THE COMPOSITE LIFE HISTORY OF PUCCINIA PODOPHYLLI SCHW.1

By H. H. WHETSEL, Professor of Plant Pathology, Cornell University; H. S. JACK- SON, Chief in Botany, and E. B. MAINS, Associate in Botany, Purdue University Agricultural Experiment Station 2

The common rust, Puccinia podo- HISTORY phylli Schw., presents certain features whicñ make it of special interest to The only previous attempt to explain the student of the biology of this great the life history of this species and the group of plant parasites. The interest sequence of the spore forms was made in this species lies primarily in the by Olive {6). Based primarily on peculiar sequence in the seasonal cytological evidence, he reached the appearance of the spore forms. This conclusion that the first crop of telia species is an opsis-form, possessing and the pycnia and aecia arise from aecia and telia only, and occurs on the an intermingled growth of the mycelium common mandrake or May-apple, of the gametopihytic and sporophytic Podophyllum peltatum L., practically generations which are independently throughout its range. and simultaneously perennial in the Puccinia podophylli produces telio- host plant. He conducted no culture spores at two different periods during work nor did he attempt to verify this the season; the first crop appears early assumption by an histological study of in the spring on the sheath leaves or the root stalk or overwintering buds. bud scales, on the stem usually at the The following summary of Olive's base and sometimes on the sepals (PL 1, work is presented in some detail, A and B) ; the second crop appears later largely by quotation, in order to bring in the summer on the under side of the out clearly his cytological observa- , fully expanded and matured leaves tions and the conclusions he drew from (PL 4, B). Between these two crops them since it will be necessary to refer of teliospores the aecia, usually pre- to these details later in this paper. ceded by pycnia, are developed on the Olive found that the mycelium in the expanding leaves (PL 2). It is also of lesions on the sheath leaves that bore interest that telia often develop in the early telia was prevailingly binu- association with the aecial clusters on cleate, as one would expect. the leaves and to all appearances on The preparations also show, occasionally, a few the same mycelium before the appear- aecidium cups on these same sheaths * * *. I ance of the second crop of teliospores, have not yet found spermogonia on the sections of which obviously develop from aecial these young sheaths; but their occasional occurrence in such situations may be expected from the fact infection (Pis. 2, 3, and 4, A). The ex- that a small amount of uninucleate mycelium planation of the origin of these early occurs, especially in the region surrounding the telial sori and the determination of the aecidial sori, there forming the meager pseudo-paren- chyma. But the rust mycelium of the sheath, in life history of this species are the objects contradistinction to that in the young leaves, is of the study reported in this paper. undoubtedly prevailingly binucleate. Further, I

i Received for publication May 24, 1924—issued April, 1925. Joint contribution from the Department of Botany, Purdue University Agricultural Experiment Station and the Department of Plant Pathology, Cornell University. 2 A report on the experiment conducted at Ithaca, N. Y., together with an interpretation of the life history essentially the same as that given in this paper, was presented before the Botanical Society of America at the Pittsburgh meeting of the American Association for the Advancement of Science in 1917 by Prof. H. H. Whetzel (not published). The problem had been freely discussed with me during the two previous years and my interest aroused. It was mutually agreed that a more detailed cultural study would be desirable and this work was begun at LaFayette, Ind., in 1917, having been planned in collaboration with my associate, Dr. E. B. Mains, who carried out the details of the cultures. It was finally decided to present the combined results in a joint paper, the preparation of which has been my responsibility.—H. S. Jackson. 3 Reference is made by number (italic) to " Literature cited."

Journal of Agricultural Research, Vol. XXX No. 1 Washington, D. C. Jan. 1, 1925 Key N. Y. (Cornell)-5 13949—25t 5 (65) Composite life history of Puocinia podophylli Sohw. Piale 1

A.—Telia of Puccinia podophylli on the sepals. Note the aeciallike character of these telia. Natural infection slightly enlarged B.—Telia on bud scales and on the stem. Natural infection. Compare with Plate 4. B. Natural size. jan. i, 1925 Gpmposiie Life History of Puccinia podophylli Schw. 67

