nutrition or humidity, but do not necessarily indicate racial dif- ference in the fungus. To determine whether various cereals, autoclaved, influence conidial length differently, plates of H. No. 1 were prepared under standard condi- tions except that in the same Petri dishes were placed shoots of wheat, rye, barley and corn. The resulting graphs of conidial length are given in Fig. M. The differences in means are as follows: On rye and wheat, 0.40 .29 On rye and corn, 0.45 .20 On wheat and corn, 0.02 .22 The mean length on rye, corn, and barley is in close agreement with that on wheat, and, apparently, under these conditions the species of shoots counts for little in its influence on conidial length. Conidial-length graphs (Fig. N) made from H. No. 1 grown on fresh wheat-stems, on young wheat shoots, on wheat leaves, and on young wheat plants, all autoclaved in test-tubes with a few centimeters of water, show a considerable increase over those under standard conditions (Graph 42, Fig. K) ; also, in Graphs 58, 60, and 61 (Fig. N), they show a much larger standard deviation and coefficient of variability, probably due to the va- riable humidity under these conditions. The small number of conidia measured, and the lack of control over humidity may be presumed to ac- count for such variation as is seen. Live wheat inoculated in rag doll showed at the 6th day 100% infec- tion. These infected seedlings were placed in a Petri dish on moist filter- 123 paper and the conidia allowed to develop to maturity. Conidial length here (Graph 64, Fig. O) was somewhat less than under standard conditions (see Graph 42, Fig. K), and the coefficient of variability was a little high. Conidial breadth.- H. No. 1 was quite constant in conidial breadth as follows: M (7 CV 6.03 .04 0.55 .34 9. 13 .57 The ratio of conidial length to conidial breadth is an important one as determinative of shape. This ratio for H. No. 1 is as follows: mean length 22.62 .05 _ o / 4 ^ . Uo mean breadth 6.03 .04 In a description of H. No. 1, written in May, 1919, for my own use, and prepared with considerably more care than is ordinarily used in specific descriptions of fungi, I noted the conidia as 3 8 septate and as 52.6 67.2 XI 9. 2 24 IJL long on wheat; and as 48 84X18 21.6 /JL on corn-meal agar, whereas my more extended study now shows the mode on wheat as 78.2 /z, the mean as 76.8 M and the range from 34 to 98.6 /r, the breadth as ranging from 17 to 23.8 /-i, with the mean as 20.4 /*; the septa with a mode of 8, a mean of 7.9, and ranging from 4 to 10. I may here note also that Bakke (6) in his description of H. teres gives the conidial dimensions as 150 [or 105*] 130X15 20 ju, and the septa as 7 14. Thus he seems to have found conidia considerably longer than I did, as also narrower ones. It should be said that the data obtained by this study of graphs of H. No. 1, though involving several thousand measurements, fail to record the longest conidium observed, and the one with the most septa, because these were both seen during observations which rendered their inclusion impossible; which is to say that to include them would have been to consciously select these unique conidia for inclusion. Anent the shortcoming of my own brief description cited above may be quoted the Saccardian de- scription of H. ravenelii: "Spongiosum; hyphis flaccidis flexuosis nodosis ramosis, inarticulatis; conidiis cymbiformibus, 3-4 septatis, fuscis, 50 ju longis, endo-chromotibus isthmo connexis." Though the mode is approx- imately at 50-54 IJL the conidia really range from 13 to 7lju (see Fig. L). Very similar errors, due to brevity of description, exist regarding many or all known species. *See Pammel, King, and Bakke (90, p. 180). 124 ETIOLOGY OF FOOT-ROT EVIDENCES OF ETIOLOGICAL RELATION OF H. NO. 1 Constant Presence of the Pathogens In all cases of American foot-rot of wheat that have come under my observation the rotten basal portion of the shoot bore and was to a large extent occupied by a mycelium, which grew luxuriantly within the wheat tissue though very sparfngly upon its surface, coursing lengthwise within the diseased cells. This mycelium was hyaline, septate, vacuolate, irregular in thickness, and, in short, agreed in all characters with those of H. No. 1 when growing in rotting wheat-tissue (page 105). Absence of other Constant Parasites No other organism which might be considered as a possible parasite was present in any large number of cases in or on the wheat tissue. Amebae and nematodes were present in great numbers in the soil, but appeared to bear no relation to the rot of the wheat. Various fungi, as Fusarium (two species), Rhizoctonia, Epicoccum, Alternaria, were occasionally found on the roots or stems, but each only rarely, in a fraction of 1% of the cases, and with no evidence of etiological relation to the rot of the stem. Identity of Pathogene proved by Culture Very numerous isolations were made by taking bits of tissue (1) from diseased sheaths, (2) from diseased stem-lesions, and (3) by stripping away the sheath, disinfecting the remaining surface with mercuric chloride and taking out diseased bits, with precautions against contamination. All such diseased bits were laid on the surface of corn-meal agar plates. Hun- dreds of these were made, with the result that in practically every instance the diseased bit gave rise to Helminthosporium conidia in general aspect like those of H. No. 1. Other organisms, as mentioned, occasionally occurred on these plates, but in only a small per cent, of instances. It seems entirely conclusive that the mycelium constantly found in the rotting basal portion of the diseased w r heat-stems is that of a Helminthosporium. Evidence of Infectiousness Several bags of soil that bore diseased wheat in 1919, near Granite City, Illinois, were brought into our greenhouse in July, 1919. In this soil was planted "Sultzer Pride" wheat, and the planting kept liberally watered. At the end of some weeks the plants were removed, and on examination all 125 showed browning and incipient rot of the basal portion of the stem. Micro- scopic examination and agar platings from these stems gave results identical with those stated above. One plant that was so badly rotted in the pot as to fall over was found bearing Helminthosporium conidia on its surface. Conidia produced in Moist-chamber Culture While stems with diseased lesions, either from the field or greenhouse, when placed in an ordinary moist-chamber rarely gave Helminthosporium conidia (or, if they did, only in small numbers), if the diseased stems were placed on wet filter-paper in moist chamber and rather closely covered with wet filter-paper Helminthosporium conidia invariably developed in quantity on the lesions, the fungus eventually spreading throughout the available wheat-tissue and producing conidia over the whole surface (cf. with page 95). Evidence from Inoculation Severed, live wheat-shoots, grown under aseptic conditions, were placed as under standard conditions (Appendix, page 180), except that the shoots were not autoclaved but put, living, upon the inoculated agar. All such shoots rotted rapidly and completely, the shoot being eventually covered by Helminthosporium conidia. Since direct examination showed no contamination, it is evident that H. No. 1 can cause rot of the wheat tissue. To determine the relative rotting power of this organism and other Helminthosporiums under these conditions, fresh aseptic shoots of corn, wheat, oats, barley, and rye were laid on washed agar with the growing tip toward the circumference of the dish, and the cut end in contact with the outer edge of the spreading mycelium of a colony about 5 cm. in diam- eter. These were examined after 2 days and again after 5 days, and the rate of browning was carefully calculated. In this way seventeen strains of Helminthosporium were tested as to their ability to produce rot in live, severed cereal-shoots. H. No. 1, the foot-rot organism, showed high rotting ability, completely rotting a wheat shoot 11 mm. long in 5 days, while H. No. 2 (H. ravenelii) produced no rot on any cereal. H. No. 1 rotted corn also, but much less rapidly than it did wheat, and its rate on oats, barley, and rye was still less. Several other numbers showed strong rotting power on wheat shoots, notably H. No. 10 (labeled H. teres), isolated by Dr. Stakman from barley, H. No. 9 isolated by him from wheat, and H. No. 13 (labeled H. sativum], isolated by Dr. Durrell from barley. 