A Study of Physoderma on Three Species of Portulacaceae

Brigham Young University BYU ScholarsArchive

Theses and Dissertations

1958-06-01

A study of physoderma on three species of portulacaceae

David L. Mumford Brigham Young University - Provo

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BYU ScholarsArchive Citation Mumford, David L., "A study of physoderma on three species of portulacaceae" (1958). Theses and Dissertations. 8095. https://scholarsarchive.byu.edu/etd/8095

This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. A STUDYOF PHYSODERMAON THREESPECIES OF PORTULACACEAE

A THESIS SUBMITTEDTO THE DEPARTMENTOF BOTANYAND THE GRADUATESCHOOL OF BRIGHAMYOUNG UNIVERSITY IN PARTIALFULFILLMENT OF THE REQUIREMENTSFOR THE DEGREEOF MASTER OF SCIENCE

DAVIDL. MUMFORD BRIGHAMYOUNG UNIVERSITY JUNE 1958 ACKNOWLEDGEMENTS

I wish to express my appreciation to all who have assisted in the completion of this project. Acknowledge- ment is made to Dr. C. V. Morton, Smithsonian Institution, for his prompt identification of host species. Apprecia- tion is expressed to Mr. Robert Eastmond for assistance with photographic work and to Margaret Mumford for typing the manuscript. I am especially grateful to my wife

Joyce 1 for her help and encouragement in connection with this project.

iii TABLEOF CONTENTS Page ACKNOWLEDGEMENTS• • • • • • • • • • • • • • iii LIST OF TABLES•• • • • • • • • • • • • • • • • V LIST OF ILLUSTRATIONS••• • • • • • • • • • • • • • 1ri Chapter I. INTRODUCTION • • • • • • • • • • • • • • • 1 II. MATERIALS ANDMETHODS • • • • • • • • • • • 4 Host Species Studies Physoderma Studies

III. EXPERIMENTALDATA AND OBSERVATIONS •• • • • 11 Host Classification Activation and Growth of Host Species Signs and Symptoms of the Disease Activation of the Parasit e Inoculation Studies IV. DISCUSSION ••••• • ••••• • • • • • 26 The Parasites The Hosts

v. SUMMARY.• • • • • • • • • • • • • • • • • 36 LIST OF REFERENCES. • • • • • • • • • • • • • • • • 42

iv LIST OF TABLES Table Page 1. Percentage of germination of three species of Portulacaceae •••••• , ••••• • • 14 2 . Average shoot growth from corms of £k_ lanceolata soaked in various concen - trations of ethylene chlorohydrin •• • • • • 15 3. Size of resting sporangia on leaves of 4 host species ••••••••••• • • • • 19 4. Measurements of resting sporangia from leaf and stem of Erocallis triphylla , • • • • • • 24

V LIST OF ILLUSTRATIONS Figure Page 1. Healthy and infected specimens of 0. py:gmaeum. • • • • • • • • • • • • • • • • Specimen of O. py;gmaeum showing infection , of the floral organs •••••••••••• 38 J. Specimens of E. triphylla showing leaf and stem infections ••••••••••••• 39 c. lanceolata, showing infection on leaves and sepals •••••••• • • • • • • 39 Longi-section through gall on stem of E;. triphylla showing hypertrophy~ •• • • • 40 Infected cells of o. py~maeum showing resting sporangia and r izomycelium~ • • • • 40 Physoderma on E. triEhylla, showing resting sporangium, rhizomycelium, and intercalary swelling ••••••••• • • • 41 Physoderma on E. triphylla, showing rhizomycelial strands and i ntercalary swellings ••••••••••••••• • • • 41

vi CHAPTERI

INTRODUCTION

Our knowledge of Physoderma on terrestrial plants dates from 1833 when Physoderma pulposum Wallroth was reported on members of the Chenopodiaceae, In general the species of Physo- derma on terrestrial plants have not been studied thoroughly, As pointed out by Karling (10) the genus now contains nearly eighty species, many which are of questionable validity because of lack of knowledge regarding them. Two species of the genus occur on economically important plants and have, therefore, received considerable attention in earlier studies, Physoderma alfalfae Pat. and Lagerh, previously known as Urophlyctis alfalfae (Lagerh.) Magnus causes a disease known as "crownwart" of alfalfa and can be very destructive when conditions are favorable for it. Physoderma maydis Miyabe causes a disease of corn sometimes referred to as "brown spot". Eddins (3) reported this as one of the most important diseases of corn in the southern United States. The first record of Physoder ma occurring on members of the Por tulacaceae is the report by Greene (5) describing Physoderma claytoni.;ma Greene on Cj.aytcmia virginic~ L. collected in Michigan and Ontario. The symptoms are described and information on resting spore measurements are given .

1 2

Sparrow (17) presents an amended description of Physoderma claytoniana Greene in which he indicates that the epibiotic, monocentric phase characteristic of the genus Physoderma is unknown for this species. Sparrow's (17) resting spore measurements are, on the average, much larger than those originally given for P. claytoniana by Qreene (5). Sparrow attributed this to immaturity of Greene's material, a possibility previously suggested. More recently, however, Savile and Parmelee (14), in a report on parasites of Portulacaceae, have reported that a difference does exist in size of mature resting sporangia. They have treated the larger-spored parasite studied by Sparrow as a new variety which they have called Phzsoderma claytoniana . R. C. Greene var. sparro~ii Savile and Parmelee. In a recent paper Karling (10) reported Physoderma on Claytonia megarrhiza Parry from Utah. The collection examined by Karling (10) is part of the material used in the present study. The host species has now been determined as Oreobroma pygmaeum (A. Gray) Howell (Lewi~ia pygmaea B. L. Robinson). The above review points out the limited number 6.f published records of Physoderma on different members of the Portulacaceae. Three species of the family Portulacaceae were collected during the summer of 1956, all showing signs of infection by what appeare.d to be a ehytrid parasite of the genus Physoderma. Since our present knowledge of Phzsoderma on members of the Portulacaceae is meager it seemed desirable to learn as much as possible about the parasite, its hosts, and the relationship between parasite and host. In addition preliminary observation in the field showed considerable difference in -reaction of the host to the parasite. One host species showed marked hypertrophy near points of infection while the second exhibited only discoloration with slight or no hypertrophy in infected areas. The thi rd host showed hypertrophy from stem infections but little or no hypertrophy from leaf infections. According to Karling (9) such differences in host reaction were used by Magnus in separating the genus Uro-ehlyctis from Ph1soder:na. A more recent Yie""Toint, however, holds that host rea,::tion is not sufficient reason for separating genera. Since the value of host reaction as a taxonomic character appears questionable a study of the above mentioned parasites was of particular note. From information based on the most thoroughly studied species Karling (9) in his monograph of the genus Physoderma has compil ed a general description of the Physoderma life cycle from wh~qh the following brief summary is taken. The par asite has two alternating phases of development, an endobiotic, polycentric phase and an epibiotio, monocentric , phase. The endobiotic phase is characterized by tha occurrence of large masses of chestnut colored resting sporangia produced on a delicate rhizomycelium. In the .spring zoospores from germ- inating resting sporangia produce the microscopic epibiotic phase. Several generations of the zoosporangia and zqospores, typical of the epibiotic phase, are produced. Later in the season these zoospores give rise to the endobiotic phase. CHAPTERII