am convinced that the aecidia which are borne on the sheaths arise, not from gametophytic cell the secondary aecia on the leaves from fusions, but only from preexisting binucleate the same binucleate mycelium, the fol- hyphae; therefore being secondary and sporophytic lowing significant statements are found : in character, and thus similar in origin to the teleutospores. Quite likely the degree of maturity of the tissues and the quality of the nourishment supplied to the parasite govern largely this phenomenon of the He found in the lesions on the leaves early development of the teleutospores, just as these that a much more abundant uninu- factors doubtless determine the cessation of the cleate mycelium is present in the production of the repeating spores and the begin- ning of the production of the teleutospores in other younger tissues and that the pycnia long-cycled rusts. The tissues making up the leaf arise from this mycelium. sheaths of young shoots of Podophyllum are un- doubtedly mature, as well perhaps as poorly nour- In those older leaves, however, in which the ished, as is shown by the character of the contained aecidia have begun to form their chains of spores, protoplasm; hence they present conditions at a very binucleate mycelium has become quite prevalent early stage not met with again until the host gains in all the sections examined, especially at the bases maturity or even old age. of the aecidial cups. These sporophytic hyphae It is obviously quite impossible to suppose that intermingle with the uninucleate mycelium, often this early teleutosporic stage in Puccinia podophylli entering the broad, caeoma like base of th» young has arisen in any other way than from hibernating aecidium, there functioning directly as basal cells sporophytic mycelium, which has grown up with of the rows of the binucleate aecidiospores. In still the infected buds in the spring. It could not come older stages on leaves, binucleate mycelium appar- from an early infection since the aecidiospores, ently prevails by the time the aecidium cups have which we assume to be the only spores from the for the most part broken open to discharge their inoculation of which the teleutospores could arise, spores, * * *. have not yet begun to form. I will have to confess, however, that I have not been able to apply His examination of the later telia de Bary's test to this species and to look for hiber- which form from aeciospore infection nating mycelium in the underground parts. shows that they arise from a localized binucleate mycelium. CULTURE EXPERIMENTS In interpreting the results of his ob- servations he assumes that the uninu- EXPERIMENTS AT ITHACA, N. Y., cleate, or gametophytic mycelium, and 1916-1917 the binucleate or sporophytic mycelium, are independently but simultaneously One of the authors of this paper perennial in the plants from which his (Whetzel) has had Podophyllum rust material was taken and that the pycnia under observation since about 1906. are the only structure borne on the During 1912 he collected and fixed uninucleate mycelium, while both the much of the material on which Olive's early teliospores and the aecia develop cytological work was based and fur- from the binucleate mycelium. He nished him without reservation all the assumes that basidiospore infection information gained from the field ob- would result in a localized mycelium servations which ha/d then been made. This assistance is freely acknowledged on which pycnia and aecia would be 4 developed, the expectation being that by Olive in the paper referred to above. this would be prevailingly uninucleate. The elaborate, though entirely or- In connection with a discussion of thodox, explanation of the situation the principle that in perennial rusts the finally made by Olive did not seem to gametophytic mycelium in young Whetzel adequately to explain the phe- nomena which had been observed in shoots grows more vigorously early in Y the season than the sporophytic, the the field. On this account it was de- following quotation is of interest: cided to test the validity of Olive's hypotheses by culture experiments. The fact that the teleutosori break out very early from the tissues of the leaf sheaths, some distance The first of these was conducted at below the tip of the stem, and that the binucleate Ithaca, N. Y., during 1916 and 1917. mycelium prevails in these sheaths almost to the The plants used for the experiment exclusion of the uninucleate hyphae does not, to my mind, vitiate the above statement, which ap- had been brought several years before plies only to the younger tissues of the shoot. I from a patch of Podophyllum in which interpret these facts somewhat as follows: the uni- no rust was to be found and were nucleate mycelium grows with especial vigor into the rapidly expanding tip and young leaves of the planted on a wooded bank in Whetzel's new shoot, growing somewhat ahead of the lagging yard far away from any other patches sporophyte. The latter apparently chooses ordi- of Podophyllum. Since the planting narily the more mature tissues for its most vigorous growth and thus early comes to predominate in the was along the path by which he reached riper tissues of the poorly nourished leaf sheaths of the road on his way to the office each Podophyllum, as well as later in the older leaf day, he was able to determine by careful tissues. inspection each year that no rust ever As a part of Olive's discussion of the appeared either on the bud scales, stems probable reason for the development of or leaves of these plants. In the sum- the early telia on the sheaths and later mer of 1916 he collected a large quantity

4 A detailed manuscript record of these experiments, together with the correspondence which passed be- tween Prof. Whetzel and Dr. Olive on the Podophyllum rust, is deposited in the library of Cornell Uni- versity under the title, " Notes on the Life History of Puccinia podophylli," bytH. H. Whetzel. Composite life history of Puccinia podophyili Schw. Plate 2