126 The results from this preliminary work indicate also a very wide dif- ference in the susceptibility of these cereals to rot by the various strains of Helminthosporium. Oats, on the whole, are less injured by them than any of the other four cereals tested. Corn and wheat were most often first in susceptibility to certain of the strains, and were also highly susceptible to more strains than were barley and rye. Seedlings in Petri dishes inoculated. Aseptic wheat-seedlings were placed on moist filter-paper in sterile Petri-dishes and were inoculated in their basal region in three ways: by placing upon them ( 1) wheat tissue rot- ted by H. No. 1 (pure culture), (2) conidia of this organism, and (3) agar bearing an abundance of growing mycelium. No difference was observed in the effectiveness of the three modes of inoculation. Each gave a 100% infection, always visible with a hand lens in 2 days (Fig. 16) as a small spot, which could usually be seen at the same time without a glass. A longer time than two days was necessary to demonstrate that this spot would develop into a general rot, but so it did in all cases when the environ- ment was favorable. Seedlings in rag doll inoculated. Wheat seedlings with shoots 2-3 cm. long were placed in a special form of rag doll (PI. XXXIII) and inoculated with H. No. 1 by placing an oese of conidia-suspension on the base of each shoot without wounding. Infection was apparent to the naked eye in every case in two days, and the results in six days are shown in PI. XXXIV. Rotting occurred in 6-12 days under favorable conditions. At 6 days the roots were often more or less blackened for long distances and the cortex filled with mycelium. Views of cross- sections showed a heavy infection of the second leaf, and the sheath completely occupied. With excessive moisture, seedlings were killed by the Helminthosporium in 6 days; but if in comparative dryness, only small lesions resulted. Seedlings similarly placed in rag-doll but atomized with conidia-suspension also gave 100% infection, and the infection was much more widely distributed. Inoculation by diseased tissue or by fungus-bearing agar was in no way superior to inoculation with conidia. Control, or check, rag-dolls, made in the same manner but without inoculum, at 2 and 6 days showed no lesions even under microscopic ex- amination. In a very small number of cases there was infection by Hel- minthosporium in the checks, and in a few instances overgrowth by a Helminthosporium similar to H. No. 29, with geniculate conidia. Roots of wheat inoculated. Conidia of H. No. 1 were placed on the root- hairs of wheat-seedlings in rag doll. At the end of 4 days all roots so in- 127 oculated were yellowish or pale straw-color, as contrasted with the white, uninoculated roots, and they had scant root-hairs. Under the microscope FIG. 16. Lesions on unwounded wheat-seed- lings two days after inoculation with conidia of H. No. 1. The shaded portion of the shoot was yellow to brown. 128 the cortical tissue was seen to be crowded with Helminthosporium myceli- um coursing mainly in the longitudinal direction of the root. The mycelial threads within the root cortex were remarkably thick 13/z. Wheat seedlings 2 cm. long, atomized with conidia suspension of H. No. 1, in 6 days were covered with infection spots over their whole surface. Inoculations in soil. Vials 12X70 mm., prepared as described on page 180, were used as containers. Wheat seeds were germinated asep- tically, and when the shoot was about 2 cm. long they were inoculated and transferred to the soil in a vial. The results differed in no essential way from those described for the rag-doll inoculations, though the plant could be kept longer under observation since it was not solely dependent on the seed for food. Aseptic wheat-grains were also planted in these vials with the inoculum placed in three different positions: (a) on the seed; (b) 1.5 cm. above the seed; (c) 1.5 cm. below the seed. When on the seed, lesions occurred low; when above the seed, they were higher; when below the seed, no lesions were on the stem in early days but the roots were heavily infected. Duplication, in pots and in benches, of all the above experiments made in vials gave identical results. Recovery of Organism After all the types of inoculation mentioned above, the organism used in the inoculation was clearly evident in the tissues and producing conidia upon them, and by dilution-plating it was recovered from them. During such recovery there was sometimes evidence of bacterial or other contami- nation, but in most cases of each type of inoculation no contamination occurred, and the pathogenic changes noted were clearly attributable to the fungus used in the inoculation. INFECTION PHENOMENA ON WHEAT Conidia of H. No. 1 and of H. No. 14 when placed on wheat in rag doll germinated from one or both ends as described elsewhere. The germ- tube grew rapidly, branching freely, and oriented itself lengthwise of the shoot more frequently than crosswise or obliquely, often following length- wise the boundary between two wheat-cells. At certain places where this mycelium touched the wheat-surface it swelled slightly, producing a round or oblong appressorium. These appresoria sometimes, probably most often, arose by the simple swelling of a cell of the main thread (Fig. 17), though frequently also from short lateral branches (Fig. 17, d) or where the terminal 129 cell of a thread abutted against the wheat tissue (Fig. 17, g). So far as observed they differed from the usual mycelila cells only in shape. The appressoria are very numerous (Fig. 17, b). They are usually produced only after the mycelium has grown to considerable length; not, as is the case with some fungi, immediately on emergence from the conidium. In FIG. 17: a, H. No. 1 on wheat, 24 hours after inoculation, showing mycelium arising from a conidium, an appressorium, and penetrating mycelium; b, c, d, H. No. 14, showing appressoria, penetrating points, and "callus"; e, f, g, h, H. No. 1: e, mycelium within cell, and with a penetrating mycelium reaching into an adjacent cell, a "callus" there resulting; /, mycelium ending squarely against a cell- surface, penetrating it and then being covered by "callus", and eventually penetrating this and the next cell-wall, the latter being thickened; g and h, similar to e. most cases the penetrating mycelium, viewed from above, appears as a minute bright point, or as if a minute hole had been pierced in the wheat cell-wall, much as is seen in the hyphopodia of Meliola (82) or on the appressoria of Gloeosporium (64) where penetration organs arise. Viewed laterally, the bright point of the appressorium is seen to mark the emer- gence of a haustorium-like strand which I shall continue to call the pene- trating mycelium. This structure is much thinner than the usual mycelium (see Fig. 18) and of different staining reactions. It penetrates the wheat 130 cell-wall, and is sometimes simple, sometimes branched. At the place where the penetrating mycelium pierces a wall and enters a healthy wheat-cell there is developed, on the inside of the wheat-cell and surrounding and covering the penetrating mycelium, a callus-like structure (Fig. 17, e-g) which for brevity I shall term the "callus". As the penetratiftg^mycelium continues to grow, the "callus" grows pari passu. Where many penetrating mycelia develop near each other this "callus" may become very large L J FIG. 18. H. No. 1: a, large "callus"-formation, with many penetrating mycelia piercing the cell walls; b, mycelium spreading over the wheat surface, and at many contact points producing appressoria and penetrating mycelia; c, penetrating mycelium of unusual form, and the "callus" rough. (Fig. 18, a) and complicated. The "callus" formation seems to be of the nature of a precipitation, probably resulting from toxic action, and a badly intoxicated cell can, in its protoplasmic disorganization, make numerous such deposits at points other than those of mycelial entrance. Thus in some instances the whole inner surface of a cell's walls may be thickly 131 studded with small dewy drops, apparently of precisely the same character as the "callus." (See Fig. 23, page 135.) The host's cell-wall at and near the point of penetration, is markedly altered chemically, as is shown by various stain-reactions. Thus, adjacent to the point of infection several different regions giving different chem- ical reactions may be distinguished, as is indicated in Fig. 19. Region 3 gives the usual chlor-zinc-iodide reaction and stains like normal cellulose. None of the other regions do this. Region 4 stains darker with the usual FIG. 19. H. No. 1 : regions of a young diseased spot: 1, mycelium; 2, penetrating mycelium; 3, normal wheat cell-Avail ; 4, region of darker staining ; 5, region of lighter staining; 6, diseased inner lamella; 7, middle lamella; 8, "callus." stains, but not so dark as normal cell-wall. The "callus" and penetrating mycelium stain faintly or not at all. The middle lamella stands out clearly in all of the diseased region, and on each side of it the inner lamella is seen to be thickened and of altered stain-reaction. Though penetration is some- times directly through the wall it is much oftener into the middle lamella, and the mycelium shows a strong tendency to follow along the line of division between two cells, thus giving a gridiron effect to the mesh. This is possibly due to chemotropic attraction by the middle lamella or, possibly, because this is the weakest place in the cuticle. No case of stomatal entrance was observed; indeed, on the sheaths of "Golden Chaff" wheat stomata are seldom present. Once within the host cell the mycelium grows rapidly, soon nearly or completely filling it (Fig. 20), and often forming a mass so dense that it resembles a pseudoparenchyma. Both longitudinal and transverse sections 132 show clearly that the mycelium is within, not between, the host cells. Penetration into adjoining live cells is attended by the same phenomena of penetrating mycelium, ' 'callus" formation, and wall-changes, though appressoria were not observed in such cases, possibly on account of the difficulty of observation. Penetration into dead cells is not attended by these phenomena. The chronological history of a lesion from a simple infection begins with the attack on one cell, which is soon overcome and occupied, and at 24, or, better, 48 hours after inoculation, observation