MATERIALSAND METHODS

Host Species Studies Specimens of three species of Portulacaceae were collected in Utah for use in this study. Diseased specimens of Claytonia lanceolata Pursh., Oreobroma pygmaeum (A. Gray) Howell, and Erocallis triphylla (S. \vats .) Rydb. were collected and dried in a plant press. Specimens of .Q.:. lanceolata and O. pygmaeum were preserved in formalin- aceto -alcohol preservative. Collections of all three species were made in the Uinta Mounta ins near the adjacent corners of Wasatch, Summit, and Duchesne Counties. Collections were made at the following five locations along Highway 150: {1} Lodgepole pine forest on the north side of the highway, 200 yards east of "Provo River Overlook," (2) East and west side of Trial Lake, (J) Broadhead Meadows, Duchesne Co., approximately thirty-three miles northeast of Kamas, Utah, (4) Bald Mountain Summit, on the north side of the highway, (5) Utah State Fish and Game Station at the base of Haydn Peak. In addition, plants of C. lanceolata were collected at the Great Basin Research Center, Ephraim Canyon, Sanpete County, Utah. Large quantities of corms of c. lanceolata were collected at the latter site and used in experimental work. 5

Because it was necessary to have living material of all three host species, an attempt was made to preserve the perennating organs of each species. Corms and roots were placed about two inches deep in flats of moist sand. The flats were covered with cardboard to prevent rapid drying .and then stored at 65° F. until used. Seeds were collected from mature pressed material of all three host species. Attempts were made to induce germination of seeds of each of the host species by the following treatments: (l) subjection while dry to freezing temperatures for fourteen days, (2) immersion in water and subjection to freezing temperatures for five days, (3) scarification by rubbing each seed against a piece of emery cloth with the finger until the seed coat had been penetrated. A fourth group was untreated. The seeds were germinated on cotton in moist chambers. With the exception of one half of those scarified, all seeds were incubated on laboratory tables at temperatures ranging from 70° to 80° F. One half of the scarified seeds were incubated at a constant temperature of 55° F. The germination experiments indicated that the scarification technique was the most efficient and attempts were made to germinate seeds in soil using this method. Seeds of C. lanceolata and 0~ EYgmaeum were scarified and planted in flats of sterilized soil. Part of the seeds were placed in the greenhouse at a temperature ranging from 65° to 6

75° F. and the others were placed in a germinator at a constant temperature of 75° F. Attempts were made to activate the perennating organs of host species by (l) chemical treatments using ethylene chlorohydrin and (2) cold treatments (described below). Using similar techniques to those employed by Denny (2) in treatment of potato tubers, corms of c. lanceolata were immersed for one hour in various concentrations of ethylene chlorohydrin. They were then plant ed in pots and placed in a constant temperature of 60° F. The following volumetric concentrations of ethylene chlorohydrin in 100 milliliters of water were used: o.a%. o.~%... 04%,and .02%. In a similar experiment cor"lls of the same species were subjected to the vapor of various concentrations of chlorohydrin for a period of twenty-four hours. This was accomplished by allowing the che mical to evaporate in a closed bell-jar con taining the corms. After treatment the corms were planted and placed in a ~untrolled temperature box for six months at a constant temperature of 60° F. The vapor concentrations of the chlorohydrin were based on amount of chemical evap- orated per liter of space in the bell-jar. The following amounts of chemical per liter of space were used: 0.05 ml., 0.5 ml ., l ml., and 5 ml.. The first of two cold treatments consisted of sub- jecting corms of c. lanceolata to freezing temperatures. One group was treated for seven days and the other group 7 for fourteen days. The corms were then potted and placed in a temperature box at 40° F.- In a second experiment, the perennating organs of all three host species were planted in pots and placed in a temperature box at 40° F. until growth was observed-

Phxsoderma Studies In attempts to germinate resting sporangia of the parasite dried material, fresh material, and material that had been "preconditioned•• were used. The general method of preparing sporangia for germination was to tease apart small pieces of disease d host material in distilled water and subject the resulting sporangial suspensions to various treatments. Preconditioni~g of sporangia was patterned after techniques employed by Sparrow (16). Infected host material of all three species was wrapped in cheese cloth and placed in perforated cans of moist peat moss. The cans were left outside adjacent to the campus greenhouse throughout the winter.

