‘II I III. IIIII'IIIIIIIHI I Hg; Imo I (00000 A STUDY OF THREE FORMS OF ORANGE RUST 0N RUBUS. WITH SPECIAL REFERENCE TO NUCLEAR CONDITIONS I. RUSTS 0N DEWBERRY. RUBUS VILLOSUS THESIS FOB THE DEGREE OF M. S. J‘ L‘ Forsberg 1932 ”v lbor. nvnuu lm-nm ~ -. A A STUDY OF THREE F0815 OF ORnI‘IGE RUST ON RUBUlé, WITH SPECIAL REFZREIICE'IO I‘iUCincLR COIIDI‘IICELS I . RUSTS ON DEIBEEEIY, amvs 313:9 egg Thesis Presented for Degree of Easter of Science michigan State College THESIS Introduction and Review of Literature.... Statement of I‘roblagg..................... Lie-t1..0d8000000000000OOOOOOOIOOOOOOOOOOOOOO Short Cycle Form Producing Four Spori-zriia. Long, cyCle 1.70m1000000000000000 Short Cycle Form Producing: {Cw Further PTOI‘lBI‘S I o o o o o o Sunnnary................ BibliogmphlV'00 O O o o 0 O O I o Ebcplanation of flutes. . O Sporidia o ACKI I (JUL-.3013 IT S The writer is greatly indebted to Dr. E. A- Bessey for helpful advice and SHEISSthNS g'ven during this work; ale for aid in translation of foreign references and criticism and correction of the manuscript. The writer is indebted to Dr. Ray Eelson .for photographic work. A STUDY OF THREE F 0913 OF Oil-£3.03 RUST OI‘I RUBUS, WITH SPECIAL REFEREKCE TC NUCLEAR CUNDITILLS. I. RUSTS CH DLHBLRRY, RUEUS VIIICbTS, AIT. lfirflyatiaa and- Bauer. .0}: .Litsaa’suata- The orange rusts of Eubgg are occasionally quite serious in most of the raspberry, dewberry, and blackberry growing regions of the United States,'in some localities making cultivation of these berries unprofitable if not altogether impossible in certain badly infected, fields. The disease is not new but was first described by von Schweinitz (37) in 1822. Since that time considerable work has been done on the problem, but many phases of the problem have scarcely been touched upon, while others need further investigation. Newcombe and Galloway (31) working on blackberry showed in 1890 that the mycelium of the fungus was perennial in the tissues of the host. Clinton (12) gave a good general description of the disease in 1893 and pointed out that it is fcmnd mostly in the southern and eastern states, having never been reported further West than Nebraska. The disease has also been reported in Europe and Asia. Kunkel's (27) discovery in 1913 that there were nore than one type of orange rust stimulated renewed investigations on the subject. Prior to this time the orange rust on 53233 was supposedly a full cycle rust in which the aeciospores germinate typically by the production of long germ tubes. These germinating aeciospores infect the same or nearby plants producing a dicaryon mycelium which later gives rise to typical teliosporee of the genus Cymnoconia. These genninate in the usual manner by the production of typical pronycelia on which arise four sporidia which give rise to monocaryon myceliun.in the new infections on the same (or related) host species. Kunkel discovered that the aeciospores in some of these rusts genninate by the production of a S-celled promycelium which gave rise to four sporidia, indicating that Vthis form was a short cycle rust. In a later article Kunkel (29) pointed out sore of the differences between, and gave a fuller descrip- tion of the long and short cycle forms of rust. Ie concluded that there are in the United States two independent, but in.part similar, rusts 03.32EEE’ one a long cycle fonn which he identified with the EEEEEEEEQELEBEREEEEIEEAEE.0f nurope, and the other a shert cycle form, for which he uses the nane Caeona nitens, first given by von Schweinitz to a collection nade in horth Carolina. as a result of Kunkel's work Arthur (5) reconnended that the name Kunkelia_nitens'be given to the short cycle form, and this name has been used to some extent in subsequent literature. Arthur (5) claimed that the long cycle form is essentially a northern species, while the short cycle form is essentially southern. This led Atkinson (6) to believe that temperature was the determining factor in the method of spore germination. He described an experiment showing that high temperatures cause production of promycelia and low temperatures cause the spores to germinate by'fbrration of germ tubes. It is possible that simultaneous infections, with the two forms of rusts, of the plants tested give the explanation of his results which no one else has been able to duplicate. (Atkinson's (5) statement was definitely disproved by further work of Kunkel (30) who, by a simple experiment, showed that temperature -3- had no influence on the method of spore germination. He also showed that the spores of the two rusts exhibit a difference in color as well as being morphologically different. Dessey (7) showed that the spores of the long.cycle form are, on the average, scnewhat larger than the spores of the short cycle form. In.a report on the distribution of the orange rusts of_gu§g§, Dodge (17) stated that the short cycle form predominates in the South and the long cycle fonn in the North, although there is considerable overlapping of the two forms. This agrees with Clinton's (12) and Arthur's (5) reports. Dodge (17) also stated that the short cycle form alone is found on the western bank of the hississippi River, from Texas to Canada. Dodge (18) showed that the aeciospore germ.tubes gain entrance into a leaf through stomatal openings. He pointed out that host stomata on normal leaves of Rubus are on the lower side, but leaves of plants which are systemically infected nay'produce many stomata on the upper side. Dodge (19) in 1923 reported an intermediate form of the rust on blackberry. It produced two kinds of aecia, one producing dry powdery spores, the other waxy spores. The dusty spores produced germ tubes and the waxy spores produced promycelia on germination. Whether this really represents a new fonn or merely an infection by the two forms will require careful cultural work to deternine. Dodge (20) showed that the sporophytic