with a 16 mm. objective shows regions with one to several cells diseased and browned, FIG. 20. H. No. 1 on wheat: a, mycelium in cells and penetrating the side walls; b, mycelium running lengthwise within the wheat cells. and the protoplasts undergoing disorganization and becoming browned. Owing to the length of the wheat-cells, the diseased regions are much longer than broad, and in many instances two diseased cells or two rows of them are seen with a quite healthy cell between them (Fig. 21). Under action of Javelle water the healthy cells plasmolize beautifully, while the sick cells show no plasmolysis. Treated with acid fuchsin in glycerine, normal cells show no stain, while in diseased cells the entire protoplast becomes pink and the inner lamella, which is swollen, also stains pink. This softening and swelling of the lamellae was extensively studied by de Bary (8), Ward (123) and Biisgen (30). De Bary, who, in 1886, was first to separate .a cytolytic enzyme from fungi (Sclerotinia libertiana), states that as the inner lamellae undergo partial dissolution they continue for a time to give the cellulose reaction, but eventually swell, disorganize, and lose this property (8, page 420) . He also describes the fungus as growing in the middle lamella. Ward (123) describes the cellulose as swelling and soft- ening under action of the enzyme produced by Botrytis. Here, too, the 133 mycelium grows in the middle lamella. Jones (71), working with Bacil- lus carotovorus , reports that the enzyme produced, attacks more strongly the middle lamella, but he noted also a softening and swelling of the inner lamella, but found that the cellulose stains (e. g., chlor-zinc-iodide) "give clear blue reactions with these fully softened walls." Van Hall (63), working with Bacillus omnivorus on Iris, reports a similar condition. The inner lamellae, swollen by Helminthosporium, no longer react as cellulose under this test. Blackman and Welsford (18), who describe in detail the entrance of Botrytis cinerea into bean leaves, state that neither before nor after penetration did the staining reactions of the cuticle give any evidence of its being softened or swollen or in any way altered chemically (though the subcuticular walls usually, if not always, swell), and no swelling FIG. 21. H. No. 1 on wheat shoots, second day after inoculation. Shaded portion was colored brown. of the subcuticular cellulose was observed before the passage of the invad- ing hypha through the cuticle. Pathogenic changes in the inner lamella precede those in the protoplast, that is, no toxin acts upon the proto- plast prior to the swelling of the lamellae. The subcuticular layer swells. Penetration of the cuticle is by pressure. Gardner (58) mentions no changes occurring normally in staining reaction of host cellulose in leaves attacked by Colletotrichum, though in cases of delayed penetration he notes that the cell-wall under the appressorium retained safranin bet- ter than did normal cell-walls. In fruit penetration, however, he found that, characteristically, the inner lamella was so altered as to retain saf- ranin. Ihe action appears to be different in both quality and quantity from that described by Newcomb (86), who, studying enzymes in seeds, states that "with all the ferments the w r alls at first become hyaline, appear 134 gradually more transparent and finally 'melt away.' ' In Colletotrichum Gardner (58) found the fungus similarly seeking the "depressions bounding the epidermal cells." This place of entrance is characteristic of many fun- gisee Biisgen (30), Rehrens (15), Ward (123), Noack (ST^Miyoshi (83), Nordhausen (88), Schellenberg (99), and Aderhold (1). The last three named, believe this to be due to chemotaxic influences. Noack in describ- ing the entrance of H. gramineum into the host mentions the appressoria. Similar structures have also been described in the anthracnose fungi by Hasselbring (64) and by Gardner (58), the pore in these structures being such as I find in Helminthosporium, though the appressorium in the anthrac- nose fungi is a mere swelling and is hyaline. Similar extreme narrowness of the mycelium at the actual point of penetration of host-walls is shown FIG. 22. H. Xo. 14 on wheat, showing fan-like mode of branching, see p. 105. also by Ward (123, fig. 57), Gardner (58, page 27), Hasselbring (64*, Biisgen (30), and Noack (87). Bakke (6) says of H. teres that the mycelium "penetrated the epidermis directly and made its way through the intercellular spaces," but he gives no further details. Conditions very closely resembling the "callus" formation are figured by Dastur (41. figs. 8, 9); depicting the entrance of smut into sugar- cane. This appears to have occurred only occasionally, and Dastur re- gards the "callus" ("plug") as probably a means of preventing infection. Conditions somewhat resembling that of the "callus" formation are de- scribed and figured by Wolff (128, figs. 2, 3) and by Brefeld (25, fig. 2) in the penetration of smuts into cereal tissue. Wolff (128, p. 20) de- scribing this says: "Es tritt hierbei der eigenthiimliche Umstand ein, 135 dass der Faden, sobald seine Spitze in das Innere der Zelle tritt, nicht frei in dieses hineinwachst, sondern von den inneren Schichten der Zell- wand, welche sich gleichsam aus stiilpen, wie in eine Scheide von bald grosserer bald geringerer, oft sehr betrachtlicher Starke eingeschlossen wird und in dieser bis zur nachsten Zellwand weiter wachst." Brefeld describes very similar conditions, including much thickening which is of yellow color, but instead of interpreting it as an enclosing sheath he regards it as wholly due to thickening of the walls of the mycelium itself. He moreover states that this phenomenon is indicative of conditions in the host, as too great age, that are unsuitable to infection, and that it is not in evidence when the host is in fully susceptible condition. Which- ever may be the true interpretation in the case of cereal smuts, I am convinced that in case of Helminthosporium^the "callus" is produced by FIG. 23. Infection by H. No. 1, 24 hours after inoculation, showing thickening of the wheat cell-walls by deposition on their inner surfaces. (Text citation at top of p. 131.) the wheat-cell, and is not part of the mycelium. Ravn (91), describing the reactions to the intercellular mycelium of Helminthosporium in cereals, states that a thickening appears upon the cell-wall of the host, resembling a drop segregated from the cell, and that several such thickenings may be seen upon one cell, sometimes filling the intercellular spaces completely. They seem to differ from those that I describe (Fig. 17), however, in posi- tion, since they are without, not within, the cell, and in composition, as those noted by Ravn take aniline stains readily. Ravn (91, fig. 23) describes an appressorium very much like that which I find and states that the mycelium from it enters the epidermal cell, where it so increases that it may fill the cell ; then makes its way to the intercellular spaces and grows there exclusively, never again entering any of the cells even by means of haustoria. It therefore appears from his statements and figures that the Helminthosporiums with which he 136 worked, differed in a very fundamental way, as pathogenes, from those which I am studying, his forms being intracellular (except as regards the first cell invaded), and not at once killing the adjacent cells. That is, the condition pictured is much like that presented by Albugo, Perono- spora, Puccinia, etc., except for the absence of haustoria. The forms with which I deal, on the other hand, though they enter through the middle lamellae, immediately become intracellular and at once kill the protoplast of the invaded cell, and proceed similarly with other cells. These differing- conditions, if substantiated by further study, probably indicate funda- mental differences in the fungi in regard to their production of toxins or enzymes, and certainly indicate an entirely different type of pathogenicity. In these early stages the disease is properly a spot and not a rot. Whether it will develop into a true, general rot depends upon conditions. Phenomena like those described under the present heading, though differing in de- tail, were noted with H. Nos. 6, 8, 9, 14, 21, 36, 39, 40, and 41. Action of various strains of Helmintkosporium on wheat shoots. Tests in rag doll, at medium moisture, with H. No. 1 and H. No. 3 gave at 2 days 100% infection for both; at 6 days there was no appreciable difference between the two; while at 10 days all shoots were rotten under H. No. 1 and some, but not so many, under H. No. 3. The test was repeated with 14 strains of Helminthosporium. All strains at 2 days showed 100% infection; the controls, no infection. The infection phenomena with all of these strains were all of the character described on pages 128, 129, showing penetrating mycelium, "callus," etc. At 6 days H. Nos. 1, 4, 5, 8, 13-16, 20, and 21 had all produced some rot. The roots also were distinctly yel- lowed by H. Nos. 15 and 16, while H. No. 20 showed less rotting than the other numbers mentioned above. H. Nos. 29 and 39 produced no rotting, and the lesions were visible only through a lens, but thus viewed, showed 100% infection, as indicated by the usual infection phenomena. H. Nos. 3, 6, 9, 17, and 18 remained local, as at 2 days. H. No. 29, a Helminthosporium with geniculate conidia, germinated abundantly from both