To study germination sporangial suspensions in distilled water were made from the dried material and from that_ conditioned throughout the winter. These sporangia were subjected to both continous and alternating cold treatments and were observed p~riodieally. Continuous treatments consisted of leaving sporangial suspensions in freezing temperatures for three, ten, and seventeen days before observing. In alternating treatments three days of freezing temperature were followed by three days at room temperature. Sporangial suspensions prepared from fresh material was studied in van tieghem cells. One set of these sporangial suspensions was incubated at temperatures just below freezing, while another set was incubated in the laboratory at temperatures ranging from 70° to 80° F. Sporangial measurements were made by mounting sporangia on slides in water. Movement of the coverslip made possible the recording of a width and length measurement for each of fifty sporangia from each host species. Similar measurements were made for sporangia of Physoderma claytoniana var. sporrowii on Claytonia virginica. This material was received from Dr. F. K. Sparrow of the University of Michigan. The width measurements were based on the tendency for one side of the sporangia to be distinctly flattened. All measurements were made at x440. Attempts were made to measure sporangia by mounting them in cedar immersion oil. By using a more viscous liquid it was easier to maintain the sporangia in the desired position until measurement could be made. This technique proved unsatisfactory, however, since it was noted that the flattened sid~ of the sporangia bearing what appeared to be an operculum, collapsed when placed in oil. The appearance of the parasite and its effect on the host were studied by super.ficial examination and by prepara- tion of microscope slides via free hand sections and the paraffin method. The stain technique employed in paraffin 9 preparations was similar to that suggested by Johanson (7). Slides were brought to fifty per cent ethyl alcohol then stained ten minutes in a solution of one per cent methyl violet in fifty per cent ethyl alcohol. After washing in alcohol the slides were stained for sixty minutes in a solution of 0.5 per cent eosin in fifty per cent ethyl alcohol. Differentiation was in a mixture of two parts turpentine, one part cedar oil, and two parts phenol. Semipermanent mounts were made of free hand sections by mounting in Amann's medium. Various attempts were made to inoculate host species growing in the greenhouse. In all cases the inoculum consisted of a suspension of resting sporangia in water. Two methods were used to inoculate plants that were actively growing and had some shoot development. In one ease the suspension was poured into the shoots so that drops collected in the axils of the leaves. The second method was to inject the sporangial suspension into the stem near the growing point with a hypodermic needle. In an effort to make experimental conditions similar to natural conditions soil inoculations were made using sporangial suspensions. The suspensions, containing at least one hundred sporangia per ml. of water, . were poured into the soil over the aorm or root. Four milliliters of this suspension were used per perennating organ. A loose soil provided for good distribution of sporangia near the corm or root. In 10

hopes of obtaining infection the perennating organs were activated with cold treatments and allowed to grow through the soil . containing the sporangia . In this experiment all three host species, and sporangia from diseased specimens of all three hosts were used . Each host was treated with sporangia from the same species in addition to sporangia from each of the other hosts . CHAPTERIII

EXPERIMENTALDATA AND OBSERVATIONS

Host Classification Specimens of each of the three host species were sent to Dr. C. V. Morton (12) at the Smithsonian Institution for indentification. In addition examination of fresh material of each host was made using Rydber g's (lJ) monograph of the Por tula caceae as a key. Based on the above information the host ._species of the present study are placed in three separate genera. Claxtonia lanceolata Pursh. is a perennial having a globose cor m f~om which one to several upright stems are produced. The ste ms are about 5-13 cm. high and have two broadly lanceolate leaves. The inflorescence is usually several-flowered with pink .petals. Oreobroma Exgmaeum (A. Gray) Howell is pere nnial and has a thick fleshy taproot from which thick basal leaves and the inflorescence are produced. The leaves and inflorescence range between 3 and g cm. in length. The flowers may be pink or white. Erocall1s triphylla (&. Wats .) Rydb. exhibits characters somewhat intermediate between the uther two species . It is perennial with a globose corm from which arise one to several shoots that range between 3 and 8 cm. in height. The stem commonly

11 12 has two or three thick fleshy leaves and one to several white flowers. The three host species are referred to by the above names throughout this paper. It seems desirable to mention that more recent keys such as Davis (l) and Harrington (6) place o. pyginaeum and E. triphylla, in the genus Lewisia. Their citations, then, are Lewisia pygrnaeum (A. Gray) Robinson and Lewisia triphzlla , (S. Wats.) Robinson. Rydberg separates Erocalli~ and .Q!:!.2- broma from Lewisa on the basis of number of sepals, the former two genera having two sepals while Lawisia has six to eight. In the classifications of Davis and Harrington, E. triphylla and O. pygmaeum are placed in the genus Lewisia, all species 0£ this genus having a cireumscissle capsule. The number of sepals becomes a specific difference in the classifications of Davis and Harrington. It might also be mentioned that E~ocallis triphylla was previously placed by Watson in the genus Cla;y;tonia, and Oreobroma P}::gmaeumwas placed in the genus Talinum by Gray, hence the synonyms Claytonia triphylla s. Wats. and Talinum pygmaeum A. Gray.

Activation and Growth of Host .sl?ecies Throughout this study the host species used have been grown in controlled temperature boxes, in the greenhouse, and in garden plots. Comparison of the three species grown under uniform conditions and observation of the plants growing in the field have provided some information on 13 activation and cultural requirements of the host.

From observations made while looking for plants infected with Ph:x;soderma it was evident that these three spe cies of the Portulacaceae are very abundant throughout the upper Provo River drainage area of the Uinta Mountains . c. l.~nceolata was found at elevations ranging from 6,000 feet to above timberline. o. pygmaeumand E. triphzlla were found only at the higher elevations above 8 ,500 feet. Field observations indicated that c. lanceolata is probably the first to begin growth in the spring although all three species are among the early flowering plants. Individuals of -c. lanceolata were found to be well advanced in growth while still under the edges of snow banks. All three species were commonly found in a moist habitat and a slightly rocky soil. Plants found in drier areas completed flowering and died back much earlier than those in locations where mositure remained longer. During the summer of' 1956 mature host pla nts were brought into the greenhouse along with the soil in which they were growing . During the remainder of the summer they flowered and produced seeds before becoming dormant. The following spring only initial shoot growth was produced before they again became dormant. In another attempt to grow the hosts in the greenhouse plants activated from perennating organs were placed in a shady area of the greenhouse. Althou gh they remained alive for several weeks little or no growth was observed. 14

As indicated in Table 1 no germination occurred in seeds subjected to cold treatments or those untreated. When treatment consisted of scarifying the seed coat up to JO% germination occurred. In attempts to grow the hosts from seeds in the soil the scarification technique was used. Seeds failed to germinate in the soil, · however, and even though seeds were germinated and young seedlings planted, growth of the hosts in this way was unsuccessful.