hyphae of the C inoconia _E? are confined to the leaves in localized areas. The ganetophytic hyphae become established in the perennial plant structures, invade the new canes, and take part in the fonnation of aeciospores. He found nycelium . He stated (0 in the roots, medullary rays, phloem and cortex of the stem that the gametophytic nycelium spreads throuyh root runners in the same manner in the lens and short cycled rusts. Dodge (21), While endeavoring to account for the occasional suppression of the spermogonial stage of the short cycle orange rust, discovered that the aeciospores of those strains of the rust omitting the spermogonial stage are uninucleated. Cell fusions do not regularly take place in the aeciun;prinordiun. The uninucleated spores develop promycelia when they germinate, but the promycelia are two-celled and each cell produces only one sporidium. He stated that if no spermogonia could be distinguished with a hand lens it would be reported as without spermogonia, when a more care— ful examination with a high powered microscope might prove that poorly developed or vestigial spermogonia were present. With the results of all germination tests reported, he nade three general statements covering the occurrence of §§§2§2.23322§.W1th‘ out spermogonia: (l) Spermogonia are always found in ccnsiderable num— bers on leaves of Ruhus infected with the long cycle rust, QEEPQEQE}? interstithzlis; (2) If no spernOponia are present on a leaf showing aecia of orange rust, the spores will be found to be uninucleated and on germination they will form two-celled promycelia; (3) In strains of the short—cycled rust whose Spores develop four-celled promycelia, spermoeonia precede or accompany the formation of aecia. Kunkel (29) claimed to have found nuclear fusion in the case of the binucleated spores of Caeona_niteng_uith subsequent reduction division in the fonnation of the pronycelium. however, Dodge and Caiser (23) contradict this report axd claim that each nucleus divides indepen- dently without previous fusion. earnest. .0}: shanks; There are still a great many problehs concerning these orange rusts of 332u§_which require further inVestigations before they can be satisfactorily explained. In this connection the vriter has nade cyto— logical studies of three forns of the orange rust on dewberry, Rubus villosus. hassles Plants for study were obtained from two different sources. Wild dewberry plants from a badly infected patch growing on a vacant lot in the west part of East Lansing were dug up in October 1930, potted, and placed in cold frames until January 1931 when they were brought into the greenhouse. It could not be detennined if the plants thus obtained were rusted or not until the leaves began to shoot and the rust appeared. When the aecia were mature the fbrm of rust was determined by germinating the aeciospores on two per cent plain agar in petri dishes. Rusted Lucretia dewberries were obtained in Pay 1931 fnom a patch near Benton {arbor, hichigan, and planted directly in pots in the greenhouse. The plants remained in the greenhouse until June 1930 when they were removed to the field. They were not removed from the pots, but the pots were sunk so their teps were level with the ground. The plants remained in the field until the last week in November when they were again brought into the greenhouse so growth could be forced. Rust symptoms appeared on infected plants about six weeks after the plants were moved into the greenhouse. The form of rust on each plant was again checked by germinating more spores on two per cent agar in petri dishes. Spore germination showed three distinct ferns of the rust on Rubus villosus- Spores obtained from different plants of wild dewberry, all dug up on the same vacant lot wnich covered less than one city block, germinated in three different ways. One lot of aeciospores germinated by the production of long germ tubes (Plate III) which would indicate that this was the long cycle form of rust. Another lot germinated typically by the production of five—celled promycelia, each promycelium giving rise to four sporidia, one sporidium to each of the upper four cells (Plate I, B)- The third lot of spores germinated by the produc- tion of three-celled promycelia which gave rise to two sporidia (Plate II). Spores obtained from rust on Lucretia dewberries were all of the type which gerndnated by the production of the five—celled pronycelium and four sporidia. Leaves showing rust in various stages of deveIOpment were killed and fixed in Formalin-Acetic-alcohol fixing solution. Other fixatives were also tried but the most satisfactory results were ob- tained with the formalin-acetic-alcohol solution made up according to the following formulazoO per cent alcohol, 90 cc.; glacial acetic acid, 5 cc.; commercial formalin, 5 cc- By leaving the material in this solution for a period of two weeks before running it through the alcohols, uniform fixation was obtained and the.material was softened s, to a degree where it was easily sectioned. The naterial was run up into paraffin in the usual way arfl sections 5 microns in thickness were cut. Several stains were tried but the one which gave the most satisfactory results was a modification of Fleaming's triple stain. The following is the technique which was used in staining: The paraffin was removed with xylol and the sections were then passed through absolute, 95 per cent and 70 per cent alcohols. They were then stained in a fifty per cent alcoholic solution of safranin for six to twelve hours. The excess safranin was removed by rinsing the slides in distilled water. The slides were then flooded with a 0.1 per cent aqueous solution of gentian violet which was allowed to remain for 10 seconds. The slide was held at an angle and the gentian violet removed by flooding with a l per cent aqueous solution of orange G. The orange G was allowed to rennin for one minute and then the excess