ends of the conidium, and on wheat produced many penetrating mycelia and an abundant mycelium within the host, though the mycelial invasion reached only a few cells, and while extending for a considerable distance lengthwise, made but little progress laterally. The appressoria were usually pyriform, as was also the penetrating mycelium, differing thus from H. No. 1 (Fig. 17). Similar tests were made with three saltants, M6, M8, and M38. Notes at 2 days showed 100% infection, and at 6 days much rot by M6, and considerable rot by the other two. 137 Though infection can be determined with certainty, I have as yet no means of accurately measuring rotting power, or of determining whether differences noted in rotting are due to environment, host, or fungus. It seams clear, however, that H. No. 29 is capable of causing only local spotting; and that the other numbers, perhaps even the saltants, vary somewhat among themselves in rotting capacity, most of them causing rot- ting to some extent under favoring conditions. The fact that so many and diverse races of Helminthosporium are able to cause rot of wheat, led me to test the ability of Alternaria to para- sitize wheat seedlings. An Alternaria, found commonly on wheat seed, was isolated and inoculation made in rag doll on wheat seedlings. At 24 hours many wheat cells showed diseased spots, being in every way like those described on pages 128, 129, including the swollen middle and inner lamellae, browning of the cell-contents, and formation of the "callus" and penetrating mycelium. The Alternaria mycelium crossing several middle lamellae, usually produced an appressorium and penetration at each middle lamella. The Alternaria mycelium was also seen to enter the wheat-cells, killing a few of them, but in no instance was this fungus observed to cause rotting or to produce a spot large enough to be visible to the naked eye. It was seen, however, to penetrate the cells of the root cortex quite extensively, causing a slight browning. Sterigmatocystis, Penicillium, and several other fungi supposed to be mere saprophytes, were treated in simitar manner, but produced none of the phenomena of infection. SUSCEPTIBILITY OF VARIOUS HOSTS TO INFECTION Tests in rag doll with H. No. 1 Corn. Three seedlings showed no infection at 2 days, though conidia were present and had germinated. At 6 days all three plants were in- fected, the infection being confined to one or two cells, though the myce- lium was clearly evident in these. Pammel, King, and Bakke (90) report negative results regarding infection trials of H. sativum on corn, but their tests were limited to leaves. Barley. At two days one plant was slightly infected, showing sev- eral lesions. In these the mycelium was abundant within the cells. Eight were not infected. At 6 days the infection showed no further progress. Rye. At two days three plants were infected; six not infected. At 6 days the infection showed no progress. The mycelium was observed with- in the cells and infection phenomena were as on wheat. 138 Sorghum (Holcus sp.). There was 100% infection of both roots and stems, with pronounced rot. T he same phenomena were observed as on wheat, including the appressoria, penetrating mycelium, and "callus." The sap of the infected cells was strongly tinged with red,_and the ' 'cal- lus" and appressorium were deep red. Adjacent colorless walls soon be- came swollen and reddened. The red coloring-matter is absorbed by the nucleus, which becomes as brilliantly colored as by an aniline dye. At six days the shoots and roots were heavily infected, the diseased regions assuming a deep red, almost black, color, and conidia formed abundantly over the lesions. When such specimens were placed in alcohol, the red color diffused to the alcohol, coloring it strongly. This red coloration by the host is a response common on invasion of either bacteria or fungi on the sorghums and sugar-cane, and on corn in the case of some diseases. Sudan grass (Holcus sorghum sudanensis) . At ten days 1 seedling gave positive and 5 gave negative results; at six days, 5 positive and 4 negative. Infection was slight on a few cells, but the mycelium was evident within the cells, and infection phenomena as on wheat were observed. Common millet (Chaetochloa italica). At two days 10 gave positive results. At six days the rot was progressing into the roots faster than into the stem, though black spots 3 4 cm. long were apparent. Infection phenomena were observed as on wheat, and much mycelium was seen within the tissue. German millet (Chaetochloa italica germanica). The results were prac- tically the same as with common millet. Amber cane (Holcus sorghum]. Results were much as on sorghum. Red top(Agrostis palustris). No phenomena of infection were observed . Beans. No rot was produced, no "callus", nor any other of the usual signs of infection; nor was it certainly determined that the mycelium entered the host-cells, though it seems probable that the fungus killed some of the bean cells. Inoculation of leaves. Pots of well-established seedlings of wheat, oats, rye, barley, corn, German and common millet, and sorghum were placed in a humid atmosphere (above 90% relative humidity) and atomized with suspension of H. No. 1 conidia. Well-defined spots occurred fre- quently on barley, less frequently on wheat. Leaf-spots due to a Helmin- thosporium, apparently H. No. 1, also occurred naturally on wheat in the greenhouse. Such spots were first pale; later with a mummified dark center surrounded by a pale zone; and were oval in outline. In rye the mycelium was seen to be abundant within the cells, and complete death 139 of the affected leaf, and also rotting without spotting, resulted. On the leaves in the humid air of the rag doll occasional spontaneous infections were noticed. In such cases the infection rapidly spread, involving nearly all of the leaf, which first turned pale, then very slightly brown. Aerial mycelium and conidia were profuse over the diseased portion. SUMMARY CONCERNING ETIOLOGY OF FOOT-ROT The evidence is conclusive that Helminthosporium is the cause of the basal rot of the wheat-stems. It is the only parasite constantly present, and has been repeatedly, and by many methods, proved capable of causing such rot. This conclusion is in accord with the findings of Beck- with (14), who as early as 1911 showed that Helminthosporium is a very common parasite within the tissue of wheat-plants. Bakke (6) in 1912 reported that when conidia of H. teres were placed on barley seeds, "At the end of two weeks' time there were not over seven seedlings to the row [originally there were twenty-five]. The roots were not in any sense indicative of a healthy state of growth." Oats and fescue-grass were not susceptible. A seedling blight of wheat observed since 1910 has been described by Stakman (113) in Minnesota, where in 1918-19 it became seriously injurious. The symptoms include dwarfing, foot-rot, and root- rot. The disease appears to be closely like, if not quite identical w r ith, the one which is the subject of this paper. She proves conclusively that the cause is a Helminthosporium. A foot-rot of wheat due to a Hel- minthosporium having quite different morphological characters is also known in Sudan (see No. 46, page 184). Certain of Ravn's experiments (91) conducted by inoculating seeds on wet filter-paper in a Petri dish, gave conditions much like those in the rag dolls. He makes no mention, however, of infection of the sheath nor of the occurrence of "rotting of the basal region. II. Evidence and Discussion of the Occurrence of Saltation within the Genus Helminthosporium INTRODUCTORY Early in my study of this Helminthosporium of foot-rot of wheat (herein designated as H. No. 1) it was noted that occasionally certain sectors of a colony growing on an agar plate differed more or less from the rest of the colony (PI. XXII, 5; PI. XXIII,!). This phenomenon is of rather common occurrence in work involving Petri-dish cultures of either fungi or bacteria, and little significance was at first attached to it; but later, when 140 the frequent recurrence of these variant sectors commanded attention, trans- fers were made from several of them to freshly poured agar-plates, and a transfer from the normal portion of the colony was added to each of these plates at a distance of about 2 cm. from the other transfer. The variant transfer was then marked on the bottom of the doubly occupied plate as M (indicating mutant), and the normal transfer as O (indicating original). In all the early transfers the M transfer resulted in a colony (Ml) of decid- edly slower growth and more profuse conidial production than that pro- duced by the O transfer. The two colonies also differed markedly in general appearance owing to minute single differences which were often difficult to analyze, but which in the aggregate constituted distinctions which were so well-marked and obvious that at first sight one would say that the two colonies were those of two distinct fungi (PI. XXIII, lower fig. ; PI. XXVII). When these colonies grew to fill, or nearly to fill, the plate, transfers from them were made to new agar plates, and later, transfers from these second plates, and so on, the series of transfers being a long one. It was found that the differences appearing in the Ml and Ol colonies were usually maintained on succeeding plates. These findings led to the tentative assumption that forms in the variant sectors were