TABLEl

PERCENTAGEOF GERMINATIONOF THREE SPECIES OF PORTULACACEAE

Species Untreated Frozen Scarified

c. lanceolata 0 0 O. pygmaeum 0 0

E. triphylla 0 0

Interesting results were obtained by attempts to activate the perennating organs of each host species with chemical treatments. All corms treated with the vapor of ethylene chlorohydrin failed to grow , but those treated by the soaking method were successfully activated. As indi~ cated in Table 2 corms soaked in 0.04% ethylene chlorohydri n attained the highest growth with shoots averaging nearly twice those in the control. There was less shoo t growth when 15 higher concentrations of chlorohydrin were used. Those treated with 0.8% chlorohydrin had shoots averaging little more than half as high as those in the control. It is also noted that fewer corms germinated in the higher concentration of chemical. The above chemical treatment required six months and was terminated on February 6, 1957.

TABLE2

AVERAGESHOOT GROWTH FROM CORMSOF C. LANCEOLATA SOAKEDIN VARIOUSCONCENTRATIONS OF ETHYLENECHLORHYDRIN

Concentration Average shoot Number of Corms of Growth* Activated Chemical out of 5

.02% 1.5 inches 4 .04% 1.75 " 5 .40% 4 .BO% .75 " 2 Control (untreated}l.O " 5 * Shoot growth was measured from corm to shoot apex.

Cold treatments were found to be the most effective way of activating the perennating organs and hosts activated in this way were grown for an extended period of time. By sub- jecting corms of c. lanceolata to seven or fourteen day periods at freezing temperatures no growth was obtained. All three host species were activated, however, when placed in a 16 constant temperature of 40° F. Growth was observed in all three host species after three months at 40° F., over fifty per cent of the plants being activated. Considerable difference was noted in the time of appearance of the three host species. c. lanceolata was the first observed followed by O. EYgmaeurn,then E. triphylla. At the time the first shoots of E. triphylla appeared c. lanoeolata had shoots averaging approximately one-half inch and o. p;ygmaeumhad shoots averaging one-fourth inch. One group of host plants activated by a cold treatment of threo months at 40° F. were grown in an illuminated temperature box at 55° F. These plants grew and many reached maturity , however, some irregularities were observed. c. lanceolata was the only species that flowered. It often - produced unusually short or unusually lon g stems and flowering commonly occurred when plants had very little shoot growth . In addition the leaves were o~casionally abnormal or lackin g . Another group of host pla nts activated by cold treatment were grown outside in an area protected from direct sunlight. Plants grown outside showed considerable variation in reaction to growing conditions. C. lanceolata withered and died within a few days after being placed outside. & triphylla grew, but produced weak-stemmed shoots that top- pled over and died back before flowering. o. ;eygm.aeumgrew very well and persisted muc·h longer than either of the other 17 species. It did not flower, however, even though it produced an immature inflorescence.

Signs and Symptoms of the Disease Physoderma on Claytonia lanceolata Pur~h: The disease was observed as early as the first week of July and could usually be found until late August at the higher elevations. Early symptoms appeared as slightly discolored areas of host tissue which later became dark brown pustules (Fig. 4,) varying from slightly raised above the host tissue to slightly sunken. The pustules were usually round and seldom exceeded 1.5 mm. in diameter. Leaves, inflorescence bracts, pedicels, stems, and sepals were found to be infected. No infections were observed on that portion of the stem below soil level. In cases of heavy infection hundreds of pustules occur~ed on each plant. In such cases the pustules occurred so close together; particularly on the leaves, that they appeared as a single infection. Close exa mination, however, showed localized infections with pust:~Les remaining distinct, having uninfected tissue between. Although the s~pals were commonly infected (Fig. 4) no instance was fou~d of infection on the petals or essential organs of the flower. In cases of light infection apparently normal growth and flowering occurred. When infection was heavy some stunting occurred and instances were found where seed production had apparently been suppressed. Generally infected plants turned yellow and died back earlier in the season ·than did healthy plants. 18

Preparations from fresh material proved most useful for studying the endobiotic phase of the parasite due to lack of young stages for perman ent preparations. Sections through the pustules showed nwnerous light brown resting sporangia ~illing many of the host cells . These sporangia are typically ellipsoidal in shape with one side being flattened. They appear to have an operculum on the flattened side . The contents of the sporangia are typically granular and are usually arranged around a large central globule+ The sporangia measure 9-14 x 18- 24 mu. (Table 3 ) and have a smooth wall measuring approximately 2 mu. thi~k. There was no evidence that infected host cells were enlarged. although at points of infection many cells were found completely filled with resting sporangia . The number of sporang1a per cell ranged from one to forty or more depending on the size of the cell. The rhi~omycelium could be observed fro m fresh pre - parations of o~rly stages of infection . It was very delicate, making observations even at high magnification difficult . Thin thread - like strands bearing hyaline swellings (Fig . $ ) were observed to extend through more than one cell . Immature resting sporangia coul d be seen developing at the ends of the rhizomycelial strands . Examination of cross-sections of the infected stems pointed out the localized nature of infection and indicated tissues invaded . At points o;f.. i :-i.fection sporangia were 19

commonly found in the cortex and epidermis. Less frequently the sporangial masses extended inward to the vascular system and appeared to be within these tissues, however, vascular infection was still somewhat questionable.

TABLEJ

SIZE OF RESTINGSPORANGIA ON LEAVESOF 4 HOSTSPECIES

Host Range of sporangial size Average sporangial size Width Length Width Length

c. lanceolat .a 14.4-19.2 mu. 11.6-24.0 mu. 16.6 mu. 21.6 mu. E. triphylla 16.0-24.0 mu. 19.9-2$.$ mu. 1$.9 mu. 24.5 mu. o. pygmaeum 22.8-27.2 mu. 25.6-36.8 mu. 23.2 mu. 29.7 mu. c. virginioa 19.2-25.6 mu. 25.6-32.0 mu. 24.0 mu. 29.0 mu.