gentian violet and orange G were reroved by rinsing in 95 per cent alcohol until no more violet could be seen streaming fron the sections. The slides were then I°Ssed quickly through absolute alcohol into xylol and mounted in balsam. This stain gave a good preparation with almost perfect differentiation of the fungus and host tissues. The nuclei in the aeciospores, pycnia, and mycelium stained a bright red, the mycelium a brilliant orange, and the chloro- plasts and host cells a rather dull violet. Careful examination cf the slides was made using the oil immersion objective of the microscope. Emaminati'n of the prepared sections showed the mycelium in the interior of the leaf tissue to be of the monocaryon or gametophytic type in all cases. In sections of leaves whichivere fixed when only pycnia appeared on the leaves and before the beginning of the development of aecia, nothing but monocaryon cells could be found. This seems to show that the mycelium which is perennial in the host tissue is all monocaryon and that a fusion of some kind is necessary for the production of the binucleate spores as will be discussed later. No dicaryon mycelium was found in the host tissue intermingled with the monocaryon mycelium, as was reported by Olive (53) working on Puccinia podophylli, §:_gbte£en§3 and grgnyge§_glycyrrhizae. various investigators on nany different species of rust have worked on the problem of cell fusions, oririn of the binucleate condition, or sexual reproduction. A review of previous investigations along these lines seems to be in order at this time. Blackman (8) was the first to describe a real fertilization as the origin of the binucleate condition in the rusts. In Ehragmiiygn violaceg§_he found the binucleate condition originating in fertile cells in the aecial prirordium as a result of simple migrations, through minute pores, of nuclei from neiphboring cells. This species was later reinves- tigated by helsford (58) who confirmed Elachnmnfs earlier observations. Blackman.aid Fraser (9) report such a nuclear migration in two other species, Uromyces poae and fuocinia poarum. Christaan (10) studied --0- -—.- .nitgng, and.Uronyces caladii. He found that the binucleate condition originates by the fusion of equal cells and later suggested that the nuclear migrations of Blacknan might be pathological. Welsford (38), how- ever, disproved the latter statement. Olive (82) observed early stages of cell fusion in Gynnoconia interstitialis and Triphragmiug'ulmariae, g. which appeared similar to the nuclear migrations of Blackman. “dams (1) showed that in 22121.:ILLMEQELEEE’ l; pvrifogie, and £;_§£igolu§ fusion occurs between adjacent fertile cells of similar size and position in two gametophytic hyphae. A dissolution of the walls occurs usually at the upper ends where they come in contact. When the lower parts of the walls have not completely disappeared, it results in a two-legFed fusion cell. Immediately following fusion there occurs a considerable increase in the size of the fusion cells. Blackman (9) ccnsiders the aecium of Phragmidiu3_viglac§ug as a sorus of female reproductive organs, each of which consists of a sterile cell above and a fertile cell below, the nucleus of an ordinary vegetative cell bringing about fertilization and performing the part which was apparently formerly taken by the nucleus of the spermatium, which he considers as functionless. He suggests that the sterile cell is reduced and that it formerly pushed its way to the surface (as it can sometimes now be observed to do} and acted as a trichogyne to bring the spermatium.into relation with the female cell below. As Colley (15) points out, the chief differences of opinion have been in regard to the nature of the two cells nhich fuse to form the basal cells and in regard to the sterile cells. After Christman's paper (10) announcing the fusion f two equal cells by the complete absorption of their Oppressed walls, blackman and Fraser (9) investigated a number of forms and stated that the binucleated condition might arise (a) through the process of nuclear migration from one cell to another as Elackman (8) first reported, or (b) by a similar nuclear migration from one vegetative cell to another below the fertile layer, or (c) by the process described by Christman. -10- During all these earlier investigations very little was said of the pycnia and pycniospores which were then mostly considered func- tionless. Smith (36) in 1884 discussed the aecial stage of a rust on the Lily of the Valley. He described and figured spermogonia and aecia. He considered the spermatia to be sale cells. He found aeciospares with spermatia adhering and suggested that the aeciospores are eggs which must be fertilized by the spernatia. Blackman (8) discussing the pycnio- spores says, ”A study of the structure of the spernatia of the Uredineae shows that they have the characteristics not of conidia but of male cells, for they exhibit a large dense nucleus, very little cytoplas , no reserve material and a very thin cell wall. These characters, totetier with Q their usual association with the aecidia, their absence of function, and tne peculiar, apparently reduced, form of fertilization to be observed in the aecidium of Ihraihidiu§;violaeeuflfl point clearly to the view that the spermatia are male cells which formerly took part in a process of fertilization in connection with the aecidiun, but now have becmie functionless." Fromme (25) working on Flax rust consents upon the close asso- ciation of pycnia and aecia which are feund on both sides of the leaves. The pycnia precede the aecia by several days or weeks. Conjugation form- ing two-legged cells takes place in the base of the aecium. It was not until Craigie's (14, 15, 16) reports and Allen's (2) studies of Puccinia_5raqini§_that the function of the pycniospores was definitely proved. Craigie (16) showed that an infection from a single sporidial