A,.—Upper surface of leaf of Podophyllum peltalnm inoculated with teliospores from stem sori. Note abundance of telia in aecial lesions and telia at base of stem B.—Upper surface of aecial lesion showing telia along the veins. Natural Infection C.—Lower surface of same lesion as B, showing aecia. Natural infection Jan. i, 1925 Composite Life History of Puccinia podophylli Schw. 69 of Podophyllum leaves which bore the season for the development not only second, or summer crop, of teliospores of another crop of early teliospores on in abundance, put them between two sheath and stem but also for pycnia and sheets of window screen and placed aecia later. this over one end of the established rust-free planting in his yard. This EXPERIMENTS AT LA FAYETTE, IND., plot had developed so that it was 1917-1921 about 8 feet long and rather narrow. The screen packet was about 12 by 18 Since this experiment was conducted inches. During the fall and winter entirely out of doors and the usual the Podophyllum leaves in the screen objections to such experiments would rotted and the spores fell with the naturally arise, it was decided to repeat débris through the meshes upon the the work with variations, in part at surface of the soil. Early in the spring least under controlled conditions, and of 1917 the empty screen was removed to conduct this work in connection with and careful observations were made on the rust investigations under way at all the plants in the plot at frequent the Purdue University Agricultural, intervals as they developed. Telia Experiment Station at La Fayette, Ind. soon appeared on the sheath leaves and EXPERIMENT 1.—During the sum- stems of nearly all the plants which mer of 1917 patches of Podophyllum came up in the area covered by the were located in the woods, some of screen packet. No rust appeared on which were heavily rusted and some of plants elsewhere in the bed during the which showed no rust. An equal entire season. A seedling in the infect- number of plants, about 25, were dug ed area also developed aecial lesions on in November from each of two plots, the leaf blade in the spring of 1917. one of which was rusted and the other Two nearby seedlings showed telia only not. The rhizomes were very care- on the stems. One of these seedlings fully washed and cleaned of all soil or stood directly under that bearing aecia dead parts and planted in a single row and when all three were removed on in a vegetable garden (Jackson's), June 4, 1917, it showed the whitish with a stake dividing the two lots. A lesions of the developing but unopened frame 18 inches by 48 inches made of summer telia. However, no summer boards 4 inches wide was placed on the crop of telia appeared on any of the ground in the middle of the row in such other plants in the patch during 1917. a position as to include 5 plants from In 1918, while leaf sheath and stem the rusted patch and 5 from the one lesions bearing the early telia were which showed no rust. A peck or less common, only a single plant developed of surface soil and débris taken from a an aecial lesion. This wTas allowed to patch of Podophyllum which had been remain, and later nearly every leaf blade heavily rusted during the previous in the patch showed a more or less summer was evenly spread over the abundant development of the summer soil in this frame. No protection was telia. Since that time this patch of given the plants during the winter. plants has been rusted more or less In the spring of 1918 all 10 plants each year, all stages of the rust appear- in the frame showed the early telia ing in the manner usual for this species. on the bud sheaths or stems and later This experiment, while not in itself about half of them showed pycnia and conclusive, strongly suggests that the aecia in fair abundance on the blades interpretations of the life history made of the leaves. No infection of any by Olive are not in accord with the sort occurred on the other 40 plants facts. It would appear from this at either end of the frame. The experiment that the infection on the experiment was not continued since sheaths and stems, resulting in the the ground was needed for other early crop of telia, arose directly from purposes. the basidiospores developed from the Were the rust perennial in the late crop of teliospores which were rhizomes some of the plants outside used as the inoculum and that these the frame which had been dug from teliospores were responsible also for the rusted patch should have been infection on the seedling leaves, result- rusted. The infection on the plants ing in pycnia and aecia. Since the in the frame presumably resulted from aecial-bearing seedling was removed the teliospores present in the top soil along with the seedling showing the used as a mulch. initial stages of the summer telia, and EXPERIMENT 2.—In November, 1917, since no other plant in the patch showed about 50 rhizomes dug from a patch summer telia during 1917, it would fur- of Podophyllum which showed heavy ther appear that the spring crop of infection on nearly every plant during teliospores was responsible the next the spring were thoroughly washed. 70 Journal oj Agricultural Research Vol. XXX, No. 1

Some of these were placed in flats, conduct some of the experiments with covered with potting soil, and placed somewhat more refined methods and out of doors with a heavy covering of under as strict control as is feasible in straw. Some were potted at once and dealing with growing plants and with placed in the greenhouse. The material a fungus which can not be grown in which was placed in flats was brought pure culture. into the greenhouse in March, 1918, EXPERIMENT 3.—The telial material when the leaves were just emerging which was used for the following in- from the sheaths. No rust developed oculation experiments was obtained in on either set of plants, though they the spring and summer of 1917 in the were grown to maturity. same patches of Podophyllum from In March, 1918, before the buds had which the rhizomes used in the previous swollen to any extent, a second lot of experiment were taken. The first rhizomes were dug from a patch which material was collected May 28, 1917, was known to have been heavily rusted and consisted entirely of sheaths and during the previous summer. These stems bearing the early crop of telia. were thoroughly washed, potted in These were placed out of doors on the potting soil, and allowed to develop surface of potting soil in flats and left in the greenhouse. No sign of rust till the following spring, 1918. On infection appeared on any of them. July 17, 1917, material of the late crop An inspection of the remaining of telia which develop from aecial in- plants in the patch of Podophyllum fection and occur abundantly scattered from which the last-mentioned rhi- on the under surface of the leaves was zhomes had been dug was made at collected and handled in the same way intervals during the spring and sum- as the sheath and stem material men- mer of 1918 and the plants were found tioned above. Finally, in the fall of to be heavily infected, showing the 1917, after the Podophyllum plants had early development of telia on the died down and the leaves were more or sheaths and stems, pycnia and aecia less disintegrated, surface soil from a later on the leaves, and finally the patch of heavily infected Podophyllum late crop of telia on the under side of was collected and placed out of doors the leaves. to winter. At the same time rhizomes Stems showing the early telial sori were again collected from a patch of and the attached rhizomes were col- Podophyllum which had shown no in- lected in the spring of 1918 and were fection during the year and were studied in an effort to determine the placed in flats, covered with potting extent of the mycelium. Free-hand soil, and left out of doors with protec- sections were made, beginning in the tion during the winter. In March, region of the sori and progressing down 1918, these rhizomes were brought into the stem into the rhizome. The sec- the greenhouse, thoroughly washed, and tions were mounted in chloral hydrate potted. Some of the pots were given and iodine, which furnishes a satis- a surface coating of the surface soil factory medium for the study of such obtained from the infected Podophyl- sections. The mycelium is easily dis- lum patch, some were mulched with the tinguished by this method and was soil and stems from the flat prepared in found only in the immediate region of May, 1917, and some with the soil and the sori. No mycelium was discovered leaves from the flat prepared in July, lower down in the stem or in the 1917, as described above. It should be rhizome. pointed out that the sheaths, stems, and This series of experiments taken in leaves had largely disintegrated after conjunction with the one made at overwintering and most of the telio- Ithaca would seem effectively to dis- spores were mixed in the surface soil of pose of the hypothesis put forward by the flats in which they had been kept; Olive that the rust is perennial in the hence the reason for using the soil as a buds or rhizomes. mulch. As the buds of the potted A number of other experiments were plants developed they pushed up considered desirable, however, in order through this mulch and became ex- to determine as accurately as possible posed to infection. just what did occur. The question The results of this experiment were naturally arose as to whether there as follows: In one of the pots mulched might be some difference in the telio- with soil, sheaths, and stems bearing spores formed early in the season on the the early teliospores, pycnia and a few stems and sheaths and those formed aecia developed on the leaves of one later from aeciospore infection, either plant and pycnia and aecia together in their ability to cause infection or in with a few telia immediately associated the sequence of resulting spore forms. with the aecia on another. In the pot s It was also considered desirable to mulched with soil and leaves bearing Jan. i, 1925 Composite Life History of Puccinia podophylli Schw. 71