Physoderma on Oreobroma pygmaeum (A. 9ray) Howell: The disease on this host was collected early in July an d until the latter part of August on plants growing in a m~:st boggy area. Early symptoms of the disease are distinct reddening (Fig. 2) of an area of host tissue the canter of which later becomes darker. Accompanying the color change is a prominent hypertrophy of host tissue. Well developed stages of the disease then appear very conspicuously as dark purple-brown; often shiny galls with surrounding red areas (Fig. 2). These galls are very irregular in shape, but tend to be round. They often measure between three and four millimeters in diameter and may be raised as much as two to three millimeters. 20

The disease was fo und on all ae ri al parts of the plant with the floral heads and lower half of the leaves most commonly infected. In cases of light infection the plants continu ed growth and ff floral heads did not become infected they completed flowering and produced seeds. Heavy infection caused marked stunting an ,.i conspicuous malformations of various parts of the plant. The larger galls on the leaves and pedicels often caused sharp bends (Fig. l) in these parts to the extent that they would alter the normal plant form. The malformations of the floral heads (Fi g. 2) were particularly noticeable, various organs being distorted al- most beyond recognition in cases of heavy infection. In contrast to the complete absence of infection on the petals and essential organs of C. lanceo1ata this species commonly had infection on all parts of the flower (Fi g . 2). Petals and sepals were found to be thick and fleshy due to infection and stamens were short, thick structures many times their normal diameter. If anthers developed they too were subject to infection. The pistil was often several times its normal size and galls present on one side would distort the ovary often bending it until the style would point in a lateral direction . Also in contrast to c. lanceolata the number of infections per plant was much less. There was rarely more than fifty galls on a heavily infected specimen and usually much less. The endobiotic phase was studied from free hand and 21 microtome preparations of infected parts. Sections made through galls showed masses of light brown resting sporangia. The sporangia were ellipsoidal with one side flattened similar to those found on c. lanceolata. A1though shape and contents of Ph;ysoderma sporangia on o. py;gmaeum (Fig. 6) were similar to those on C. lanceolata the sporangial measurements, 23-27 x 26-37 mu. (Table 3) were consistently greater. ._The cells of the hypertrophied areas were found to be filled with sporangia and the hypertrophy apparently resulted from enlar~ement of parenchyma cells thus filled with sporangia. Host cells were found containin g one to seventy or more sporangia. Although masses of sporangia made observation difficult no proliferation of host tissue was observed. Fresh preparations of early stages of the disease showed the delicate rhizomycelium. No significant difference was observed in the vegetative system of the parasite on the three host species. Hyaline swellings on the mycelial strands were of various shapes and were either ·one- or two-celled. The rhizomyoelium extended through several host cells and resting sporangia of various sizes, depending upon age, were developing at the ends of mycelial strands. Slightly immature sporangia appeared to have several branched, horn-like structures arising from the sio .e opposite the flattened surface. Althou gh somewhat longer and quite slender, they resembled the haustoria described on sporangia of Physoderma (Urophyctis) 22

alfalfae by Jones and Drechsler (8) and on ~hysoderma claI- toniana by Sparrow (17). Sections through mature galls on the leaves and pedicels indicated all host tissues were subject to infection, the parenchyma cells of the cortex were commonly invaded. Masses of sporangia occurred from upper to lower epidermis of the leaves even in areas containing a vascular bundle. Cross-sections through galls on the pedicels showed sporangia in the phloem and in parenchyma cells of the xylem. Ph1soderma on Erocallis triphylla (S. Wats .) Rydb: Diseased mate rial was collected early in July and until late in August on plants growing in moist areas near springs. The symptoms on this host ware unusual in that they exhibited the two types previously described on c. lanceolata and O. EYgmaeum. Infections on the stem were similar to those described on o. pygmaeumin that marked pypertro phy occurred, resulting in a dark purple~brown gal l. Stems were commonly infected and hypertrophy often caused this structure to become more than twice its normal size. (Fi g . 7). The occasional infections on the leaves showed only slight variation from those described on the leaves of c. lanceolata. Number of infections per plant was less and both hypertrophy and diameter of pustules were slightly greater. Occa$ionally infections were found on the floral parts of E. triphylla. Such infections were similar to those on C. lanceolata in that sepals were infected while petals and essential organs 23 were uninfected. On material examin .ed, only few infections occurred per plant. The number usually being less than twenty-fiva. Light infection apparently did not prevent growth and flowering of plants, but as in the other species heavier inf'ections may have caused some stunting. It was also noted that infected plants usually yellowed and died back earlier in the season than those not infectad. Fresh mounts of immature infections parmitted study of the vegetative phase. The rhizomycelium (Fig. 8), and the shape and con½ents df the resting sporangia were not different from those already described on the other hosts. Measurements 9t spora~g"a• 16-24 x 20-29 mu. (Table J) were somewhat intermedi te betwe~n sporangia on o. p.x,g awn and c. lance-ola~a. Counts of sporangia per host cell ranged from one tc forty or more. · Due to the difference in host raacti-0~ on ·stems a~d leaves of this host (Fig. 3), it was considered advisable to measure resting sporangia from stem and le f infections and compare sporangial size from the two areas . The measure - ments of fifty sporangia £rem stem and leaf infections appear in Table 4• Cross-sections of the stem at points of infection provided information on which host tissues were invaded by the parasite. The common pattern was for sporangia to occur inward from the epidermis to the thick walled cells surrounding 24 the vascular system. In areas showing marked hypertrophy, however, the vascular tissue was also infected (Fi g. 5). As with infections of o. pygmaeum, the phloe m and pare nchyma cells of the xylem were invaded.

TABLE4

MEASUREMENTSOF RESTING SPORANGIA FROMLEAF AND STEM OF EROCALLISTRIPHYLLA

Source of Range of sporangial size Average sporangial size sporangia Width Length w!cttn Lengtfi

Leaf 16.0-24.0 mu. 19.9-28.8 mu. 18.9 mu. 24.5 mu. Stem 19.2-24.0 mu. 24.0-J2.0 mu. 21.2 mu. 27.2 mu.