inoculation failed to produce aecia but pycnia were formed. He also -11- showed that the monosporidial pustules (pycnia) are of two sexes, (+) and (-). ihe (f) pustules are about equal in number to the (-) pustules. This fact indicates that in all probability the sporidia which are pro- duced by the promycelia and to which the pustules owe their origin are divisible into two groups, (+) and (-), which are about equal in number. By intennixing the nectar of monosporidial pustules he induced the for— mation of aecia in the pustules so treated within 5 or 6 days. He showed that in nature flies are the active agents in carrying the nectar of one pustule to another and thus affectinn the transfer of pycniospores from (+) pustules to (-) pustules and of (-) pustules to (+4 pustules. This role of insects in the fertilization process in the rusts was probably first supeested by Rathay (35) in 1882. He listed 135 species of insects that he had observed visiting the spernogonia of about 20 species of rusts to collect the nectar drops exuded. He found that most of these insects are also visitors of extra-floral nectaries as well as of floral nectaries. he tested the drops exuded from the spermogonia and proved the presence of reducing sugars. He believed the spermatia to be male cells and the sweet exudation a means of en- listing insects in their transportation. It remained for Andrus (4) working on Urozyces gppandiculatus and H;.Xl§fl22.t° work out the mechanism of fertilization. He describes the aecial primordia as at first appearing as tangled masses of hyphae but careful focusing on thin sections revealed definite structures. a large number of two-legged uninucleate cells was noted setting at all angles. After fertilization a reorientation took place due to e.pansion and elongation of basal cells, spore mother cells, and aeciospores. If r) - 4e fertilization does not take place at the maturation of the first fertile cells, development within the princrdiun results in a chain of cells, each of which could function as a fertile cell. From each cell of the series there extends a lateral, basal stalk which night be interpreted as a trichogyne. Hyphae emerging through the epidermis provide a ueclanisn for fusion of spermatia with gametOphytic mycelium. There are several ways in which the gametophytic mycelium may make contact with spermatia. Organs may emerge through open stomata. Spernatia may enter through Open stomata and come to lie in contact with mycelium under the epidermis. Hyphae may extrude directly between epidermal cells. No such erumpent hyphae was found in the telial stage. Assuming that fertilization takes place by means of trichogenous hyphae, Andrus concludes that fusion of cells of parallel hyphae cannot constitute fertilization. The r w of binucleate basal cells present in the nature aeciun, and stated to be two—legged fusioh cells by previous observers, are not necessarily fusion cells but are structures which may be present in the primordium previous to fertilization, that is, in the uninucleate condition. There should be present in the priuordium egg cells, each with a branch representing the trichogyne and probably a second stalk representing the foot cell. These are represented by the row of two-legged basal cells in the aecium. It is with this later paper especially in mind that the writer has made careful studies of conditions to be found in the orange rusts of Rubus. -13- seawrls £912.13. badgers; -.‘:13...it’.0..}§.£§i This form of the rust produces an abundance of pycnia prior to and during the formation of aecia. The aeciospores are typically binucleate although in a few cases plurinucleate spores were also found. The monocaryon mycelium was quite extensive throughout infected leaves and was especially abundant anong the host cells just below an aecium. In these regions single strands of myoelium could be traced .running fron the base of the aecium to the opposite epidermis. The mycelium seemed to be only sparingly branched and usually each strand was separate, although occasionally two hyphae could be seen lying side by side between two mesophyll cells. However, just under the epidermis the myceliun.was much nore dense. The nycelium seemed to form a tangled mass between the epidernal layer and the first row of mesophyll cells. These masses of Lyphae were especially dense near the edges of an aecium. The haustoria were egg-shaped, the tip which penetrated the host cell appearing to have enlarged a great deal, in some cases fillirg about one- fifth of the host cell. As this fonn of the rust typically produced binucleate spores a very careful study was nade of the base of the aeciun to determine, if possible, the origin of the binucleate condition. Many cases of the two legged "fusion cells" (Ilate IV, fig. C and D) which have been so fre- quently referred to in previous literature were found. All strands of mycelium below these cells were monocaryon and the spores above were binucleatc. This showed that a fusion of some kind must have taken place but gave no evidence as to the origin of the "legs" of the "fusion cell". Lndrus (4) found numerous hyphae extruding through stomatal -14- openings or between adjacent epidermal cells. These hyphae he interprets as being trichogynes or receptive hyphae with which the pycniospores make contact in the fertilization process. The Opposite end of the trichogyne thread was considered to make one leg of the so called "fusion cell" or as he now refers to them, erg cells. The other leg wrmld then represent a foot cell or stalk cell. Dr. R. F. allen (3) reports in a very brief note that she has found essentially the sane structures in EEEEEQEZ _t£iticina_as described by Andrus for EESElfiéfin Fer detailed paper is in press but has not yet becore available for study. With these things in mind the writer node careful exlrifiation of his preparations to determine if similar conditions existed in the