mutants or saltants of a more or less permanent nature, and a more serious study of this phe- nomenon was undertaken. In their origin the variants or Saltants always appear as sectors which differ from the portion of the parent colony adjacent to them (see PI. XXII XXV) . To the naked eye the most common deviations from the orig- inal type are in density, color, and rate of growth. Closer observation, with the microscope, frequently shows variation in the grouping, size, and shape of the conidia, and in the branching of the mycelium. Quite often many small sectors of divergent character appear at the edge of a large colony, especially on a plate that is beginning to dry. Many of these divergencies are merely modifications due to local environmental changes, and whether they are more can be determined only by close study of their behavior in subsequent transfer or transfers. Closer consideration of the characters involved in these saltations is best deferred to the following topic. In following this discussion it must be borne in mind that M refers to the variant sector on the plate on which it originated; Ml, to the colony re- sulting from the first transfer from M; M-2, to that resulting from the first transfer from Ml, and so on; and that O refers to the original colony in which the M arose. It is my custom to give the saltant a serial number (writing this on the plate in which it was found), and, usually, to transfer 141 both the saltant and the original to the same plate so that they may have the same environmental conditions, the identical quantity and quality of agar, and by growing close together may render comparison easy. Notes on the origin and subsequent behavior of the saltants were made under the eerial number, ard the transfers were designated by additional numbers Thus, IVI98-7 refers to saltant No. 98, transfer 7. CHARACTERS OF SALTANTS AS SHOWN IN TRANSFERS General appearance. The colonies of the saltant and of the original when grown on the same plate were usually so strikingly different in gen- eral appearance (Fl. XXII, XXIII) that a mere glance sufficed to give the impression that they were colonies of two different species. This dif- ference in general appearance is, on analysis, referable to one or more of the individual differences mentioned below. Pate cf linear growth. Frequently the saltant was of much slower growth than the original, resulting in an M colony of much less diameter than that of the O colony, being often less than half of it (see two ex- amples: one given in Fl. XXII and one in PI. XXIII). In some instances, however, the M colony grew faster than the O colony. Ccnidial production. Frequently the M colony, especially when slow- growing, was much more productive of conidia than the O colony, so much so as to give the colony a decidedly perceptible darker color. In several instances, however, the M colony was of the opposite character, producing few conidia or, in some cases, going to the extreme of appearing to pro- duce none at all. Generally speaking, rate of linear growth was in inverse ratio to that of conidia-production ; while those saltants that were pale and possessed much aerial mycelium were usually of rapid linear growth and low conidial production. Ccnidial clusters. Some saltants varied strikingly from each other and from the originals in the mean number of conidia borne per conidio- phore. Ccnidial length, breadth, septation, and shape. These characters, as evidenced by casual observation or by a study of graphs and the data derived from them, are shown to be strikingly different in various saltants. For clearness I present in this connection records concerning only a few saltants, giving graphs and data for others later. Graphs of ccnidial length of saltants M35, M36, and M40, those which show greatest deviation from originals in this regard, are given in Fig. P with the essential data. It is to be observed that the modes of M35 and 142 M40 are 57.8 and 64.6 n, respectively, far below the mode of the original, which was 81.6 M- M36 shows less striking difference, but this is still marked. Comparison of the means shows those of M36 and M40 to be approximately 17 and 18 -divisions (1 division = 3. 4 /*), while the original was 23 divisions. In other words, the conidia of M36 wese-only about three fourths the length of the normal conidium of H. No. 1. Such dif- ferences as they appeared in the microscope are shown in PL XXVI. The difference in variability is also strikingly large. Striking variation in conidial breadth, both relative and absolute, was observed. Graphs and data of the more pronounced cases are presented in Fig. Q and others are given later. In connection with Fig. Y (Graphs 114-138) are given summary data on the conidial length of saltants in- cluded in this study. It is to be noted (Graph 6A, Fig. B) that whereas the mode of the ordinary conidium stood at 20.4 /i and no conidia exceeded a thickness of 23.8 /z, the modal thickness of M8-7 (Graph 75, Fig. Q) is 23.8 ju, with many conidia 27.2 ^ in thickness, one even 30.6 /z. Such dif- ferences between saltants and the parental form are presented to the eye in PI. XXVI. The ratio of conidial length to conidial breadth is perhaps still more striking than the mere variation in length. In such variants as M6 (PI. XXVI, b) and M8, while increased greatly in thickness the conidia were at the same time absolutely shorter, thus emphasizing to the eye both differences. The ratio of length to breadth in H. No. 1 is as follows: mean length _ 22.62 =*= .05 _ # mean breadth 6.03 .04 while in a sample of one of its saltants this ratio is mean length _ 20.67 .22 ^ mean breadth 7.82 =*= .11 and in another sample of the same saltant it is mean length 19.58 .30 mean breadth 7.30 = t .06 2.67 .05 = *Probable error was computed according to the above formula kindly furnished me by Dr. J. A. Det- r> lefsen, where a <= probable error of A; b = probable error of B; and E = probable error of A 143 Variations in septation were also noted; thus in Fig. R, Graphs 79-82 are quite different from Graphs 83 and 84, while all of these are lower than the results gotten from H. No. 1 in Graph 35, Fig. J. I attach but little value, however, to these variations because they seem inconstant. Variation in conidial shape is common, some saltants showing the sides more nearly parallel than others, and the conidium as a whole less elliptical or fusiform. Variation in submerged mycelium. Aside from rate of growth and variation in branching which resulted in changes in density of the colony, differences in the submerged mycelium were observable in but two cases, most strikingly so in M26. In this saltant certain hyphal threads near the edge of the colony appeared to be much more vigorous than their neighbors, becoming a trifle thicker, and lengthening with such rapidity as far to outstrip the others, reaching out as single strands to a consider- able distance beyond the usually even frontier of the colony, beginning then a dense, bushy branching in all directions, reminding one of witches'- brooms in trees. Numerous outposts of this kind give a peculiar lumpy appearance to the colony as seen by the naked eye. This peculiar mode of branching was clearly to be seen in M26, where it originated, and was frequent throughout subsequent transfers. Instead of single threads reaching out in this way, the mycelium sometimes formed fascicles which would grow out rapidly into new territory without branching, then sud- denly branch profusely, forming a dense brush. These two rare characters were striking in effect both to the naked eye and under the microscope. Nearly every transfer from M26 or its descendants gave colonies with very strikingly marked sectors, characterized essentially by abundant conidial production, and therefore dark in color. The other sectors bore few conidia, were pale, and being a trifle less rapid in growth they were usually crowded out (PI. XXX, lower fig.). These characters were main- tained through many transfers. (See PI. XXXI.) Variation in aerial mycelium. Saltant sectors and their progeny often differed from the originals in the abundance and character of the aerial mycelium. In some cases it was so scant as to be unnoticeable; in other cases so abundant and floccose as to obscure from vision the colony beneath. In character it varied from loose and fluffy to "ropy," the latter term indicating a tendency of many mycelial strands to twine together (Fig. 5, c, p. 104) . In other cases it collected in clumps, the process being attended by peculiar distortions (Fig. 6, e) . In some saltants these clumps were abundant and aggregated; in others few and scattered (PI. 144 XXII, XXIII). The occurrence of clumps of mycelium upon the surface of the cultures has been stressed by Ravn (91) as of taxonomic importance (cf. also with PL XI, XIII, XXIII (below), XXVIII). H. No. 13, in one small sector, showed eight white clumps; the balance of the plate, none. In transfer M71 the clumping character seemed lost, but the following transfers were pale in type. In other cases the clumping habit seemed to be fixed and characteristic (PI. XXVIII). Variability in colony color. The color is mainly due to abundance or scarcity of conidia or to abundance or lack of aerial mycelium, or to both. The white aerial mycelium is practically without conidia. Transfers from sectors with much white, sterile aerial mycelium were not always constant in these characters, but