Activatio n of the Parasite Attempts to induce germination of the resting sporangia were generally unsuccessful. On one occasion examination of a sporangial suspension of fresh material that had been incubated at a temperature of approximately 40° F. for four days contained what appeared to be typical chytrid zoospores, but

' their origin was not determined. Occasionally sporangia were observed in which the large central globule had broken up and movement of the homogenous contents could be seen. In addition to the above, empty sporangia were often observed, but they were never seen in the process of germination. 25

.!!!£culation Studies All attempts to inoculate host plants were unsuccessful . Several observations in the field, however, are worthy of mention in connection with this aspect of the present study . At the Utah State Fish and Game Station in the Uinta Mountains all three host species were found growing together . One population was of particular interest because it was noted that although all three species were growing side by side, C. lanceolata was heavily infected whi le E. tri phylla and O. pygmaeu~ were uninfected . This was true even though the leaves or stems of diseased and non-diseased plants were in contact . In the same general area a population was examined

in which all three species were infected by Physoderma . c. lanceolata having its typical non-hypertrophied pustules, o. pygm?eum with prominent galls, and E. tri EhXlla with both pustules on the leavas and galls on the stems. CHAPTERIV

DISCUSSION

The Parasi tes Karling (9), in his monograph of the genus Physoderma, has discussed the status of this genus and for the first time merged the genus Urophlyctis with Physoderma. This concept had been suggested earlier by Sparrow (15) and is the most accepted viewpoint at the present time. As Karling (9) indicates the genus Urophlyctis was originally separated from Physoder ma by Schroeter on the basis of sexuality and the presence of ephemeral, epibiotic zoosporangia. Schroeter believed that the resting sporangia developed after sexual fusion of turbinate enlargements. Karling (9) also indicates that Magnus l~ter distinguished Physoderma and Urophlyctis on the basis of host reaction. In Urophlyctis were placed all species causing a marked hypertrophy of the host, while those causin g only discoloration and slight or no hypertrophy were maintained in Physoderma. Karling (9) further points out that investigation of species in both genera has dis- proved one by one the characters separating them. Although the difference in host reaction does exist he maintains that ~bis is not sufficient basis for separating the genera . Li ngappa (11) supports the merger of Physoderma and

26 27

Urophlyctis in his studies with two new species from India, Physoderma corchori Lingappa and Physoderma commelinae Lingappa. As he points out these two species show the two distinct types of host reaction used by Magnus as basis for separating Urophlyctis from Physoderma. On the other hand these new species have the same type of germination of the resting sporangia. Lingappa (11) therefore points out that there is no correlation between host reaction and ger mination of the resting sporangia, thus minimizing the importance of host reaction as a basis for generic distinction . The parasites and hosts reported in the present study provide excellent material from which to study the significance of host reaction as a taxono mic character. As pointed out in the description of Physoderma on c. lanceolata and O. pygmaeum two very different types of host reaction to the parasite occur . Hypertrophy and malformation of plant organs are common on o. 12ygmaaum, while c. lanceolata exhibits discoloration and occasional slight hypertrophy of host tissue . Both types of host reaction are found on E. triphylla . Infectio ns on the leaves cf E. triphylla are similar to infections on c. lanceolata . On the other hand stem infections on E. triphyll§ usually showed hypertrophy similar to infections on o. pygmaeum. It was noted, however, that apparently mature infections having no hypertrophy often occurred on the stems . In contrast to the above differences in host reaction microscopic examination showed the vegetative phases of the parasites of all three hosts to be quite similar. The above brief comparison of Physoderma on three hosts points out how well this material lends itself to a study of host reaction. This comparison also indicates that a problem exists with regard to assigning the three Physoderma parasites to species. This problem is discussed in the following paragraphs. The basis for designating new species of Physoderma is not too well defined. As Karling (10) mentions the tendency is to name a new species of Phvaodern:.a with each new host. A review of the literature shows that new species have been described on the basis of nearly any combination of the following characters: host reaction, host range, size, shape, and structure of the resting sporangia, size

and shape of zoospores, methodI of . germination of the sporangia, and general structure of the vegetative phase. In the present study only host reaction and the endobiotic phase of the parasites have been successfully studied. There is as yet no conclusive data on the method of germination of the sporangia or oross ...inoculation studies. In my opinion such information is essential to assigning these parasites to species. For this reason the parasites studied are not assigned to species until further information is obtained. The similarities of their vegetative phases, including general shape of resting sporangia, suggest that the Physo- 29

derma parasites on the three hosts under study might be regarded as one species. This of course, assumes that no difference exists in method of germination of the sporangia. To regard these parasites as a single species, however, one would have to mini mize the importance of resting spore size and host reaction. These two characters have so far played an important role in the designation of species in the genus Phx:soderma. For the present the parasites are there- fo ne considered separate entities and are re .ferred to through• out this report as Physoderma on c. lanceolata, Phxsoderma on 2.!, pxgmaeum, and Physoderma on E. triph:tlla. Whether they are best considered as distinct genera, species, or varieties should await further study .

The endobiotic phase, including size 0£ sporangia, and host symptoms of the Physodez:nY! on c. lanceolata are very similar to those of Physoderma claytonia11.,a Greene. The \ vegetative phase and host symptoms of Physoderma on f.:. lanceolata are also similar to those illustrated by Sparrow (17) for what he thought was Physoderrna clax:toniana Greene. The parasite Sparrow (17) studied has more recently been designated as a variety of that species, Physoderma claytoniana var Sparrowii (Savile and Parmelee (14), because of its larger sporangia. The size of sporangia of Phx:soderma on c. lanceolata being similar to PhysodErrma claytoniana Greene, are therefore smaller than the variety. Sporangial shape and position of haustoria along with structure of the JO