the late crop of teliospores, four plants showed only aecia and with which no showed pycnia and aecia on the leaves pycnia were observed. The inoculation in numerous spots, some of which on was made on the expanded leaves, and all four plants in later stages of develop- fully mature telia developed on June 9. ment showed accompanying telia. In The second sowing was made on May the pots mulched with soil from the 3, 1918, using aeciospores from plants infected patch one plant showed a few collected in the field on which the pycnia and abundant telia on sheath aecia were accompanied by pycnia. and stem, together with pycnia and Sowings were made on one mature aecia on the leaves in numerous spots plant and three younger plants. An in association with which telia were not attempt was made to inoculate the uncommon, and the other plant showed sheaths and stems as well as the numerous spots with pycnia and aecia, leaves. Telia appeared, however, only with some of which were associated on the leaf blades of the three younger telia. In all cases where telia accom- plants, being fully mature on May 20. panied the aecia on the leaves they were A third sowing was made on May 10, most commonly found on the veins 1918, using aecia obtained from the which were involved in the areas of inoculations described in Experiment aecial infection. A control series of 3. The inoculation was made on pots in which the rhizomes were several plants in various stages of mulched with ordinary potting soil maturity. Telia developed only on showed no infection of any sort. the leaves of the younger plants. EXPERIMENT 4.—In this experiment In all of these sowings the telia ob- overwintered teliospores from the late tained wTere of the type occurring gre- crop on the leaves, which by previous gariously in spots on the under side of test were known to be germinating, the leaves, commonly found in the were sown at three different dates on field in midsummer, and referred to in Podophyllum plants taken from an this paper as the late crop of telia. uninfected patch. This work was all These experiments confirm those of done in the greenhouse. The first Arthur (1) and effectively dispose of the sowing was made on April 27, 1918, on idea that repeating secia might be pres- leaves and stems of four plants, three ent in the life history of the Podophyl- of which had the folded leaves well out lum rust. of the sheaths and the fourth just LATER EXPERIMENTS.—During 1919, emerging. The result showed devel- 1920, and 1921, other experiments were opment of pycnia and aecia on leaves conducted, which confirmed the results of all plants and telia on the stem of one. of those outlined above in all details. Epiphyllous telia developed in a num- In a few cases telial material which was ber of the aecial areas, mostly on the observed to be germinating in hanging- veins. The second sowing was made drop tests was transferred to the plant. on May 6, 1918, on six plants of The results were the same as where less Podophyllum all of which were well refined methods were used. out of the sheath, with the leaves It may be noted that only a few partly expanded. Pycnia and aecia records of infection on the sheath were developed on the leaves of all leaves are made in these experiments. plants. Some of the aecial lesions on This, it should be explained, is due to each plant showed telia in immediate the fact that some difficulty was en- association. In one case telia were countered, under rather dry greenhouse developed in association with pycnia conditions, in preventing the sheaths without aecia being present. In the from drying up before the telia devel- third sowing on May 11, made on the oped. In many such cases, however, leaves of more mature plants, numerous evidence of infection was observed, and lesions bearing pycnia and aecia devel- in the later experiments of 1919 to 1921, oped on two of the plants. Telia this phase of the problem was given were also developed later in association special attention. with the aecia on a majority of the spots. On both plants a few spots SUMMARY OF RESULTS OF containing aecia developed in associa- EXPERIMENTS tion with which no pycnia were observed. A careful analysis of the results of EXPERIMENT 5.—This experiment these experiments shows that the writers dealt with the infection from aecio- have obtained the development of telia spores. Three sowings were made on sheaths and stems, and pycnia and under control conditions in the green- aecia with associated telia on the leaves, house. The first sowing was made on by inoculating with germinating telio- May 28, 1917, using aeciospores col- spores of both the early and the late lected in the field from plants which crops. 72 Journal oj Agricultural Research Vol. XXX, No 1