in many instances they were so; for example, M72, derived from single conidium Cl-1, and M78, derived from M26. Differences quite comparable with these were noted by Crabill (36) . Zonation was well marked in some saltants and almost entirely lacking in others (PI. IX, 3, 4, 5, 20). Some saltants formed sclerotia abundantly though the originals did not do so. Density of colony also differed, some saltants producing colonies of much denser growth than others. Variability. -Variability itself was a distinctive character in certain instances. Thus, while the original of any given saltant is usually fairly constant in its characters and only occasionally gives rise to saltants, one saltant, M26 (PI. XXX, below; XXXI), was definitely characterized by the fact of its inconstancy (see page 143). Many saltants were tested as to their infecting power and their rotting power, but no real difference in these respects was noticeable between the different saltants, or between the saltants and H. No. 1 . Since many species of Helminthosporium can infect many cereals this power may be rather fundamental in the genus and thus not be so readily subject to saltation as are less fundamental characters. I have no means as yet to measure slight differences in either virulence or rotting power. It may be mentioned here that Ravn (91) states that culture upon dead substrata diminishes the vir- ulence of Helminthosporium. Whether such diminution was permanent or merely a temporary modification he did not determine. Edgerton (51) reports that different races of Glomerella differ in virulence. Correlation of characters in saltation. Certain correlations of characters are noticeable; thus, colonies of slow linear growth were usually high in 145 conidial production and rice versa. Correlations observed are indicated as follows: Slow linear growth* >high conidia-production Much aerial mycelium >low conidia-production Pale colony* ^rapid growth Thickening of conidia ^shortening of conidia Pale colony >low conidia-production Clumping of mycelium low conidia-production 1 he differences in colony-color and growth-rapidity here noted, are much like those described by Edgerton (51) in Glomerella plus and minus strains. Crabill (36) notes also a correlation in that his minus strains were always of more rapid growth than the plus strains. TENDENCIES IN SALTATION Saltants showing very low conidia-production, verging on sterility, coupled with paleness of colony, occurred with the greatest frequency. A type with increased conidia-production and of slow growth was next in frequency. The latter of these types was the most frequently thrown dur- ing the early period of my work though it has been rare recently. On the other hand, the former type, which rarely appeared at first, is now the most common. A type characterized by thickness of conidium, as M6, M8, etc., has been frequent all the time. These three types were by far the most common, and may be said to show the three tendencies. Markedly short- conidia saltants were few, as were also clump-bearing types that possessed permanence. Strains that threw either of the two types first mentioned above were very likely to continue to throw similar types. The same may be said of clump-bearing types. STABILITY OF THE SALTANTS Many saltants have been tested in various ways to determine, to some degree, their constancy. Through numerous transfers on corn-meal agar the O colony and the M colony of many saltants have been carried side by side. Under such conditions, though the original may give rise to new saltations or the saltant may saltate further, the main portion of both the O and M colony, as a rule, maintains its characters. It is manifestly impossible to test all the saltants to ascertain what their future behavior will be. All that can be done at present is to record certain observations concerning them. Several saltants possessing strongly distinctive characters have been repeatedly transferred and have maintained their characters through all of these transfers ; and as far as can be foreseen 146 are as stable in their present form as are other fungi. Thus, saltants with short conidia (as M35 and M40) and saltants with broad conidia (M6 and M8) have been cultured and graphs of conidia repeatedly made, the sal- tant maintaining its character. For example, a determination_of_measure- ments of conidia of M35 made after several transfers and the lapse of some weeks gave the following data: Mo- CV 17.31 .25 2.51 .17 14.50 1.05 Comparison of the above data with data of Graph 65, Fig. P, shows that this saltant not only remains far below H. No. 1 in length but is also constant. It is particularly to be noted that all comparative conidial meas- urements were made under standard conditions. Other characters ex- hibited by saltants, such as color, zonation, and aerial mycelium, are sim- ilarly permanent when strongly marked. Saltants are, however, subject to further saltation and indeed in some instances are exceptionally liable to it, for example, M26. Not all suspected examples of saltation afforded by variant sectors proved to be permanent in character, and some lost their distinguishing marks after one or a few transfers. Such instability was not observed in cases of conidial length and breadth, or of pronounced pale colony-color, but was more commonly noted in cases of slight differences of aerial mycelium, slightly pale color of colony, clumping, etc. While all cul- tures were carried, for convenience, on corn-meal agar, and their differences were observed on this medium, all that were studied critically were passed through other media autoclaved wheat-shoots and live-wheat to deter- mine whether such passage would alter the character of the saltant. The saltant characters were apparent on other media, as green-wheat agar or beef agar, though the general colony-character of both original and saltant was changed by the medium. After passage through these conditions, or through wheat, they were inoculated under standard conditions for all graphic comparisons. There is no evidence of alteration of the characters of the saltants by such procedure. In other words, the saltation is not a phenomenon associated with the medium and ended when the fungus gets back to its normal habitat. STABILITY OF THE SALTANTS THROUGH THE CONIDIA Dilution platings of conidia of well-marked saltants gave colonies all alike and with all the characters of the saltant, showing permanence of these characters through the conidia. 147 APPARENT REVERSIONS In several instances where colony color, aerial mycelium, or partial sterility was the saltant character, small sectors of the colony were so changed as to resemble closely the originals, and as far as tests were applied could not be distinguished from them (PI. XXXII); in no case, however, where true saltant character was proved by constancy through several trans- fers did the whole stock revert; what appeared as reversion was limited to occasional sectors of the colony, and in no case did such change occur in the entire margin of a colony. SUPPOSITITIOUS CAUSES OF THE VARIANT SECTORS Several alternative suppositions other than that of saltation may be briefly discussed as possible causes of the variant sectors. The mycelium at a certain point may become weakened, or die, and the change in equilib- rium resulting may cause the variant sector. Spores of another Helmin- thosporium or of some other organism may fall into the colony from the air, and the variant sector may represent merely a contamination. The in- oculum used on a plate that shows saltation may have consisted of more than one strain or elementary species of Helminthosporium. The first supposition is open only to crude experimentation, while the second, if valid, implies a wonderful Helminthosporium-richness in the air of my laboratory as well as very faulty technique. Since saltation occurred after the fungus, H. No. 1, had been transferred many times by lifting a small bit of agar from the edge of a colony, the presumptive evidence that no mixture then existed is very strong. The following experiments bearing on these suggestions may, however, be worth recording. Wounding. A culture of H. No. 1 on corn-meal agar was allowed to grow to a diameter of about 4 cm. Then by means of a hot iridium wire the mycelium was killed at the points indicated in PI. XXIX, above. In all cases the uninjured parts soon entirely outgrew the wounds, and the whole colony presented an entire, normal outer border with no evidences of saltation. In some instances a clear straight line extended from the point of wounding nearly to the edge of the colony. Evidently disturbance of equilibrium such as this did not cause saltation. Mixed planting. Acting on the 'knowledge that the saltants were frequently slow-growing, and thinking that possibly ordinary transfers might be mixtures of two or more races, of which the slower-growing one ordinarily remained masked, M8, a well-characterized saltant, was planted 148 on a corn-meal agar plate and allowed 24 hours to grow, by which time a vigorous mycelium had developed. A goodly quantity of conidia of H. No. 1 was then placed in the midst of this young but well-established M8 colony, but it remained uniform to full occupation of the plate^hpwing no saltations. Implanting conidia of H. No. 1 in a partly developed colony of the samz strain. This experiment was conducted like that of wounding except that instead of using the