rhizomycelium, however, agree very olosely with Sparrow's illustrations of these structures. The similarities between the Physoderma on C. lanceolata and Physo~2rma claytoniana suggest the possibility of these belonging to the same specie$. Resting sporangia of the Physoderma on o. pygmaeum (Table 3) are quite similar in size to resting sporangia of P. claytoniana var. sparrowii. This similarity is so despite 6 - • marked differences in host reaction to these parasites . If emphasis is placed on host reaction as a basis for distinction these parasites might be regarded as separate species . On the other hand to regard them as one species host rea ction would need to be discounted entirely. In either ease much would depend on comparison of the other characters such as germination of sporangia and host range . Host reaction t o the Bhysoderma on o. pygmaeum is quite pr.onounced and is worthy of mention at this point, As indicated in the data host symptoms include extreme malformation of all floral parts . This is interesting especially when compared with symptoms caused by Physoderma c1:aztoniana, Physoderma on c. lanceolata, and Physoderma on E. triphylla. In all cases the parasite has been found infecting the sepals but never the petals or essential organs . In th~ case of Ph;x:soderma on O. 12ygmaeum, however, the floral parts are commonly infected and results in the most characteristic symptom of this disease. The Physoderma on E. triphylla present an unusual prob- · Jl lem in that there is a marked difference in host reaction to infections on the leaves and on the stems. Sparrow (17) reports similar reactions on Claytonia virginica although differences are less pronounced. In addition to the host reaction difference on E. triphylla, there is a slight difference in size of resting sporangia from leaf and stem tissue (Table 4). There is a possibility that some measure - ments (Table 3 and 4) are based on immature material although c~re was taken to avoid such an error. When these differences in host reaction and size of spor·angia are considered in connection with similarities of vegetative phase and gene ral sporangial structure, the question arises as to whether there are one or two parasites on E. tri{?hYlla. An answer to thts question would be governed by the amount of emphasis placed on host reaction, host range, and spore size. This is assum- ing, of course, that the method of germination of the sporangia is the same in both cases. It might also be noted in Table 4 that the difference in average size of resting sporangia on leaf and stem material of Ke. tri;ehylla is approximately 2 mu. in width and 3 mu. in length. This difference is just as great as the one between size of resting sporangia from infected leaves of E. triphylla and c. lanceolata. Much of the above taxonomi .c discussion is based on the assumption of similarity in germination of the resting sporangia. It does, however, point out the significance of the information obtained in this study, particularly when it can be coupled 32

with further information on the parasites here considered. Additional knowledge of the method of sporangial germination and host range of these parasites will undoubtedly contribute to a better understanding of the taxonomy of the genus Physoderma.

The Hosts It is evident from the data that the host species in the present study can be grown under controlled conditions for . experimental uae . A critical step in inductng seed ger~:lnation .of' all three host species is apparently penetra- tion of the seed coat. This is suggested by the successful germination (Table 1) of all three hosts only after scarification of the seed coat. The testing of a wider range of conditions would likely make possible the growth of the host species from seeds in the soil . During the present study perennating organs of all three host species were succQssfully stored for a year's time. With regard to activation of the parennating organs

by using chemicals, the data suggest a possible growth governing mechanism in the use of ethylene chlorhydrin. While higher concentrations of the chemical definitely had an inhibitory effect on both activation and growth, low concentrations appeared to stimulate growth slightly. A second experiment using a wider range of low concentrations was not completed because of insufficient time , but may indicate 33 better the value of such a treatment in governing host growth . It is evident from the data that a eold treatment of above freezing temperatures for three months was the most successful method of activating host perennating organs. This method was used to activate the host plants that were used in inoculation studies. It was also used to activate plants grown outside in garden plots and in illuminated temperature boxes. Cold treatments involving freezing temperatures appear to have been too severe. A knowledge of the natural growing conditions of the hosts offer a possible explanatio n for some of the unusual reactions ndted when grown under experimental conditions. Atte mpts to grow the host to maturity in the greenhouse met with failure. A similar difficulty is reported by ThomatS {le} in work with Clayto nia la nceo ljta. The low I temperatures at which these plants normally gr-ow, particularly O. pvgmaeum and E. triphylla, would suggest green - house temperatures as being too high • .It is ditficult to explain the failure of flowering in O. pygmaeum and!:,. triphylla. It is noted , however, that c. lanceo+ata the only species that did produce flowers was also the only species found at lower elevations. In the host plants under study the epidermis and cortex were m.ost commonly infected by the E.b¥..s.od.ermaparasite. As pointed out in the data, however, vascular infection was 34 observed on o. pygmaeum and E. triphylla. Infection of the vascular system of c. lanceolata is still questionable. In the two hosts having vascular infection, sporangia were observed in the phloem and in the parenchyma cells of the xylem. This was particularly true near hypertrophied areas of o. py~maeum and E. triphylla. These examples of vascular infection are additions to a relatively limited number of such infections by chytrids. Gopalkrishnan (4) reported the infection of the vascular system of Agropyron repens L. Beauv. by Physoderma graminis (Busgen) Fischer as the first record of such an infection by a chytrid. More recently Lingappa (ll) made a similar report of Physoderma corchori Lingappa in the vascular system of Corchorus olitorius Linn. Future experimental work will likely point out the conditions necessary for germination of the resting sporangia of the parasites under study . Gopalkrishnan (4) suggests that drying delays germination by possibly causing imper- meability of the exosporangium. Sparrow (16) indicates preconditioning of sporangia may be required for germination. Even though this information was employed in attempts with fresh and preconditioned sporangia, germination was not obtained in the present study. In not being able to obtain germination of the resting sporangia some of the most logical methods o.f inoculation could not be attempted. When germination of resting sporangia is accomplished cross- inoculation experiments will likely be more successful. 35

This, in turn, will provide information for assigning the parasites under study to species. In addition, it should add to our knowledge of host reaction and spore size as taxonomic characters. CHAPTER V

