Bulletin 11.1.1140X8 a>tixx*a.l History A Comparative Study of Two Components of the Poinsettia Root Rot Complex )F ILLINOIS tTMENT OF REGISTRATION AND EDUCATION RAL HISTORY SURVEY DIVISION NA, ILLINOIS NOV :^1971 LIBRARY THEUBRARYOFTHE SEP 1 f b/1 UNIVERSITY OF ILLINOIS AJ. URBANA-CHAMPAIQN VOLUME 30, ARTICLE AUGUST, 1971 age 437, Figure 3 age 439, Figure 4 age 440, Figure 5 age 441, Figure 7 ERRATA Should have caption from Fig. Should have caption from Fig. Should have caption from Fig. Caption should read: The colony on the righ was exposed to constant light for 14 days, while the colony on the left was maintaine in the dark for the same period. 5, ILLXN'OIS a>tui:*a.l History Su.z"v-ey BXTLLETIM A Comparative Study of Two Components of the Poinsettia Root Rot Complex tobert S. Perry OF ILLINOIS RTMENT OF REGISTRATION AND EDUCATION JRAL HISTORY SURVEY DIVISION ^NA, ILLINOiS VOLUME 30, ARTICLE AUGUST, 1971 STATE OF ILLINOIS DEPARTMENT OF REGISTRATION AND EDUCATION BOARD OF NATURAL RESOURCES AND CONSERVATION William H Robinson, Chairman; Thomas Park, Ph.D., Biology; L. L. Sloss, Ph.D., Geology; (Vacant) Chemistry; Robert H. Anderson, B.S.C.E., Engineering; Charles E. Olmsted Ph.D., Forestry' W. L. Everitt, E.E., Ph.D., Representing the President of the University of Illinois; ROGER e! Beyler, Ph.D., Representing the President of Sonthe— '" "-Illinois University. NATURAL HISTORY SURVEY DIVISION, Urbana, Illinois SCIENTIFIC AND TECHNICAL STAFF George Sprugel, Jr.. Ph.D., Chief Alice P. Campbell, B.A., Secretary to the Chief Section of Economic Entomology William H. Luckmann, Ph.D., Entomologist and Head Willis N. Bruce, Ph.D., Entomologist Wayne L. Howe, Ph.D., Entomologist STEVENSON MOORE, III, Ph.D., Entomologist, Extension HOWARD B. Petty, Ph.D., Entomologist, Exten- sion James E. Appleby, Ph.D., Associate Entomol- ogist EDWARD J. ARMbrust, Ph.D., Associate Ento- mologist . Marcos Kogan, Ph.D., Associate Entomologist JOSEPH V. Maddox, Ph.D., Associate Entomol- ogist RONALD H. Meyer, Ph.D., Associate Entomol- ogist Robert D. Pauscil Ph.D., Associate Entomol- ogist RALPH E. Sechbiest, Ph.D., Associate Entomol- ogist George L. Godfrey^ Ph.D., Assistant Entomolo- gist Clarence E. White, B.S., Assistant Entomolo- gist Keun S. Park, M.S., Assistant Chemist Sue E. Watkins, Supervisory Assistant RoscOE Randell, Ph.D., Assistant Professor, Extension Donald E. Kuhlman, Ph.D., Instructor, Exten- sion Tim Cooley, M.A., Assistant Specialist, Exten- sion Jean G. Wilson, B.A., Supervisory Assistant Martha P. Nichols, M.S., Research Assistant Keturaii Reinbold, M.S., Research Assistant Lowell Davis, Technical Assistant Na.ncy' D. DeWitt, B.S., Technical Assistant Marcia Janes, B.S., Technical Assistant Lii-PiNG Kan, M.S., Technical Assistant Marie Monkman, M.S.. Technical Assistant Stephen Roberts, B.S.. Technical Assistant Douglas K. Sell, B.S., Technical Assistant JOHN T. Shaw, B.S., Technical Assistant Section of Botany and Plant Pathology " J. Cedric Carter, Ph.D., Plant Pathologist and Head ROBERT A. Evers, Ph.D., Botanist Junius L. Forsberg, Ph.D.. Plant Pathologist Eugene B. Himelick, Ph.D., Plant Pathologist R. Dan Neely, Ph.D., Plant Pathologist D. F. Schoeneweiss, Ph.D., Associate Plant Pathologist J. Leland Crane, Ph.D., Assistant Mycologist Walter Hartstirn, Ph.D., Assistant Plant Pathologist Betty' S. Nelson, Technical Assistant Gene E. Reid, Technical Assistant Section of Aquatic Biology George W. Bennett, Ph.D., Aquatic Biologist and Head D. Homer Buck, Ph.D., Aquatic Biologist R. Weldon Larimore, Ph.D., Aquatic Biologist William C. Starrett, Ph.D., Aquatic Biologist Robert C. Hiltibran, Ph.D., Biochemist William F. Childers, Ph.D., Associate Aquatic Biologist Donald F. Hansen, Ph.D., Associate Aquatic Biologist Richard J. Baur, M.S.. Research Assistant Dennis L. Dooley, Technical Assistant Mary Frances Martin, Technical Assistant Robert F. Randall, Ph.D., Technical Assistant Kenneth R. Walker, Technical Assistant C. Russell Rose, Field Assistant Warren U. Brigham, M.S., Junior Technical Assistant Section of Faunistic Surveys and Insect Identification PHILIP W. S.MiTii, Ph.D., Taxonomist and Head Wallace E. LaBerge, Ph.D., Taxonomist Milton W. Sanderson, Ph.D., Taxonomist Lewis J. Stannard, Jr., Ph.D., Taxonomist Robert W. Poole, Ph.D., Assistant Taxonomist John D. Unzicker, Ph.D., Assistant Taxono- mist Donald