hot wire conidia of H. No. 1 were implanted at the points indicated in PI. XXIX, below. All implants within the colony grew sparingly and resulted in small clumps 1 2 mm. in diameter and highly sporiferous (PI. XXX, above). Implants at the edge grew poorly, but those a few millimeters outside the colony became established and grew well, each implant developing as an independent colony and inhibiting ad- vancement of the old colony, but bearing no resemblance to a saltant. In one case, however, such implants showed marked change in characters and are still under culture as saltants (M70, PI. XXX, upper fig.), though efforts to produce other saltants in this manner were fruitless. Implanting other Helminthosporiums. In a way similar to that of the last experiment numerous other species or saltants (e.g. H. No. 2 and M6) were implanted in an H. No. 1 colony, and always with the result that the implant either failed utterly to establish itself or developed as an entirely independent colony that did not blend with the main colony, being in this unlike a saltant sector in character. If implants were put about 3 mm. be- yond the tips of the advancing mycelium, the conidia were observed to germinate before the mycelium of the H. No. 1 colony arrived, but even such implants became entirely submerged and lost. Two entirely distinct types of Helminthosporium, found intermingled on a single grain of wheat, were planted together an oese of suspen- sion of the mixed conidia on an agar plate. The resulting colonies gave the two types of Helminthosporium, but did not give the sectors so char- acteristic of saltants. Saltation not due to parasites. The saltant sectors and their transfers often differed so strikingly from their originals, particularly when they bore few conidia and had much white aerial mycelium (see PI. XXVII) as to suggest that perhaps the great difference w r as due to a parasite growing in the Helminthosporium colony. Close microscopic inspection of saltant sectors showed that there was only one type of mycelium present, that it was all indistinguishable from Helminthosporium mycelium, and 149 that no conidia indicating contamination were present, therefore, if the colony were parasitized it must be either by a mycelium like that of Helminthosporium and without conidia, or by some virus of un- known character. To test this possibility well-established colonies of H. No. 1 were inoculated with such striking saltants as M84. Transfers of M84 were also made to points near the circumference of the H. No. 1 colony. If M84 bore a parasite of any kind this parasite might be ex- pected to invade and overgrow the H. No. 1 colony. This it did not do, but the two colonies halted a few millimeters apart in the manner char- acteristic of two Helminthosporium colonies. It is quite clear that the idea of colony parasitism is untenable in this connection. Position of inoculum. Since it was possible that the differing appear- ances presented by the various sectors might be due to the position of the mycelial strands in or on the agar, that is, on top of it, in it, or below it, tests were made in three ways: 1, by placing conidia in an oese of w^ater on the surface of poured agar; 2, by similarly placing conidia, without water, in a shallow scratch made in the agar; 3, by so cutting the agar that a flap about a square centimeter could be lifted and inoculated on the lower side, that is, the side in contact with the glass, the flap being then put back in place. These three modes of inoculation resulted in colonies of indistin- guishable character. SALTATIONS FROM SINGLE CONIDIA Eight separate pure cultures were made from single conidia. The eight colonies were under careful microscopic control from the time of planting the conidia, through germination, and until the colony was well developed, and it is certain that in each instance the colony was from a single conidium. These pure strains, all alike in colony character, were labeled Cl, C2, C3, etc. Well-marked saltants appeared in four of them as follows: 72 Cl 110 119 C2.. 120 108 109 121 122 128 150 68. .95 114 115 116 C3 36 117 111 112 113 . 128a 106 f 126 107 ..125 } 127 C5.. 37 Thus, it will be seen that single conidium 3 gave rise to sixteen clearly denned saltants; C5, to one; Cl, to two; and C2, to seven demonstrating absolutely that these saltants were not due to impurity of cultures. Evi- dently the saltant sectors do not result from contaminations. FREQUENCY OF SALTATION It is impossible to give any mathematically accurate statement as to the frequency of saltation in Helminthosporium. One hundred and twenty-six variant sectors were selected, transferred, and more or less studied; and this number could easily have been doubled or trebled. It is not probable that all the forms in these sectors were truly saltants; doubtless some of them were mere modifications, but the number that were permanent in character is large. How many of these saltants agreed with each other in observable characters it is also impossible to say, but since they arose independently it may be that they do not often agree absolutely. The percentage of saltants, based on those theoretically possible, is, how- ever, small even in races that are most actively saltating. Thus in a colony 6 cm. in diameter there are probably more than 5,000,000 cells, and theo- retically it appears probable that saltation occurs in a single mycelial cell, or perhaps by the union of two cells, yet saltations occur with even less frequency than one to each 6-cm. colony, therefore less than once out of 5,000,000 possibilities. In this connection, though no direct comparison is possible, it may be noted that East (47) considers the occurrence of twelve inherent variations in observations made on 100,000 hills of more than 700 varieties of potatoes, that is, about 1:10,000, as an unexpectedly high rate 151 of frequency. In tobacco only one bud variant was noted in 200,000 plants. Benedict (16) regards the production of 50 new Boston ferns in fifteen years as rapid. East (46) notes that all of the asexual variations have been losses of characters. The pedigrees of the various Helminthosporium saltants which I have studied are indicated in the following table: PEDIGREE TABLE OF SALTANTS 65 S o / 81 59 \ 82 53*, 56*, 57*, 58 3 ...26*....- 60,61 78 89* f 52. 74 90* 86 96 H.No. 7...-! 85 4 73 76 104 103 69 f 98 5* 9* \ 92 H.No. 8...^ 99 93 I 100 , 94 :QQ f 69 00 88a.. 88b 88c H.No.9...-* VO \O H- H- If H- O o ^ J ill c G Jc W Oo to tO ts3 ** * ^ ^ 0^8 > > oo ^^ NO If H- 00 4^ ii rr P ^ 3 ^3 O (D ^^ "* * &3 ro to 3^ o ? ^ 3 p I/Q p i ^ OTO 3 p to g -i - P ( _ k ^ 3 j^ C*o \o KJ 3 3 n . . & & SO 00 NO O Oo Oo ^ 'p CL u a .1 u <* P i 5j 3 4^ g 3 On On O 00 P rt 0. 3 FIGURE A GBAPH 47.6 51. 54.4 57.5 61. 64.6 868.0 o 74.6 76. 8 f.6 66.4 q 1.6 96.6 \ \ 7 FIGURE B Conidial breadth of H. No. 1 grown on corn-meal agar made at different temperatures: Graph 5, on agar made at 60; Graph 6, on agar made at 43. Graph M a . CV 5 5.50 .04 .22 .03 4.06 .61 6 5.50 * .03 .18 .02 3.31 == .40 Graph 6A, conidial breadth of H. No.l grown under standard conditions. (See app., p. 180). Graph f M ? fs : **a O ^ ^* O\ C/J ON H^ If H- tf If |fi g,| SB to - to O 00 00 Cx> to to to Cyi * 4- to >- O ^J ^* H- H- Ni l- o c*> 0\ p NJ H* i * ^^ H^ O^ P o 3 ^ x. o P* U C^ ? S 3' B ^3 pj "S-o 'S n _ o " 10 n ^ O p* CL 3 5' o o 5T P P 5'^^ fr fr O Ov *- ON OO to CA> H- ^ x /^ P 1M g 3_ p P n era orq O FIGURE C GBAPH 27 2 30.6 34.0 37.4 40.8 44.2 47.6 51.0 54.4 57.8 61.2 64.6 68.0 71.4 74.8 78.2 81.6 85.0 88.4 91.8 95.2 CD FIGURE D Conidial breadth of H. No. 1 grown on green-wheat agar of differ- ent compositions: Graph 13, on washed agar 34, green- wheat agar %; Graph 14, on washed agar %, green-wheat agar y. Graph f M a CV 13 14 6. 10 .06 0.38 .04 6.32 .80 14 44 5.98 .07 0.71 .05 11.87 .86 Conidial septa of H. No. 1: Graph 15, grown on washed agar M, green-wheat agar %; Graph 16, grown on washed agar %, green- wheat agar }4. Graph f M 4*- 2. 3 X CL O --' sr 3 H- H- " 3 O 3 FIGURE F 5 S 1 3- . . n 3- (T> _ O ^ rt- O 3. C c/) jy CL i _ i _ > cr V *o 53* f? *** JH Q* I ' fD - - Orq K) Cn CA> ON O ^I H- H- 3 8 9 3 . o- Cn O K> 4^- 3 -t 3 II 3 CL GRAPH NUMBECS 8 37.2 30.6 34.0 37.4 40.8 44.2 51.0 54.4 71.4 74.8 78. 2 j SI. 6 85.0 88.4 91.8 102.0 108.8 112.2 \ \ to to to P to o & g- -* Cn C/i to K* o H- H- If I ? CTQ O ^ ^ ^ P "" O _k 3 H- ff If ** 00 O ON OO H- If H- Oc n if 8 1 pj q^ 1 o cr Si FIGURE G W GBAPH 44.2 476 51.0 54.4 57.8 61. a 84.6 68.0 71.4 O 74.8 JO : 78. a i 81.6 85.0 88.4 91 8 95.2 98.6 102.0 105. 4- 108.8 I 18.2 \ \ \ \ ooooooootototototo OotOi-'O'OOO-^JONOn ON On _^ H-H-H-H-H-H-H-H-ffH- i-^>-^ootototototototo \O v Orf^Oo->JO'~'i^ l ~'ON i-^ to Oo Oo bo < Oo Oo On ffH-H-H-H-H-H-H-H-H- S'g. ^ CL re CL fl c o ^ *^% Oo -. to 3 - II O <* en !l s o il P 3 CL re CL ?r re" o 333 3* en n> -. P p P <""*" CL *~ CL RfEf CL -t, C 3 O 3 oi 5 P a^ ?-g:8-^ oo re CL ^ 3-c ,|?* P "O &s: 3* _ 3" " ^ NJ [g orq ^D ^"^ ^T *^ ^i a. oo ps o 3 O 11 f i a ^ ^ X 3 SSisu H- 3 -3 CL p^ -. . M /S El- "-* t- 1 !-- tO 00 J ON 00 ON ^4 OO On 4^. o ^ Oo OO i . - to SO ON to -J OO Oo Oo t ' -M On H-H-H-H-H-ffH-H-H- o - = 8 1 1 n ! 3^0,^ & & ^" 2.5.