orange rusts of Rubus. Onlv an CCCiSiOHfll hypha extrudiuf ttrOUvE a stoma could be 'found and very few hyphae could be found pushinv their way up betwee adjacent epidermal cells. Put another condition which is worthy of note was observed. Ln the side of a leaf opposite an aecium and in test cases directly over it a strand of myceliun was often found running under the epidermal layer with the tip of the hypha penetrating an epi- dermal cell. These epidermal cells were in many cases completely dis- solved, leaving a gins sass in place of the original cell. This gummy appearing substance has spread over the surface of two or three adjacent epidermal cells and contained very small round bodies which stained a brieht red. The two adjacent epidermal cells had crowded together, partly fillirg the gap made by the dissolved cell. Such a condition is illustrated in Plate VII, figure 1. In other cases, as shown in Plate VII, figure C, the hyphae did not seem to enter the cell directly but a somewhat enlarged portion touching an epidermal cell seemed to cause the dissolution of the epidermal cell. Plate VIl, figure A, shows several strands of hyphse running under the epidermis. kany hyphee could be traced coming from these receptive places and running nearly to the base of the aecium but they became lost in the complex mass of cells which was always present there. It would appear that this issolution of the epidermal cells might ls caused by the secretion of an enzyme by the fungus mycelium and that this gummy use» may be an ideal receptive spot for the pycnio- spores which could then fuse with these receptive hyphae in the process of fertilization. The base of the aecium.exhibits a very complex mess of fungus cells but careful study reveals the presence of many two-legged cells. In aecia which are just in the first stages of development these cells are not so clearly oriented and someahat more spread out. They are somewhat triangular in shape and seen to be connectec to two strands of hyphse ccninc from different directions. After fertilization these cells become oriented sore distinctly in rows and are swollen so they beccme crowded into a rather compact mass. These cells in the base of the aecium exhibit a very'marked similarity to those described by Andrus (4) and interpreted by him as "egg cells". The fact that numerous "egg cells" were found in the base of the secium correspondinr very closely to those described by Andrus and also that similar though not identical receptive structures were found would seem to indicate that in this short-cycle rust on EEELEJ these 8 with one leg representing the 3...] two-leesed cells may be oogone cel trichogyne and the other representing a fcet cell as described by Andrus in the rusts investigated by him. Long—Cycle Fogg As far as the monocaryon mycelium in the interior of the leaf is concerned, this form of the rust Was found to be quite similar to the shortecycle form producing four sporidia. The mycelium is quite abundant throughout the tissues of infected leaves. It is sparingly branched and shows the same peculiar habit of running in a bundle of several strands between the epidenris and the first layer of nesophyll cells. The haustoria are very similar in size and shape to those of the short—cycle rust. The aecial primordia also showed the sane charac- teristic COnplex structure of uninucleate cells. llate VII, figure G, shows the structure of a young aeciun with a row of spore initials above (a) and a complex of OOgOue cells below (b). Figures d and F sh w sons of these cells somewhat more in detail. So far no marked difference in this fOHL and the short-cycle form already described have been shown. But w“en sections showing mature aecia were etamined, the writer found a condition which has not been reported by previous investigators. as reported by Kunkel (27, 28, 29, SO) and Dodge (21) this form.of the rust is supposed to be the typical full cycle -ust in whic‘ the aeciospores gerudnate by a long germ tube (Plate III). An abundance of pycnia and pycniospores were also produced on both sides of leaves infected with this type of nret. All previous investigations showed the aeciospores to be typically binucloate. Teliospores are also supposed to be produced in this form of rust, but the writer failed to find any tcliospores on plants infected with t‘is fornzof the rust, either in 1930 or lfibl. This may have been -17- due to the fact that both of these seasons were hot and dry which was probably unfavorable to the development of the rust. When sections of this rust were examined by the writer, the aecia were found to be nade up of chains of long narrow aeciospores, each containing a single nucleus. Interspersed between these rows of uninucleate spores a few chains of binuclcate spores could be seen (Plate IV, figure a). Ey carefully studying the base of these chains to determine their origin, two-legged or so—called ”fusion cells" could be found in many cases. The base of a mature aecium did not stow the complex mass of cells that was characteristic in the other fonn of rust, but appeared to be rather shallow with only a few strands of.mycelium between the first row of nesophyll cells and the spore chains (Plate IV, figure a). Some of the hyphae seemed to turn up at right angles into the aeciun as will be shown later was the case in the short-cycle form producing two sporidis. No cases of dissolution of epidermal cells could be found, but a few hyphal strands could be seen projecting up between two adjacent epidennal cells. In one case a hypha was seen projecting clear to the surface and this hypha showed a cap-like structure on the etposed end (Plate VII, figure D) similar to illustrations by Andrus. .The mycelium from this projecting tip could be traced as running into a tangled mass of nycelium at the base of a young aeciun which was forming on the sane side of the leaf. Only three epidermal cells separated