W. Webb, M.S.. Assistant Taxonomist Roderick R. Irwin, Research Affiliate Bernice p. Sweeney, Junior Professional Scientist Section of Wildlife Research Glen C. Sanderson, Ph.D., Wildlife Specialist and Head Frank C. Bellrose, B.S., Wildlife Specialist Richard R. Graber, Ph.D., Wildlife Specialist Harold C. Hanson, Ph.D., Wildlife Specialist William L. Anderson, M.A., Associate Wildlife Specialist W. W. Cochran, Jr., B.S., Associate Wildlife Specialist William R. Edwards, M.S., Associate Wildlife Specialist JACK A. Ellis, M.S., dissociate Wildlife Special- ist RONALD F. Labisky, Ph.D., Associate Wildlife Specialist ^ Charles M. Nixon, M.S., Associate Wildlife Specialist Stanley L. Etter, M.S., Assistant Wildlife Spe- ROBERT E. Greenberg, M.S., Assistant Wildlife Specialist G. Blair Joselyn, M.S., Assistant Wildlife Spe- GEORGE B. rose, Ph.D., /lssisfa?!t Wildlife Spe- David R. Vance, M.S., Assistant Wildlife Spe- cialist RONALD L. WESTEMEIER, B.S., Assistant Wildlife Specialist RONALD E. DUZAN, Technical Assistant ; NORMA J. Hubbard, Technical Assistant ; Mary Ann Kjos, Technical Assistant , Helen C. Sciiultz, M.A., Technical Assistant Hilda Wiesenmeyer, Technical Assistant Eleanore Wilson, Technical Assistant ROBERT D. Crompton, Field Assistant JAMES W. Seets, Laboratory Assistant Section of Administrative Services ' ROBERT O. Watson, B.S., Administrator and i Head ' Supporting Services WiLMA G. Dillman, Property Control and Trust Accounts ROBERT O. Ellis, Assistant for Operations Lloyd E. Huffman, Stockroom Manager J. William Lusk, Mailing and Distribution « Services . , „ Melvin E. Schwartz, Financial Records JAMES E. SERGENT, Greenhouse Superintendent ' Publications and Public Relations Owen F. Glissendorf, M.S., Technical Editor ROBERT M. Zewadski, M.S., Associate Technical'. Editor , . , _,.. Shirley McClellan, Assistant Technical Edt- RiciiARD M. SHEETS, Technical Illustrator WiLMER D. Zehr, Technical Photographer Technical Library DORIS F. DODDS, B.A., M.S.L.S., Technical Li- JEAN ICKES, B.A., M.S.L.S., Assista7it Technical Librarian CONSULTANTS: PARASITOLOGY, NORMAN D. Levine, Ph.D., Professor of Veterinary Parasitology and Veterinary Research, University of Illinois; Wildlife Research, Willard D. Klimstra, Ph.u., . Professor of Zoology and Director of Cooperative Wildlife Research, Southern Illinois Univer- sity; STATISTICS, Horace W. Norton, Ph.D., Professor of Statistical Design and Analysis, Univer- sity of Illinois; ENTOMOLOGY', GILBERT P. WALDBAUER, Ph.D., Associate Professor of Entomology, University of Illinois. CONTENTS ACKNOWLEDGMENTS 420 LITERATURE REVIEW 420 MATERIALS 422 Fungi 422 Media 422 Poinsettias 423 METHODS 423 Microscopic Examinations - 423 pH Studies - 423 Temperature Studies 424 Light Studies 424 Pathogenicity Tests 424 RESULTS - 425 Morphology of Thielaviopsis hasicola 425 Colony Characteristics of T. hasicola 429 T. hasicola on Poinsettias 430 Morphology of Chalawpsis thielavioides 430 Colony Characteristics of C. thielavioides 433 C. thielavioides on Poinsettias 434 Comparison of Growth of Chalawpsis thielavioides and Thielaviopsis hasicola on Various Media 435 Comparison of Growth Rates - 435 Effect of pH 436 Effect of Temperature - 438 Effect of Light on Growth Rates - 439 Results of Pathogenicity Tests - 442 DISCUSSION 446 SUMMARY 447 LITERATURE CITED 450 INDEX 452 This paper is published by authority of the State of Illinois, IRS Ch. 127, Par. 58.12. It is a contributi07i from the Section of Botany and Plant Pathology of the Illinois Natural History Survey. Robert S. Perry is a graduate student in the Department of Plant Pathology, University of Illinois, and a Junior Professional Scientist in the Section of Botany and Plant Pathology, Illinois Natural History Survey. (30346—2M—8-71 ) ''s^\U'LmJ-^ Frontispiece. — Healthy poinsettias and plants showing disease symptoms. In the upper picture the plant in front was inoculated with Thie/av/opsis basico/a when about 90 days old by the stem-incision method. It shows the curling and browning of leaves and the loss of leaves resulting from the disease caused by this fungus. The other plants were not inoculated and remained healthy. In the lower picture the stunted plants on the right were inoculated with T. bos/co/o when about 30 days old by the soil-probe method. The control plants on the left were not inoculated and show normal, healthy growth. A