No basis was found for the idea that The basidiospores fall upon the por- the rust is in any sense perennial or tions of the plant which are exposed that any condition* approaching an in the early stage of development and "unlimited infection" is evident. It infection results. The sheath leaves would appear that all of the structures or bud scales are the first organs ex- develop from a localized mycelium and posed. As the aerial stalk pushes forth that this mycelium in all cases (except from within the enfolding bud scales, the late crop of telia) arises from basid- both the base of the stem and the leaf iospore infection. It is also evident blade are also exposed to infection. that the first spore structure borne on The upper part of the stem is rarely this mycelium may be either telia, or infected as it long remains protected pycnia accompanied by aecia, and in by the enfolding leaf blades. The two rare cases aecia without pycnia. sepals of the flower are, however, not These experiments when taken to- uncommonly infected, for they are usu- gether with field observations made at ally partly exposed as the young plant Ithaca, N. Y., and La Fayette, Ind., emerges (PL 1, A and B). would seem to make necessary an inter- The period of basidiospore produc- pretation entirely different from that tion (that is, teliospore germination) made by Olive. apparently is relatively early and short. Very numerous infections occur on the LIFE HISTORY AND SEQUENCE OF scale leaves and on the base of the SPORE FORMS stems. This is perhaps due to the fact that they are nearer the soil surface It seems desirable, therefore, as a and in the more effective zone of basis for further discussion, that the basidiospore dissemination. At Ithaca writers here set forth in some detail it has been noted in some seasons that what they conceive to be the regular only certain scale leaves, such, for ex- sequence of events in the life history ample, as the second from the top, show of this rust. infection. This observation (Whet- In the spring, at the time when the zel's) was referred to by Olive as a buds of the Podophyllum plant are "curious fact." The explanation is, emerging from the ground, the telio- however, comparatively simple. These spores of Puccinia podophylli are pres- "second" scale leaves were the only ent in the surface layer of soil around ones exposed in the particular patch the developing buds. These telio- under observation at the time of in- spores are derived both from the spring oculation. There are three or more of crop of telia on leaf sheaths, stems, and these buds scales at the base of the sepals and those associated with aecial stem. The top scale had not emerged lesions, and from the summer crop pro- from within the protecting outer second duced on the leaf blades. They have scale and the shorter third scale was either fallen to the ground during the still covered by leaf mold. It has previous summer, as the telia are of the since been observed, in the same patch, pulverulent type, the pedicles breaking that all the scales and the base of the close to the spore, or they have been stem may be infected. It would appear liberated by the rather complete disin- that these differences are the result of tegration of the host tissues. The seasonal variation in the coincidence spore wall is beset with sparsely scat- of basidiospore production and de- tered rather long spines which would velopment of the host and of variations serve to prevent the spores from being in the extent of exposure of the bud carried too deeply into the soil or scales above the surface of the leaf washed away during heavy rains. mold. At the time when the sheathed leaf From the basidiospore infections on buds begin to push their pointed tips the scale leaves, the base of the stem, above the surface of the soil, these telio- and the sepals, telial sori are developed spores (of both crops) begin to germi- almost exclusively. Pycnia and aecia nate in the normal way for teliospores, are occasionally found intermixed with that is, by the development of a typical the telia, the latter usually occurring promycelium and sporidia. The scale singly. Both are, however, rather rare, leaves form a sheath about the base of and by far the greater number of the stem, through which the young lesions include telia only. In one case plant emerges. These scale leaves noted at LaFayette an isolated lesion usually extend slightly above the on the stem bore a group of pycnia ground level. The lobes of the two surrounded by a ring of telial sori, peltate leaves of the flowering stalk exactly similar in appearance to many are folded about the stem in a convolute true microforms of Puccinia. manner and the single blossom bud is The occasional development of pyc- partly exposed. nia and aecia among the telia on sheath Jan. i, 1925 Composite Life History of Puccinia podophylli Schw. 73

leaves is readily understandable when similar situations aecia may occasion- one has the fresh specimens in hand. ally be found with a few teliospores in These always occur on the thicker, the margin. In one specimen studied fleshier parts of the sheaths. These a sorus was found, one-half of which parts mature more slowly than the consisted of aeciospore chains and one- margins, thus simulating the conditions half of teliospores. obtaining in the longer-growing tissues Occasionally aecial lesions may be of the leaf blade where aecia normally found in which pycnia do not develop develop from this primary infection, as and quite commonly only a very few noted below. pycnia are found. When infection The infection on the sepals is not takes place on leaves which are rela- commonly observed because they soon tively quite mature the number of drop off. If a search is made, however, telial sori formed may be greater than at the right time, they can usually be the number of aecia (PI. 3, A and B). found in considerable abundance on In one instance noted in the culture the ground among flowering stalks, and results, telia accompanied by pycnia, if infection is abundant in the patch on but with no aecia present, resulted from stems and sheath leaves these fallen basidiospore infection on the leaves. sepals will almost invariably be covered Such a condition exactly parallels the with telia. Infected sepals still at- normal result when infection occurs on tached to the plant have, however, the sheaths and stems, and is compa- been found both at La Favttte and rable to the usual condition of a micro- Ithaca (PI. 1, A). Puccinia. When basidiospore infection occurs It has been observed at Ithaca, on the blade of the leaf, aecial lesions N. Y. (Whetzel), that a very much almost invariably result. These show smaller number of leaf blade infections at first pycnia on the upper surface, fol- resulting in aecia occur than of infec- lowed shortly by the aecia below. It tions on scale leaves and stems result- should be emphasized that the basidio- ing in telia. Patches of Podophyllum spores which cause this infection may have often been noted in which the result from the germination of either early crop of telia on sheaths and stems the early or late crop of teliospores of was almost universal, but in which no the previous season. aecial lesions on the leaf blades devel- The aecial lesions are rarely evident oped during the season. In that local- until after the teliospores on the sheath ity no cases have been observed where leaves, base of stem, and sepals have more than a small percentage of plants begun to mature. Fully developed showed aecial infections. This, how- aecia are seldom found earlier than a ever, is not the case at La Fayette, Ind. week after an abundance of mature Aecial lesions on the leaves are usually teliospores appears on sheaths and found in considerable abundance in in- stalks. It is obvious that these early fected patches. It would be interest- telia could not have been produced ing to have data on this point from from aecial infection. The delayed ap- other sections of North America, par- pearance of the aecia is perhaps due ticularly from regions at the northern to the slower development of the fungus limit of the range of the host species. in the less rapidly maturing tissues of The aeciospores are scattered by the the leaf blade. wind and cause infection of the sur- As the aecial lesions begin to mature, rounding expanded leaves of the Podo- that is, when the cups are open and the phyllum plants. This infection is usu- spores are being discharged, one often ally very profuse and general, and after finds, usually on the upper surface, a period of ten days or two weeks angu- about the margins of these lesions or lar yellowish spots appear, on the under more commonly on any larger veins side of which are developed the grega- which may be included in the lesion and rious, pulverulent telia bearing the sec- even in the center of the spot among ond or summer crop of teliospores the pycnia, a rather abundant produc- (PI. 4, B). These mature and fall to tion of telial sori along with more or the ground to mingle with those of the less development of epiphyllous aecia early crop, which have already been (Pis. 2 and 3). These telia are like largely disseminated. those developed on sheath and stem and Reference has repeatedly been made are certainly developed on the same to the two crops of teliospores. Those mycelium as the aecia. A careful study which appear early in the season on the made by one of the writers (Jackson) sheaths, stems, sepals, and in associa- of the sori in such lesions has revealed tion with the aecia on the leaves form the fact that occasionally a greater or the first crop, and those which arise less production of chains of aeciospores from the infection of aeciospores form may be found in these telial sori. In the second crop. There is, however, 13949—25t 6 Composite life history of Puccinia podophylli Schw. Plate 3