SUM.MARY

l . Thr e species of the Portulacaceae, each infected by a parasite of the genus Physodgrma, have been colleeted at s ,veral locations in Utah . 2. The host species have b en identified as Olax:t:onia lan ceolata Pursh ., Preobto a 2xsaeum (A. Gray ) Howell , and Erocallis triphylla (S. W ts . ) Rydb. The report or fhy -soderma on c. lanc~ol@t. and E. t~iRhYlla are new re cords and xtend our knowledge o! the- host r nge ot this par sit . 4. By scarific tion of these d coat , seed germination has been induced in a.11 three hot speci s . , . Plants activat d by treatment 0£ perennating organs with near free zing temperatures have been grown tom turity in experimental conditions . Tr atm nt with high concentra - tions ot ethylene chlorohydrin inhibited act1vat1on and growth of host sp ci s . On the other h nd lower concentra - tions appeared to have a slight sti mulatory ff ct ~ 6. A des cription is giv n of syn.~toms and ndobiotic phase of th disease on each host and is accompanied by photographic illustrations . Vascular inf ct1.on is report d in o, RYQ¥!eumand& , tr1phllia . Comprative spor angi al measurem nts have be n made for 11 thr parasit s . 37

7. Two distinct types of host reaction are reported. Infections on o. pygmaeum and stems of E. triphylla exhibit marked hypertrophy, while those on c. lanceolata and leaves of E. triphylla show slight or no hypertrophy. S. Although attempts have been made to germinate resting sporangia of each parasite, no germination was obtained. 9. Attempts to make cross-inoculations on the three hosts have been unsueeessful. 38

Fig. 1. Healthy and infected specimens of O. pygmaeum.

Fig. 2. Specimen of O. pygmaeum showing infection of the floral organs. 39

Fig. 3. Specimens of E. triphylla showing leaf and stem infections.

Fig. 4. C. lanceolata, showing infection on leaves anct sepals. 40

• •

Fig. 5. Longi-section through gall on stem of E. triphylla showing hypertrophy. Rf'f' rox.. sox

Fig. 6. Infected cells of O. pygmaeum showing resting sporangia and rhizomycelium. Rpr rr:,x. ,zsx. 41

Fig. 7. Physocterma on E. triphylla, showing resting sporangium, rhizomycelium, anct intercalary swelling. flffYOX.. 2000>t.

•

Fig. 8. Physoderma on E. triphylla, showing rhizo mycelial strands and intercalary swellings. Rppro1<. 2ooox LIST OF REFERENCES

1. Davis, R. J. Flora of Idaho. Wm. C. Brown Co., Dubuque, Iowa. 1952. 2. Denny, F. E. Hastening the sprouting of dormant potato tubers. Boyce Thompson Institute 1: 59.65. 1929. Eddins, A.H. Infection of corn plants by Physoderma zeae-maydis. Jour. Agr. Res. !I:£: 241-25J. · 1933. Gopalkrishnan, K. s. Development and parasitism of Physoderma graminis (Bus.) Fischer, on Agro,~ron repens (L.) Beauv.' Phytopath. _il: 1065-10 • 1951. 5. Greene, H. c. Notes on Wisconsin parasitic fungi. IV. Farlowia 1: 569-581. 1944. 6. Harrington, H. D. Manual of the plants .of Colorado. Sage Books, Denver, Colorado. 1954. 7. Johansen, D. A. Plant Microtechnique. McGraw-Hill Book Co., New York and London. 1940. $. Jones, F. R., and Drechsler, C. Crownwart of alfalfa caused by UroEhlyctis alfalfae. Jou.r. Agr. Res.~: 295-324. 192 • . . 9. Karling, J. s. The genus Physoderma. Lloydia ,U: 29-71. 1950. 10. Karling, J. S. Unrecorded hosts and species of Physoderma. Bull. Torrey Club,[!: 292-299. 1950. 11. Lingappa, B. T. Two new species of Ph soderma from India. Mycologia ltJ_: 109-112. 193.3 12. Morton, C. V. Personal letter to the writer. Washington 25, D. C. August 17, 1957. 13. Rydberg, Per Axel. Portulacaceae. North American Flora -21: 279-3J6. 1932. Savile, D. B. o., and Parmelee, J. H. Some fungal parasites of Portulacaceae. Mycologia 11:&:573-590. 1956.

42 43

15. Sparrow, F. K. Aquatic Phycomycetes. Univ. of Michigan Press, Ann Arbor, MiQhigan. 1943 16. Sparrow, F. K. Observations on ehytridiaceouis parasites of phanerogams. I. Ph soderma menianthis deBary. Amer. Jour. Bot. l,l: l 12-118. 194. 17. Sparrow, F. K. Observations on chytridiaceous parasites of phanerogams. III. Physoder ma claytoniana and an associated parasite. Amer. Jour. Bot. J1: 325-329. 1947. 18. Thomas, Kathryn. The floral development of Claytonia lanceolata Pursh. Unpublished Master's thesis, Depart- ment of Botany, Univ. of Utah. ABSTRACT

Three species of the Portulacaceae each infected by a Physoder ma parasite were collected at several locations in Utah. The hosts have been identified as Claytonia lanceolata , Pursh., Oreobroma pygmaeum (A. Gray) Howell, and Erocallis triphylla (S. Wats .) Rydb. Since our pr esent knowledge of Physoder ma on members of the Portulacaceae is meager , it seemed desirable to learn as much as possible about the parasite, its hosts, and the relationship between parasit e and host. Plants of all three hosts activated from perennating organs were grown for extended periods of time under uniform conditions. Cold treatments were most successful in activating host perennating organs and scarification of the seed coat was necessary to induce seed germination. From field and laboratory studies a description was made of the appearance and the effect of the parasite on each host species. Marked hypertrophy of host tissue and frequent distortion of pla nt organs were characteristic of infections ,.on O. :eygmaeum and the stems of E. triphylla. Discoloration and slight or no hypertrophy were characteristic of infections on C. lanceolata and the leaves of!.!. triphylla. Microscopi c study of the endobiotic phas e indicated similarities in the vegetative phase of the parasites on the three hosts. Comparative sporangial measurements, however, showed differences in the size of resting sporangia from each host. The present report of the occurrence of Physoderma on c. lanceolata and E. triphylla are new records and extend our knowledge of the host range of this pa r asite. The report of vascular infection on O. pygmaeum and E. triphylla are additions to a limited number of such infections reported for chytrid parasites. Various attempts to germinate · resting sporangia of the parasites and make cross-inoculations on the three host species have been unsuccessful. Such information is important in the classification of the parasites here involved and will undoubtedly contribute t o an evaluation of taxonomic characters in the genus Physoderma.