Comparative Study of Two Components of the Poinsettia Root Rot Complex Robert S. Perry THE MOST IMPORTANT patho- logical problem encountered by com- mercial growers of poinsettias ( Euphor- bia piilcheirima Willd. ) is caused by a complex of fungi composed of Thie- laviopsis basicola (Berk. & Br.) Ferr., Rhizoctonia solani Kuhn, and Pijthium ultimum Trow. Collectively these three organisms cause a condition referred to as the poinsettia root rot complex. Each of these fungi is capable of pro- ducing a disease, or they may occur in combination, producing a disease com- plex. R. solani and P. ultimum gen- erally attack young poinsettia cuttings during the early stages of propagation, and early death usually occurs. Symp- toms of the disease caused by T. basic- ola usually appear later in the season, often near poinsettia marketing time, resulting in root deterioration and leaf drop. Tlie presence of symptoms of any of the diseases caused by these fungi is not restricted to a specific phase of the plant's life cycle but may appear anytime. Since the three patho- gens involved respond differently to fungicides and environmental condi- tions, control of this complex is difficult. T. basicola is restricted to attacking the roots of poinsettias, but it is ca- pable of producing infection at almost any point on the roots. An extreme in- fection may cause the entire root ball to turn black and deteriorate. An ad- vanced symptom of Thielaviopsis root rot is a longitudinal splitting of the stem near the ground line. P. ultimum commonly produces infection sites near the root tips and causes extensive root deterioration. In the later stages of the disease caused by this fungus, its effects and those produced by the T. basicola disease are indistinguishable. R. solani attacks poinsettias either at the ground line or at the transition region between the roots and stem. A ground-line in- fection usually produces girdling of the stem. An infection at the root-stem transition region usually spreads down- ward through the root system and re- sults in dead and decaying roots with intact white tips, a condition distin- guishable from the other tsvo root rots. It has been shown that each fungus in this disease complex is strongly in- fluenced by soil temperature (Bateman & Diinock 1959). Thielaviopsis root rot is favored by the 13° to 26° C. and Pythium root rot by the 13° to 21° C. temperature range. Although these two diseases are restricted by higher temperatures, Rhizoctonia root rot is favored by the 21° to 30° C. range. The temperature ranges favorable to these three pathogens coincide with tlie temperatures favorable to growing poinsettias. Therefore, poin- settias are susceptible to one or more members of the complex at all temper- atures suitable for poinsettia growth. Soil pH appears to have as much in- fluence on the development of the disease complex on poinsettias as has temperature. Thielaviopsis ( Bateman 1960) and Pydiium (Bateman 1962) root rots are most destructive in alka- line, neutral, and slightly acidic soils, but they are considerably restricted in liighly acidic soils. Soil pH, however, appears to have little, if any, influence on Rhizoctonia root rot (Bateman 1963). The diseases caused by these fungi usually have been investigated individ- ually rather than as a complex. Be- cause of the complications involved in \\'orking with three pathogens that fre- quently occur in different population ratios, obtaining consistent, meaning- 419 420 Ilunois Natural History Survey Butlletin Vol. 30, Art. 7 ful data for the complex is diiEcult. Controls have been found for P. ulti- mum and R. solani (Tompkins & Middleton 1950; Raabe & Hurlimann 1970). Although a control has recently been suggested for Thielaviopsis root rot of poinsettias (Manning et al. 1970), a single control for the entire complex has not been found. Since more research has been con- ducted on P. ultimum and R. solani than on T. basicola, the present research was originally designed to investigate some of the environmental factors affecting the growth of Thielaviopsis and the development of the root rot caused by it. However, another fungus, Chalaropsis thielavioides Peyronel, fre- quently was obtained in isolations from diseased greenhouse poinsettias. Since C. thielavioides had not been reported as being a part of the poinsettia root rot complex, the author decided to