A.—Upper mrteM of leaflet inoculated when quite mature with overwintered teliospores o late or summer crop. Note abundant production of telia and scanty infection B.—Lower surface of same leaflet shown in A. Note development of telia almost to the exclu- sion of aeoia C.—Upper surface of leaflet inoculated when young with overwintered teliospores from the early crop on the stems. Note abundant production of telia among the pyenia and the vigorous infection D—Lower surface of leaflet shown in C. Note abundant development of aecia with an occa- sional telial sorus. Contrast with B jan. i, 1925 Composite Life History of Puccinia podopJiylli Schw. 75

in addition to the difference in seasonal GENERAL DISCUSSION appearance, a noticeable difference in the character of these two sorts of telia, It is evident from the above that the difficult to describe but easily recogniz- writers' interpretation of the life history able when one has the specimens before differs radically from that of Olive. him. In the early crop, the telia are It now becomes necessary to attempt usually somewhat larger, less definitely to correlate this new interpretation circular in outline and somewhat with Olive's cytological observations. longer covered by the cinereous epi- The writers have at present no basis for dermis. They also cause a slight but criticizing these observations nor do distinct hypertrophy of the host tissue they see any reason, for purposes of similar to that produced by the aecia. discussion, for not accepting them as When on succulent tissue they also matters of fact. appear to have their origin much It must be admitted, however, that deeper in the tissues. This difference in view of the experimental results is perhaps best brought out by com- recorded above, it would be highly paring Plate 1, A and B with Plate 4, desirable that the cytological situation A and B. in Puccinia podophylli be reinves- It seems probable that this differ- tigated. It is apparent that we are ence in type of sori is correlated with dealing with a rust showing consid- the fact that the early crop of telia on erable plasticity in its development sheaths, stems, and sepals, as well as since from basidiospore infection telio- those which occur in association with spores are the first spore form produced the aecia, have a gametophytic origin, under some conditions, while under while the late crop which develops others, pycnia followed by aecia occur. from aeciospore infection is sporo- It will be recalled that Olive found phytic in origin. a small amount of uninucleate mycelium with the developing telial sori on the The writers have been unable to obtain any evidence either from observ- sheaths and an abundance of such ation or cultures to show that this mycelium in lesions on the leaves, but species possesses secondary or repeat- that the aeciospores as well as the ing aecia. Cultures conducted both teliospores developed on a binucleate in the field and in the greenhouse under mycelium. No so-called sexual fusions control conditions have shown that the were observed to take place at the base of the aecial cups. It was assumed that aeciospores give rise to the summer crop all the telia and aecia were formed on a of telia. perennial binucleate mycelium and the pycnia were formed on a perennial SUMMARY OF LIFE HISTORY uninucleate mycelium. The aecia were then assumed to be secondary in nature The teliospores of both the early and and sexual fusions would not be late crops are functionally and morpho- expected. They were to be expected, logically indistinguishable and the according to this view, in the primary basidiospores developed on the promy- aecia which would originate from basidi- celium of either sort may, after ospore infection. These Olive assumes overwintering, cause infection on any he had not seen, and evidently con- exposed portion of the Podophyllum sidered it doubtful whether they would plant. When infection takes place on be found to be present in the life the sheath leaves, stems, or sepals, history. telia are at once produced. They may In view of the evidence already or may not be accompanied by a few presented it would hardly seem neces- pycnia or aecia. When, on the other sary to consider seriously the possibility hand, infection takes place on the blade of a systemic perennial mycelium in the of the leaf, pycnia followed by aecia case of this parasite. However, the are developed. Aecia may develop paper by Olive has given this theory without accompanying pycnia. Telia such general acceptance that it seems similar to thoáe on sheaths and stems necessary to emphasize the fact that may or may not accompany the aecia the writers have not been able to on the leaves, and when present develop obtain the slightest evidence that the from the same mycelium as the aecia. rust is in any sense perennial, or even The late or summer crop of telia systemic, in the host plant. develops from aeciospore infection. According to the view of the writers, There is no evidence of repeating aecia. accepting the cytological findings of Composite life history ot Puocinia podophylii Sohw.