the edge of the aeciun from this hypha. Several binucleate cells in short chains appear- ing to be spore initials could be found in this young aecium. This -18- would lead one to believe that a fertilization had taken place here and given rise to the binucleate condition. The facts that the young aecium primordium shows the sane characteristic structure as the form typically producing binueleate spores and that binucleate spores were found in scne cases semas to indicate that a fertilization and development of binucleate spores is normal for tilS form.cf the rust. But the fact that rows of uninucleate Spores seem to arise from these "egg" cells seems to indicate that if no fertilization takes place spores are able to develop parthenogeneti— cally, in which case each spore would contain but a single nucleus. Whether or not these spores will produce infectiOn was not deternineu, but it seems evident that they will germinate as good germination was obtained of spores from other leaves of the same plant. Pheaiflaeaaioai Passage Ifiiflql'fiéia Plants infected with this fonn of the rust produced only a very ‘few scattered pycnia. The pycnia which were formed seemed to be of an undeveIOped or vestigial type, producins no or in some cases only a few pycniospores (Plate VI, figure B). This fully afrees with Dodge's (21) report on this form of the rust. The mycelium in this form seews to be almost identical with that of the other two forms except that it does not seem to be quite as extensive in the lea tissues or quite as abandant just under the epi- dermis although it is frequently found there. he receptive structures of any kind could he found in this form of the rust. Dodge (21) showed that this form of the rust produces chains of uninucleated aociosyores which arise from uninucleate cells of the -19- sorus primordium. In sections nade by the writer the rows of uninucleate aeciospores were seen to arise directly from the nonocaryon hyphae at the base of the aecium. a single hyphal thread may be traced coming up be- tween two nesophyll cells of the host, turning and running along the base of the aecium for some distance and then suddenly turning altost at right angles and terminating in a row of uninucleate aeciospores. The base of the aeciun seens to be Very characteristic in that it is nade ur of only one or two rows of mycelium, many strands of which could be seen turning up into rows of aeciospores (Plate V). There seem to be no fusion cells of any kind in this the of aeoium and the spores produced are all of the uninucleate type. The spores in the chains appear rather long and slender as compared to the stores of the binucleate type, but they round up some- what when they become free from the chain. Keasurements by Dr. E. A. Bessey in 1929 of spores of the two short-cycled forms on Wild dewberry showed that the spores of the two sporidial form averaced about 4 microns narrower than the soores of the four sporidial form. The lengths of the two kinds of spores were about the same, the figures being respectively 23.95 microns x 17.5 microns and 24.6 microns x 21.7 microns. The ran es of spore measurenents for these lots were respectively 15.4 microns to 29.9 microns x 14.5 mi: one to 23-2 microns for the two sporidial form and 14.5 microns to 57.7 microns x 13.0 microns to 30.8 microns for the four sporidial form. It would appear that this type of the rust is a form which develops spores parthenogenetically and goes through its entire cycle in the uninucleate condition. It may be that the condition in this rust is similar to the -80.. monocaryon race of oat smut (Estilsge_aXene§_Pers.) reported by Flerov (24‘. The spores of this fungus obtained in;yjtrg_arose from a uninuc- leate cell without any fusion of nuclei. The fungus grown in vitro had no true dicaryon phase, aoconplishirp its life cycle in the nonocaryon condition. It produced uninucleate teliospores which gave rise to two- celled pronycelia, the sporidia of which genxinated to produce nonocsryon mycelium without any fusion of cells. In the dicaryon race of the oat smut also studied by Flerov the binucleate condition arises by the fusion of two pronycelial cells, or of two sporidia, or by division of the nucleus of a sporidium into two nuclei and elongation of the cells. It seems quite possible that this short-cycle form of the orange rusts may develop in much the same manner and live its entire life cycle in the monocaryon condition. On the other hand it may be that a condition exists ccnpsrable to that described by Oort (54) workins on Conrinus dimetarius in which _. _“_“_-,_ two types of fertile sexual combinations occur, AB x ab and Ab x aB. Clamp connections occasionally occur between Ab x AB and a3 x ab. The forner will produce ra.ner abnormal fruits, apparently consisting of intermingled hyphae of the two sexual phases, for the basidta produced are fron the beginning uninucleste and produce but two besidiospores, both being either Ab or nB, the basidia of these two types being about equal in number. This represents a parthogenetic monocaryon develop- ment occurring in the absence of the rust of the opposite sexual phase. Enrther lroblens fl the observations node in these studies have thrown ne new light on the orange rusts of Rubue which at once opens up nore new problems but at the same time indicate possible netbods for their solu- tion. lwo of these problems which should be solved by inoculution experiments ure (e) whether the two sporidiul short—cycle forn repre- sents merely a +-or - strain or the short cycled rust or a partheno- genetic mutation; and (b) whether the long~cycle form where no fertili- zetion has taken place is really normal or whether it is~e new nutetion which develops parthenogehetically. Some other probleis to be followed out are as follows: 1. The question of varietal strains. All three forms of the rust have been found on the wild dewberry. Do other forns of the rust show a host preference or will they all attack various species of Rubus? Dr..n. A. Bessey reported to the writer of finding a field of Cultivated blackberries with two varieties yrowing near one another. One variety showed only short cycle rust en* the other only long cycle a field of blackberries or raspberries may be surrounded by rusted wild 33h3§_nnd yet STOW only an occasional rusted plant. wild Ruhus ollegheniensis collected by E. A. Bessey near exford, Lhio, showed only short cycle rust while in nine years of collection in hichigan the lonr cycle rust has been with few exceptions the prevalent fonn on wild specimens of this species. Cultivated dcwberries are only occasionally rusted While wild dewberries are very frequently rusted. 