in- vestigate its importance as a pathogen on poinsettias. Numerous similarities between C. thielavioides and T. basi- cola were evident. The object of this work was to compare the two fungi. Prior to undertaking such a study, the pathogenicity of Chalaropsis on poin- settias had to be established. A com- parison of Chalaropsis and two isolates of Thielaviopsis was made to determine the effects of environment on the growth of the fungi and the ability of the two fungi to produce disease symp- toms on poinsettias. ACKNOWLEDGMENTS Much of the work on this project was conducted through the facilities and with the cooperation of the staff of the Illinois Natural History Survey. I would like to express my greatest ap- preciation for the guidance and as- sistance offered throughout the project by Dr. J. L. Forsberg, a Plant Path- ologist at the Survey. I am also espe- cially grateful to Dr. J. C. Carter, Head of the Section of Botany and Plant Pathology at the Natural History Sur- vey, for his many helpful suggestions and for providing me with laboratory facilities and equipment at the Natural History Survey. Other members of the Natural His- tory Survey staff who deserve special recognition for making this project successful are Dr. W. A. Hartstirn, Dr. E. B. Himelick, and Dr. J. L. Crane. Dr. Crane was especially helpful in providing assistance in photomicros- copy. Invaluable contributions in the forms of technical advice and assistance in the propagation of the poinsettias for the host investigations were made by Dr. M. C. Carbonneau, Mr. D. C. Saupe, and Mr. R. C. Chancellor, all members of the Division of Floricul- ture and Ornamental Horticulture of the University of Illinois. They also provided all of the poinsettia plants, potting materials, and greenhouse fa- cilities used in this study. I gratefully acknowledge the assist- ance of Dr. W. M. Bever, Professor of Plant Pathology and Head of Depart- ment, University of Illinois; Mrs. Betty A. Nelson; and Mrs. Mary A. Sponsky in the preparation of this report. Richard M. Sheets, Natural History Survey Technical Illustrator, drew the graphs, and Wilmer D. Zehr, Survey Technical Photographer, provided some of the photographs. The paper was edited by Robert M. Zewadski, Asso- ciate Technical Editor of the Survey. LITERATURE REVIEW Thielaviopsis basicola (Berk. & Br.) Ferraris is pathologically important as a member of a root rot complex and as the fungus responsible for the black root rot disease and the internal collar rot disease on more than 120 species of plants in 30 families. T. basicola has been reported as a component of a root rot complex on poinsettia (Baker, Davis, & Thomas 1953; Bateman 1962 and 1963; Bateman & Dimock 1959; Dimock 1951; Keller 1954; and Keller & Shanks 1955), citrus (Tsao & Van Gundy 1962), tobacco (Alb'son 1938; I Aug., 1971 Perry: Two Components of Poinsettia Root Rot 421 Stover 1950), and cotton (Blank, Leyendecker, & Nakayama 1953; King & Presley 1942; Leyendecker 1952; Presley 1947; Sherbakoff 1940; and Staffeldt 1959). T. basicola is known to produce a root and crowTi rot on phlox (Peterson 1967). Keller & Potter (1954) have reported that T. basicola causes a general root decay of cyclamen, be- gonia, scindapsus, cineraria, and ger- bera. Lloyd & Loclavood (1961 and 1963) reported this fungus on the roots of peas and beans. Ostazeski ( 1966 ) reported the sttuiting and death of bird's-foot trefoil resulting from root- deterioration caused by T. basicola. The influence of various environmen- tal factors on the growth of T. basicola in culture and the incidence of disease produced by this fungus have been studied extensively. Bateman ( 1963 ) suggested that pH is probably the most influential factor affecting the growth of the pathogen and definitely the most important factor aflFecting disease de- velopment. He reported that Thielaviop- sis grew best in a range of pH 4.7-5.5 and that cultural growth was retarded on neutral or alkaline media. All of the reports indicate that Tluelaviopsis is favored by a slightly acid condition. Lucas ( 1955 ) found pH to be more in- fluential on the growth of the pathogen in culture than in soils and reported a pH range of 3.9-6.2 for the two media. Keller ( 1954 ) found the optimum pH range to be 6.2-7.2 for disease develop- ment on poinsettias. Earlier findings by Rawlings (1940) do not agree with Bateman's