A.—Upper surface of leaf showing aeoial lesion which involves the disk and the -veins. Note the pycnia and the abundant development of telia. Natural infection B.—The late or summer crop of telia on upper and lower surface of leaves. Compare with A and with Plate 1, A and B. Natural infection jan. i, 1925 Composite Life History of Puccinia podopJiylli Scliw. 77

Olive, the conjugate condition may be by the mycelium during the develop- assumed to arise on the mycelium before ment of the aecia and aeciospores, or the primordium of the sorus develops, directly to the more mature condition or at least in a very early stage of its of the tissues. Perhaps, however, development. In connection with the their formation is best explained by a sori on the sheaths, stems, and sepals, combination of these two influences. the change from a uniucleate to a In some cases the telia that develop in binucleate condition would be expected association with the aecia appear prac- to take place soon after infection, tically simultaneously with them. This which would account for Olive having is especially true wThen the lesion in- found uninucleate mycelium only in volves the midrib or one of the larger small amount. On the leaves, how- veins. In such cases the explanation ever, the change would be expected to is similar to that given above to ac- take place later, after the pycnia are count for the development of telia only formed, and hence, as Olive found to on stems, sheaths, and sepals. The tis- be the case, a considerable mass of sues of the veins and midrib are quite uninucleate mycelium would be "ex- comparable in the character of the pected. food supply to that of the stem. It Once the binucleate condition has has already been noted in the discus- been established in the mycelium there sion of the life history that when ma- are no difficulties from a cytological ture leaves are infected with basisio- point of view as to the spore form spores, telia may predominate over the produced, since both aeciospores and aecia in the resulting lesions and in teliospores arise from binucleate cells. rare cases may be formed exclusively. It is very likely that the type of spore It has been repeatedly observed in produced is determined by the nutri- connection with many long-cycle rusts, tional conditions prevailing in the host. that as the hosts approach maturity However, it should be borne in mind there is a gradual reduction in the de- that the tendency to an unstable con- velopment of repeating spores and a dition is quite certainly inherent in corresponding increase in the develop- the rust itself and the nutritional ment of the teliospores either in the factors are best interpreted as second- same or separate sori. It would seem ary, serving primarily to explain the that this situation exactly parallels situation as we find it and in view of that in the Podophyllum rust except our present, perhaps incomplete, cyto- that since there are no repeating spores logical knowledge. the teliospores developed in association That telia are the first structures with the aecia normally occur in sep- developed from infections on the arate sori. As noted in the discussion sheaths, stems, and sepals is perhaps of the life history, however, teliospores correlated with the influence of the may rarely develop in the aecial cups. rapid maturity of the tissues of these It is important to note, however, organs and the nature of the available that only a few other cases have hitherto nutrient. This influence was recog- been observed where aecia have been nized by Olive. He used it, however, replaced by telia. In most rusts the ma- to explain why telia were formed turity of the tissues affects aeciospore instead of " secondary " aecia on the production largely by reducing the "perennial" sporophytic mycelium. number of aecia produced or the The occurrence of telia in associa- quantity of aeciospores. It has been tion with the aecia on the leaf blades, repeatedly observed in the laboratory which has been repeatedly mentioned, at La Fayette in connection with is readily correlated with Olive's cyto- heteroecious culture work that when logical observations and can best be over-mature leaves or those in a poor explained also by the factor of food re- growing condition are inoculated, while lations. Olive finds that the aecia are pycnia may be formed to some extent, borne on a binucleate mycelium. It few if any aecia develop. The fact would appear quite logical to expect that aeciospores are replaced by telio- that this same mycelium might produce spores in the Podophyllum rust is telia when the factors which determine therefore another indication of the teliospore production become opera- plastic and unstable condition of this tive. species. The development of telia in associa- The assumption that the conjugate tion . with aecia usually occurs some- coixdition arises at an early stage in time after the majority of the aecia are the development of the mycelium can mature. It would seem reasonable to also be used to explain the fact that suggest that the development of telia aecia may occasionally be developed under these conditions may be due to without the pycnia. In such cases the depletion of the local food supply the change may have taken place 78 Journal oj Agricultural Research Vol XXX, No. 1