2. Dr. E. A- Eessey described to the writer a rust which he found on wild black raspberries. The aeciospores gernineted by producing four-celled pronycelie on the ends of moderately long germ tubes. These plants were transplanted fron rand Ledge, hichigan, to the College at East Lansing and showed rust the next year but the spores all gerbinated by very long germ tubes of which perhaps 1 in 1000 developed a prony- celium 60 or 70 microns long and nearly twice as thick as the 500 or 400 micron long germ tube at the end of a ich they arose. This phenome- non has been observed subsequently a few tines and in 1927 or 1928 was observed in rust on cultivated black raspberries near Coloma, hichifan. This conditi n has never been explained. 3. Does the same plant produce aeciospores germinating to form different numbers of sp ridia in different years? Can a short cycle rust change over to long cycle or vice versa? In 1929 rusted Lucretia dewberry plants collected near Benton Harbor, Kichiran, by Dr. E. a. Bessey {errjnated by promycelia which, almost without exception, bore but three sporidia but in 1928 and 1931 Lucretia dewberries fro: the some vicinity bore spores which produced promycelia with four sporidia. The 1931 collections were rade by the writer. In 1929 Dr. hessey collected in Lichigan State Park at High- land, hichitan, six rusted plants of wild dcwberry. Five of these were of the long cycle type but the remaining plant had long cycle and diort cycle (four sporidial) aeciospores on the same leaf. In June and July 1928 in Benton Harbor and vicinity Dr. Bessey collected thirty specimens of rusted cultivated Cumberland black rasp— berries all of which showed, when the aeciospores were germinated, the deve10pnent of long genn tubes except one leaf on one of the plants in which the aeciOSpores germinated by a promycelium. The renainder of the -35- leaves on the sane plant showed long cycle rust. 4. On rusted plants why are scne leaflets only partly rusted; in other cases all the leaves are entirely rusted. 5. What are the northernmost limits of the short cycle rust in Michigan? It has never been reported from the Upper Peninsula, but the long cycle fonn is not rare there. The short cycle fonn Les been found near Charlevoix on cultivated blackberry, the one leaf sent in from there having intermingled spores, sore of which produced promycelia and others gern tubes. Sum- r: a rv ———-—— - —~ .- 1. Three ferns of orange rust are described on Rubus villosus nit. 2. Sections of rusted leaves show that the overwintcring mycelium is monocaryon in all cases. 3. The short cycle form producing four sporidia has binucleate aeciospores; the short cycle fonn producing two sporidia has uninucleate aeciospores; both kinds of spores were found in the long cycle form. 4. Dissolved epidennal cells forming an ideal receptive spot for ycniospores were found in the four-sporidial fen; of the rust. ”*phae which night be interpreted as receptive trichogynes were found extruding between two epidennal cells of leaves infected with the long cycle rust. 5. Cogone cells, as described by Andrus (4) were found in the four sporidial short cycle and the long cycle ferns. 6. No oogone cells or receptive hyphae were found in the two- sporidial slert cycle form. 7. a list of further problems to be followed out is given. (1) (Q (4) A C) v (7) (9) (10) (ll) (15) (14) 2111119322551 Adams, J. F. Sen ual Busions and DevelOpnent of the Sexual Organs in the keridermiuns. Pennsylvania agricultural Experiment Station Bulletin 160:31-77. 5 plates. 8 figuresx 1919. Allen, Ruth F. The Cytology o Heterothallisl in Puccinia era; inis. *c-“’—.—— Journal of Agricultural Research‘ggz583——611o 1721332§° 1930. Heterothallism id Puccinia triticina Science n.s. 7 :162-163. 193 . and us, C. Frederico The Kechanisn of Sex in Uronyce_ and U viLnae. Journal of gricultuial Research 42:5' 11 figurOes. 1931. endicu1atus {1140) 'tE“; 3: Arthur, J. C. Orange Rusts Of.32h§§f Potanical Gazette 63: 501-515. 1 figure. 1917. atkinson, George F. Selected Cycles in Ggynoconia peckiana. inerican Journal of Botany‘§;78-83. 1918. BesseV, E. 1. Notes on the Crange Busts of Rubus. Papers of the hichigan loadeny of Science, nrts, and Letterslgzdl-65. 1 figure. laggté. ' Blacknan, V. E. On he fertilization. 1ltera: tion of Generations, and Gen neral Cytoloety of the Uredineae. annals of Botany 1§1ono-373. 1 plates. 1904- Elackman, V. H. and Fraser, Felon C. I. Further mdie es on the Sexuality of the Uredineae. Annals of Eotany 20: o-13. 2 plates. 1906. Christman, a. E. Sexual Reproduction in the Rusts- Botanical Gazette ga:2€7+°75. 1 plate. 1905. ~1‘_--‘_-_ The alternation of Generations and the horphology of the Spore I‘orrns in the lists. Botanical Gazette 11:81-101 1 311:9. 19(17. Clinton, 3. P. Orange Rust of Raspberry and Blackberry. Illinois Agricultural Experiment Station Wulletin 29:273-300. 1. I_lates. 1893. Colley, R. H. Parasitism, Lorph0109y, and Cytology of Qronartiur ribicol a. Journal of igricultural Research 1§:%l9~350. 12 elites. 1918. ‘*"‘*-**r Creieie, J. V. Discovery of t e Function of Pycnia of the Rust Fun-gji. Hatire 1‘30: 735-707-1927. 1.--..