- o o'" 1 ON ON "-* OO Oo ON Oo t i 00 H- KJ FIGURE H QBAPH 20.4 238 27 2 30.6 34.0 374 40.8 44.2 47.6 51.0 54.4 257. n 061.2 Z 64.6 68.0 71 4 74.8 78.2 81.6 85.0 884 91 8 95.2 98.6 102 ^ & ro r\j N OD \ \ \ FIGURE I Length of 1646 conidia of H. No. 1 grown on corn-meal agar. See also pp. 120-121. Graph M a CV 34 14.34 =t .08 5.35 =*= .06 37.35 =*= .70 FIGURE J N0.35 VO b- oo O) O v5EPTA- Conidial septation of H. No. 1. Graph f M - TD f 3* J rl ^ 1 3 i on various cc Conidial 1 "I a K tO tO tO o 3 : 8 8 o i CD 3" O H- H- H- H- 2 On 1 pc lo i- i. O ON 9J ^ '^ C/) "t * o' QfQ 3 f ag I? c 3 4^ d PC O ** Oo Oo to to 3 P 3 80s QC 4i> Oo sT cr; C 3 ^ If H- H- H- q ?r o. 'In H-. h- O o p 3 3 3 M Q =T C ^ , 1 4_ 1 1 H- P0 O . U5 3 C- 00 O' (yp o ^" to H- H- 7 o | 3" 5' CO - C" O\ On N3 o o o o 3 X C FIGURE M NUM5EE5 238 27.2 30.6 34.0 O Cn 3 7.4 [y 40.8 44.2 47.6 51.0 54.4 7578 ! }6I.2 > S646 68.0 71.4 74.8 78.2 8 1 .6 - 85.0 88.4 91.6 95.E 98.6 lo^o FIGURE N o o oo-o o H- c, i-t i_t i_k tO O -4 (t o 3 2 O f^ i i 3- vS IV "O t/Q CO CC 00 Cn o p o CS GEAPH NUMBELC^ s ^ 54.0 37.4 \ 51 .0 KA 4 \ j 57.8 \ 61 .2 ; \ <[g8 O / \ O 10 71 4 \\ x Q l.t 2 0> 74 s / \ \ U / 78.2 fc \ \ ^-^^ 81 .6 A / 85.0 oo 4 / / \ / / ^^-^^ 91 .8 QC / 1 \ 9 C i / / / \ \ 1 / 1 / 108.8 \ 1 12.2 Z 115.6 \ / FIGURE O o ON ON O^ "t W Qrq 4" C*> to P . O TD Q H 3 =r o sr tr H- H- vo to ON -i oo n' D. ON h- \0 ^ TJ g. - * a c> s o ON ^ 3^ p n 2 t ON 55* 2. s, IxJ CM i; NO 00 3 E o ^ o sr 3 o ? rt- * KJ to 10 to ^ to t^i ON ' ON O Cn TO* ON to H- If 1 If If S 2 CN) Cn Cn SP 8 -^ '- o oo g ^ O g CU c r: " TD 3" 3 a* PI - to .^ f .^ CK) ON S ^ ? ON CN* ^1 it Cn - o tf If If If If * g ^ ON rt-" 8 5 to to" It to 3" O sf v l ~ r o o K p 4- 4^ to to 3- ON pc to to b to 3 Cn --a ^ If H- If If .'< g ON Cn 4- oo 9 to ? FIGURE P 23.8 27.2 30.6 34.0 374 40.8 44.2 476 51.0 544 57.8 Z6I.2 646 68.0 71.4 748 78.2 81 6 850 88.4 91.8 952 986 GUAPH 8 a \ \ \ \ O ^ O vO ON ^r 885 If H- -* C*> Cn 4*- to O to * Cn to H-H-H-fflflflflf 8 35 4^ Cn Cn \o Cn Cn OO O > . ON OO Ol o t^> H- If ks (fH-H-H-lflflflf 4^C*j^"-^J4^OjC/iCn e:a if H- In C*i ON OO vO to ^j ^^ to *-j 4^O> 'C^joovoooo >-*4a.OoOi-*vOONCn H-lfH-H-H-H-lflf ON4^VOVOONONOOON 00 ^-O K- 3 1^ "^ ET 2 "O B -* I , r s l- 8 ^ o g S-o 2 3* PJ ON 5" 2. s z s ft s I CD i p Cn Q. * a .? ^ | o^ ^ L >c C/l ^^ r; W p "L o O ^ ^Q Q ^ 9 ^ to >-. o ^o "o 2 "- -- r 2-:.8 to r} -o Aj 2, 3- . fa vi 4^cyi4^NiC>j_ K >rr c^i c/i v o c/i to '^ y ^ 1 .2 1 00 ^ g 4i. H-. 4^ Oo to to o Oj H->. 4^. oo oo 00 O If If H- H- H- H- If 1.1 * ~ O g p '^^ "^. 00 to o > ffH-H-H-H-H-H-o 3 ^ OOOOO'-'i-' -n ^jsO'OOOOU.O 3 "" O oo ~ C*J t^> - &, sO 00 4^ -4 O 00 ^J C/i *O OJ C\ 4^ OO CA> H-H-H-lfffH-lfQ O ^O OO H-. C*J OO C/i n FIGURE R 00 PJ <^ *0 frS 8 2 n H- < : TJ - I I " 5- c/j c^ X FIGURE S Gc 27.2 30.6 34.0 37.4 40.8 44.2 47.6 51.0 54.4 57.8 ^r o 6L2 64.6 O> 68.0 71.4 74.8 78.3 81.6 85.0 88.4 91 8 APH NUMBER CD H* Cn O O OJ H- If H- If If If o o ^ o o > -J 00 O -J 4^ 4^ OO i ' Co O Cn K3 ON NO ON tsi f^*i ^ If If H- If H- If 8S H- H- H- H- If NO O - p .g ffi"S- s 1 Z 2S| ,, 1*1 1 -^ ^ r w H ? < 1 r o ? E ffi'3" . 00 S' *** 9 E g Cn O 2. O ^ - 3 -9 x g. O c 2 3 o ^ a. FIGURE T FIGURE U Graphs of conidial length of several Helminthosporiums under standard conditions: Graph 93, H. No. 14; Graph 94, H. No. 13; Graph 95, H. No. 15; Graph 96, H. No. 16; Graph 97, H. No. 17; Graph 98, H. No. 18; Graph 99, H. No. 19; Graph 100, H. No. 20. Graph f M a CV 93 404 22.04 =t .10 3.03 .07 13.76 == .33 94 597 24.78 =t .09 3.53 .06 14.24 .28 95 461 22 54 .10 3 .49 .07 15 .51 .35 96 445 23 75 =*= .12 3 .80 =t .08 16 .03 .37 97 252 24 39 .15 3 .63 .10 14 .88 .45 98 97 23. 03 =b .28 4 .10 =*= .19 17 .81 .88 99 205 24. 59 .19 4 .15 .13 16 .89 .57 100 315 18. 84 =t .12 3 .30 .08 17 .53 .48 FIGURE U FIGURE V Graphs of conidial breadth of several Helminthosporiums under standard conditions: Graph 101, H. No. 11; Graph 102, H. No. 20; Graph 103, H. No. 13; Graph 104, H. No. 14; Graph 105, H. No. 15; Graph 106, H. No. 16. Graph f M o- CV 101 39 5.19 d= .04 .40 .03 7.74 ^ = .59 102 58 5.11 .14 .52 .03 10.27 =i = .65 103 79 5.97 d= .08 1.06 .05 17.85 d = .98 104 32 5.59 d= .08 .74 .06 13.30 =" = 1.14 105 88 5.39 d= .04 .56 d= .02 10.50 d = .53 106 45 5.88 .05 .56 .04 9.61 d = .68 FIGURE V ? SSSSS =0 sf o n OJ O H- i- 1 K) KJ K3 O o ^* H- |f H- H- S " TO ffi 5- ts> NS OO ON Os 0\ (f ff H- H- o o *-* '** OO on I K> NJ O 00 Q H- H- H- < -i- H- ?1 Si s| a ? FIGURE W QBAPH NUMBEES I FIGURE X Graphs of conidial septation under standard conditions of H. No. la (111); H. No. Ib (112); H. No. Ic (113). Graph f M Or CV 111 49 6 .71 .10 1 .12 .07 16. 75 =t 1 .17 112 65 6 .61 =*= .09 1 .15 .06 17. 52 1 .08 113 17 6 64 .09 ,58 =*= .06 8. 85 1 .02 FIGURE X FIGURE Y Saltant Graph f M a CV Ml-5 114 160 23, 40 == .18 3. 53 == .13 15. 11 .58 M2-4 115 154 21. 77 .17 3. 17 .12 14. 56 =*= .57 M3-4 116 131 22. 66 * .20 3. 50 == .14 15. 48 .66 M4-6 117 168 22. ,72 .21 4. 04 .14 17. 80 =*= .67 M5-5 118 145 23. 72 .19 3. 52 .13 14. 86 .60 M6-5 119 166 23. 65 .20 3. 85 * .14 16. 28 .61 M8-8 120 156 23. 92 .18 3. 38 .12 14. 35 .55 M12-3 121 148 23 64 =t .20 3. 73 .14 15. 81 =t .63 M13-3 122 173 22 .73 .18 3. 61 .13 15. 90 .69 M14-3 123 126 22 ,32 .17 2. 98 .12 13. 37 .57 M15-3 124 134 23 ,51 .20 3. 51 =*= .14 14. 93 .62 M36-2 125 156 22 .31 =b .16 3. 14 =t .12 14. 09 .54 M37-2 126 131 22 03 .21 3. 64 .15 16. 55 .70 M32-2 127 86 23 00 .16 2. 33 .12 10. 14 .52 M33-2 128 155 22 ,37 .16 3 02 .11 13. 52 .52 M40-2 129 97 17 .70 =*= .17 2 51 .12 14. 19 .70 M41-2 130 148 22 .92 .19 3 .45 .13 15. 05 .60 M42-2 131 125 22 .36 .21 3 .53 .15 15. 81 .69 M43-2 132 139 22 .00 .19 3 .36 .13 15. 29 =t .63 M44-2 133 134 22 .18 .15 2 .72 .11 12. 27 .51 M45-2 134 142 22 .88 =*= .21 3 .76 .15 16. 43 .67 M46-2 135 158 22 .84 .17 3 .34 .12 14. 66 == .56 M47-2 136 112 23 .53 .22 3 .56 .16 15. 12 .69 M48-2 137 146 22 .00 .16 3 .02 .11 13. 78 .55 Ml 7-3 138 128 22 .56 .14 2 .38 =*= .10 10. 56 .45 FIGURE Y 114 fl5 116 117 116 1 19 120 121 \Z 123 124 S l25 I l26 I . 130 131 13 133 134 135 36 1381 X 7 \ X \ \ \ \ N \ \ V x \ \ X ^-oo in GO t co w ^ (O CU PLATE VII Several wheat stems showing characteristic diseased spots; also diseased portion at the node in one shoot. PLATE VIII Diseased plant, showing numerous dead leaves and leaf-sheaths, also more than a dozen new shoots issuing from below the diseased portions. These shoots varied in height from a few millimeters to several centimeters. PLATE IX ^22 H. Nos. 1, 3, 4, 5, 20, and 22, growing on corn-meal agar. PLATE X H. No. 36, showing very floccose mycelium. PLATE XI H. No. 3 (left) and No. 1 (right) as grown in Piorkowski-flask culture. PLATE XI PLATE XII H. No. 1 as grown in Kolle-flask culture. PLATE XII PLATE XIII H. No. 3 as grown in Kolle-flask culture. PLATE XIII PLATE XIV Petri-dish cultures of H. No. 1 on different amounts of agar: 14, on 12 c.c. ; 15, on 30 c.c. PLATE XIV 14 15 PLATE XV H. No. 1 growing in tubes of rice with different amounts of water. Note abundance of sclerotia in the drier tubes at the left. PLATE XVI Tj Braz.il- nuts TflUDl H. No. 1 grown on washed agar with nutrients added as indicated fragments of Brazil-nuts, rice, tapioca, and corn-meal agavy (Circles indicate approximate limits of growth at ' PLATE XVII Photomicrographs of H. No. 1, showing attachment of conidia to conidophores. PLATE XVIII Photomicrographs of H. No. 1, showing the fragile nature of the outer brown"] spore-wall and the gelatinous texture of the hyaline mass enclosed. (Three different magnifications.) PLATE XIX ;; v > Y' : * '**-> J5*/ \ &* * a */ ' *K^ - ' ', - * v * * \ *? f 3MH'g: ^- -' * Photomicrographs of H. No. 1, showing conidia under different magnifications. PLATE XX Photomicrograph of conidia of fl". ravenelii. PLATE XXI Conidia (a] of H. No. 36, showing variation in size and shape; b and c, conidia and a conidiophore of H. No. 39. PLATE XXII Two saltants: upper one showing origin of M5; lower one showing a white clump and slow growth. PLATE XXIII Two saltants: upper one showing origin of Ml; lower one of slow growth and bearing clumps. PLATE XXIV Upper figure showing origin of M2; lower figure showing origin of M30-M34. PLATE XXV Saltants growing with their respective originals. PLATE XXV PLATE XXVI Photomicrographs (same scale) of conidia of several Helminthosporiums: a, H. No. 1; 6,M6; c, M35; d, H. 20. PLATE XXVI PLATE XXVII Saltants growing with their respective originals. PLATE XXVIII M34, characterized by abundance of sclerotia and white mycelial clumps, the latter a constant character of this saltant. PLATE XXIX Above, H. No. 1 wounded by hot wire at points shown; below, H. No. 1, with H. No. 1 implanted at various distances within and without the colony. PLATE XXIX PLATE XXX Above, two implants of H. No. 1 one of them the origin of M70 in H. No. 1 colony, showing some white floccose aerial mycelium; below, M26, with origins of M53, M56, and M57. PLATE XXX PLATE XXXI \126-1 as it appeared on two separate plates. PLATE XXXI n 26-r PLATE XXXII M125. Pale colonies, showing dark sectors which were apparently reversions to the original form. PLATE XXXII PLATE XXXIII Showing method of using rag doll in inoculations (a, b, c) and also (d) vial and paper cylinder for soil inoculation: a, the rag doll unrolled in sterile Petri-dish, and aseptic wheat seedlings in place, ready for inoculation; b, doll in place in tube and seedlings growing; c, showing development of root hairs in condition for inocu- lation below the doll; d, as stated above. PLATE XXXIII PLATE XXXIV Rag doll opened for examination 6 days after inoculation. All the seedlings show beginnings of foot-rot. U.C.BERKELEY LIBRARIES 881261 THE UNIVERSITY OF CALIFORNIA LIBRARY