very early, before a sufficient mass of the general mycelium appears to exhibit single nuclei, but the mass of mycelial hyphae round mycelium has been formed to enable about the teleutospore sorus as well as those directly the pycnia to develop. Either aecia connected with teliospore formation appear to have or telia might then develop on the conjugate nuclei. mycelium as the first spore form fol- They did not find how or where the lowing infection, dependent on the change took place. Moreau (4), how- available food supply or the maturity ever, found all the mycelium uninu- of the tissues. cleate and the binucleate phase begin- It is well to recall at this point that ning in lower cells of the telial primor- the telial sori on stems, sheaths, and dium. Kursanov (3), on the other sepals strongly suggest the normal sit- hand, more nearly confirms the find- uation in microforms, and it is enlight- ings of Blackman and Fraser (2). He ening to review briefly the cytological finds, however, only a small amount of situation which has been foutid in the uninucleate hyphae, the vegetative species of short-cycle Puccinia and mycelium being prevailingly binucleate. Uromyces which have thus far been A more detailed review of the litera- studied. ture of this subject would show that in It is convenient to consider these in the first group there was considerable two groups. In the first the primor- variation in the amount of sporophytic dium of the telium is made up of uninu- tissue developed between the origin of cleate hyphae, and the conjugate con- the binucleate condition and the forma- dition arises as a definite process, by tion of teliospores. cell fusion or nuclear migration, in a We apparently have, therefore, in manner similar to that found in aecia. some forms of short-cycle rusts a very This is the general situation in the short sporophyte, while in others the following species: Puccinia (Nephlyctis) origin of the conjugate condition has transformans Ellis and Ev. investi- arisen at varying distances back from gated by Olive (5), Puccinia (Polythelis) the mother cell of the teliospores, some- fusca (Pers.) Wint. by Pavolini (7), P. times only a little way, sometimes far, malvacearum Bertero by Werth • and possibly even near to the point of infec- Ludwigs (8), P. buxi DC. by Moreau tion by the gametophytic basidiospore. (4) and Uromyces scutellatus (Schrank) It seems to the writers that this Lev. and Puccinia rossiana (Sacc.) cytological situation, especially as found Lagh. by Kursanov (8). Uromyces in the second group of species men- laevis Körn., studied by Kursanov, tioned above, parallels very closely that probably also belongs in this group, described for P. podophylli by Olive. but the actual process of the formation It is unfortunate that in these short- of the conjugate condition has not cycle forms the fusions have been ob- been observed. served only in the cases where the In the second group the primordium change takes place at the base of the of the telium is made up of binucleate telium. This circumstance, however, hyphae and the vegetative mycelium serves to point out the wealth of prob- is also primarily binucleate. The tran- lems awaiting solution in this field, and sition from a uninucleate to a binucleate it is hoped that students of cytology will condition in these forms has unfor- soon give us the necessary link to make tunately not been observed, but it is the chain of evidence complete. assumed by the authors that it occurs very early in the vegetative growth of PHYLOGENETIC CONSIDERATtONS the mycelium. This group includes Puccinia fergussoni Berk. & Br., P. It is pertinent to ask what is the asarina Kze., P. aegopodii (Schum.) significance of the peculiar life history Mart., Puccinia sp. (P. confería Diet. & as shown by the Podophyllum rust. Holw.?) on Artemesia, and Uromyces Without attempting at this time to go gagae Berk, as investigated by Kursa- into the evidence which has determined nov (3) and P. adoxae Fuck, and their position, it may be stated that the Uromyces scillarum (Grev.) Wint. by writers of this paper are among those Blackman and Fraser {2). who believe that, regardless of what It should be pointed out that, except the most primitive rust may have been, for Puccinia malvacearum, P. buxi, and the long-cycle species in the group P. rossiana, the species of the first group Pucciniaceae at least are the older and normally develop pycnia; while in that the short-cycle forms as exempli- those of the second group, without fied by the microforms of Puccinia and exception, pycnia are normally absent. Uromyces are derived or reduced forms Uromyces ficariae Wint. is especially and are relatively more recent. interesting in this connection. Black- It follows, then, that during the man and Fraser (2) record that— process of development from a long to Jan. i, 1925 Composite Life History of Puccinia podophylli Schw. 79 a shorter type'of life history some inter- 8. When mature leaves are infected grading conditions are to be expected. telia may predominate over the aecia, It is not at all surprising also to find with or without the development of evidences of a profound readjustment pycnia. and an apparently unstable condition 9. Puccinia podophylli exhibits evi- in the nuclear phenomena in some dences of being in an unstable or plastic condition as to life history. The writers believe that Puccinia 10. It is suggested that the food con- podophylli is a form which still exhibit ditions of the various tissues invaded evidences of the sort "bf changes which have an important influence on the may have taken place in the develop- spore form developed. ment of the reduced forms from the 11. This species is believed to be a more complex. form which still exhibits evidences of the sort of changes which may take SUMMARY place in the evolutionary development from the complex to the simpler forms ' 1. Puccinia podophylli shows no evi- of life history. dence of being perennial or systemic in the host. 2. The early crop of teliospores which LITERATURE CITED occjir on the bud scales, stems, and [JB,J. C. sepals preceding the aecia arise directly from mycelium produced by basidio- Mycol. 11: 60-67. spores from overwintered teliospores !) BIACKMAN, V. H., and F and are usually not accompanied by pycnia. 3. The aecia which normally develop 1922. on the blade of the leaf also arise in a Na similar manner from the same source. (4) MOBEATJ, g. Pycnia are usually found associated. LES UEÉDIN 4. The late or summer crop of telia (5) OUVE, B.\ ' 1908. SEXUAl are produced on mycelium developed NUCLEAB DO from infection by aeciospores. 22:331-360. 5. There is no evidence of repeating (6) 1913. aecia in this species. AND QAMETOPHYTIC 01 6. Basidiospores from either the early P. OBT«—-., ..-.- or late crop of teliospores may result in . Ann. Mycol. 11:297-311,11108. the production of either the early telia or the aecia. 7. Telia may develop in association with the aecial lesions and arise di- rectly from the same mycelium.