- 1.... _ “m“ On the Occurrence of Pycnia and Aecia 1n sax-min Rust Fung i. Phytop ethology $§:lOOS-1015. 3Hfi cure . 1929. 3 3__ .An Experimental Investigation of Sex in the Rust Funéi- Phytopatholoey 33:1001-103'. 11 figures. 1951. Dodge, 5. O. The Distribution of the Grange Rusts of -b1§; Phytopathology 33361-71. 1923. _‘ ._.u _ Effect of the Oranfe Busts of Rubu_s on t11e Developzzert and Die tributior' of Sto11ata- Journal of 1 ricultural Research 25:195-bOO.1 slate. 1 figure, 1923. - __ .A New Type of Orange Rust on Ilackberry. Journal of lgricultural Research 25:191—191. 1923. “a Systenic Infections o Rusts. Journal of agricultural Research 2 4‘ . a '9 7 livurss. 1923. f‘E2u J*51;:1U1 t11e Grants 5:209-21. ELJEE£E§§° Uninucleated nocidiospores ir Caeona nitens, and associated Ihenomena. Journal of airioulim1a1 Research 25: 1015-1058. 5 plates. 1921- -3__~__ Cytological Evidence Bearing on the Sexuality and Origin of Life Cycles in the Uredineae. Iroceedings of the International Congress of Plant Sciences3§51751—1766. 1929. -n__“_ and Caiser, L. 0. The Question of Nuclear Fusions in the Blackberry Rust, Caeona nitens. Journal of Agricul- tural Research.§§;1003-1024. 4 REEFERL. 1923. Flerov, B. K} fiksitologii Ustila3o avenae Pers. podannim kulturi in vitro. ('fhe Cytology cf Lstilalo avenae Pers. on the Basis of Cultures in vitro) Trudi Se r>ktsii po Iikolog ii i Fitopatologii Russkavo Botanitcheskauo Obshtchestva trudi Loskovskovo Ctdeleni (Acts of the Section of kycology and Ihytopa thology of the Russi Botanical Society. Acts of tiie hoscow Subdivision) 'If23-36. _1_2_1__a__t_e__3_- 1923. Fromme, P. D. Sexual Busions and Spore Development of the Flex Rust. Bulletin of the Torrey Botanical Club 59:113-131. 2 Ilates. 1912 ”“ ”'" The Kerphology and Cytolopy of the lecidium Cup. Botanical Gazette §§:1-35. 2 Filtes. 8 firures. 1911. “MW.“ Kunkel, Ctto. The Production of a Iromycelium by the Aecidiospores of Caeoma nite 1s Burrill. Bulletin of the Torrey Botanical Club 195361-333. 1 fMJEZT 1913. U“ ()3 .* Nuclear Behavior in the Promycelia of Caeone nitens Burrill and Elc_ciniu peckiana Howe. American Journal of Eotany };5 ~47. l rlutE $1914- .«_~ Further studies of the Orange Rusts of Rubus in t} e United States. Bulletin of t11e Torrey Botanical Club 45:5:39- 53 9. 5 fgigggg. 1913. __ Further Dots on the Grange Busts of 1bus. Journal of 1gricu1turel Research 131501-512. 4 ylete_. 19307 hewcombe, F. C Fungus of the . and Galloway, L. i. Perennial Lycelium of the E 2 FEA‘tBSo 189 00 lackberry Rust. Journel cf chology'gglOB-lCV. Olive, 3. J. Sexual Cell Fusion and Vegetative nuclear Divisions in the Rusts. Annals of Botary 22:551-560. 1 plate: 1908. Intermingling of Perennial Sporophvtic and Guueto— phytic Generations in }ucciniu podcgh"1li, E. obtcgens, and Uronyces tlycyrrhizue. Annalee lycolog iaLll: 297-511. 1 nlatg. Oort, S. J. P. Die Sexualitét von Coyrinus finieturius. Recueil des Traneux Botaniques Iéerlandais 3;:35-148. flutes 4-6. Figures 1-4. 1950. v ” ' n ‘ Ratb.ay,ner1ck. LnterSLIChunten uber die arermogonien der Qostpilze. Denkschr iften der k. Skademie der Wiesenschaften .ien 46: (52 Ia.es). 1892. . Disease of the Lily of the Velley- Gardener's 2:12-15. Figures 2-5. 1884. M Smith, J. C Chronicle 2 von Schweinitz, Ludovicus David de. Synopsis Fungorum Carolinae Supeii ioris. Schriften der Esturforschendeu Gesellschaft zu Le ipz igv;:20-151- 2 n ates; '1822. Welsfoxu, E. J. Nuclear Kirrations in 1hrar.1'1uy violeceun. Annals of Lotuny 29:295-299. 1 plute. 1915. I. Germinating lipure C. .r Ekplenatio: of Plates spores of the hort cycle form producing four syoridia. Promycelium before sporidia are formed. Figures A, L, D. more advanced stages of germination. Figure 2. II. Germinatin A germinated spore in which the prcmycelium has cone in contact with the agar. In this case secondary gerr tubes are forwed instead of sporidia. g q:ores of the short cycle fbru.rrcdu ing two Sporidia. Figures a, B, C, D, H. Nonial gerxdnation. Figure F. III . Gemina ti IV. Biif‘slre A 0 Figure L. Figures C V. Figure A. Figures 3 VI. Pycnia Figure A0 *3 o _ . igure B. VII. Fifme 4-1. Fi{ure C. n gens tube has formed on the end of the prcuycelium where the promvcelium,cane in contact with the agar. on of spores of the long cycle type. Cross section through an aecium of the long cycle flarm sh wing chains of two kinds of spores. Cross section through an aeciun of the four sporidial short cycle fonh. & D. Chains of spores of the four sporidial short cycle form showing two-legged "fusion cells” at the base. Cross section through an aecium of the two sporidial short cycle fonn. & C. Chains of aeciospores which arise on the ends of nonocaryon mycelium which turns up into the aecium. Pycnium from the four sporidial short cycle form. Vestiqial pycnium from the two sporidial short cycle forn- Strands of hyphae running under the epidermis. a dis— solved cell is shown between two epidgrual cells. The end of a h; *pha wldch his turn d up into an epidermal cell wlich has d ‘issolved leaving a gunmy Kass. A swelled hyphu which is seen lying in contact with a dis- solved epidermal cell. Figure -. A receptive hypha protruding between two epidermal cells. Zipure E. "Cosmic" cell (b) with spore initial (a) stove. Figure F. Group of "oogone" cells (b) with spore initials (a) aLOVe. 'on of a young aecium of the long cycle form. initials; (b) complex of oogone cells. a, B, and C are from the four sporidial short cycle form. D, E, F, and u are frOn the long cycle forn. VIII. lhotomicrographs of the base of 3 yo n" aecium of the long cycle “L, form showing tie complex arran enent cf the cells. IX. fhotomicrograph of the base of a young aecium of the two-spcridial sfnrt cycle font. Plate I Plate II r. -31- Plate III ti *7— HI Plate IV "47—- +, a.- Plate V ~5‘l'- Plate VI -55- Plate VII -56- Fla te VIII -37- Pla ize IX i? L I... N 0 E S “U M 0 8 R 3 1293 03056 5968 ”I, H I" H H S” " "I H " H u