sugges- tions on the importance of the pH fac- tor. Rawlings found pH to exert little influence on the colonv type. He re- ported that pH had a greater influence on the amount of growth (based on colony diameter) than on the colony type or components of the colony. Rawl- ings also stated that growth on basic agars (pH 7.0-8.0) was greater than growth on acidic agars (pH 4.0-5.0). More chlamydospores were produced on acidic agars than on basic agars. The effect of temperature on the growth of Thielaviopsis in culture and on the development of the disease brought about by this organism have been studied by numerous investigators. Lucas (1955) and Tsao & Van Gundy ( 1960 ) reported that the best growth of the fungus in culture occurred at 21° to 28° C. Bateman & Dimock (1959) reported 21° to 24° C. the optimum temperatures for growtli. King & Pres- ley (1942) found the best growth to occur at 30° C, whereas an early re- port by Rawlings (1940) stated that Thielaviopsis grew better at 20° C. than at either 10° or 30° C. Optimum temperatures for disease development are reported to be slightly lower than optimum temperatures for die growth of this fungus in culture. Peterson ( 1967 ) declared 16° to 20° C. to be the optimum range for the devel- opment of the disease. In this temper- ature range stem lesions developed in 4 weeks. At slightly higher temperatures lesions developed after 8 weeks. Keller & Shanks (1955) determined the ap- proximate optimum temperature for disease development to be 10° C. Sev- eral researchers have attempted to cor- relate average soil temperatures with soil types in predicting the incidence of disease (Baker, Davis, & Thomas 1953; Bateman 1963; Johnson & Hartman 1919; and Staffeldt 1959). As a result of their work, it is knowTi that certain ty]^)es of soils are more conducive to Thielaviopsis root rot than are others. There are few reports on frequency of occurrence of Thielaviopsis root rot in a field situation. Presley (1947) re- ported that in one field of cotton in Mississippi 20 percent of the plants were killed by T. basicola. Reports in tlie literature concerning the pathogenicity of Chalaropsis thie- lavioides and the incidence of the disease it causes are scant. This fungus has been reported as a pathogen on the roots of roses ( Baker 1953 ) , walnut trees (Hamond 1935), Chinese elms (Wright 1942), agave (De La Isla 1962), and lupine (Longree 1940). In general, Chalaropsis has been reported 422 Illinois Natural History Survey BtrLLETiN Vol. 30, Art. 7 as a fungus that causes the failure of bud and graft unions. Often referred to as the "black mold fungus," Chal- awpsis frequently produces a fungal mat over the cut surface of the stock to be used in a graft. The impeding pres- ence of die mycelial mat prevents the successful fusion of the stock and scion. Other accounts contain reports of black mold causing grafting failures on roses (Baker & Thomas 1946; Longree 1940; and Milbrath 1946). Baker & Thomas (1946); Lamb, Wright, & Davidson (1935); Wright (1942); and Hamond (1935) stated that Chalaropsis is a wound parasite. Studies on the effect of environ- mental factors are lacking. Baker & Thomas (1946) reported that the op- timum temperature range for growth of Chalaropsis in culture was 18° to 30° C. Boerema (1959) suggested that C. thieJavioides is an occasional surface contaminant on carrots packaged in perforated polyethylene bags. Lloyd & Lockwood (1962) reported Chalaropsis to be a common contaminant on pack- aged carrots. Since carrot slices are used as an isolating medium for obtaining Thielaviopsis from diseased tissues, they warned against mistaking the con- taminant Chalaropsis for Thielaviopsis. Longree (1940) reported numerous similarities between Chalaropsis and Thielaviopsis. She prepared a good morphological description of Chalarop- sis and compared several of the differ- ent isolates reported prior to 1940. MATERIALS Fungi Two isolates of Thielaviopsis basicola were used for testing the effects of en- vironmental factors on the growth and pathogenicity of the fungus. One strain, referred to in this paper as TBT, was obtained from Dr. Peter Tsao of the University of California at Riverside. This culture had been isolated from citrus roots. A second isolate, TBB, was received from Dr. D. F. Bateman of Cornell University. This culture had been isolated from tobacco and was known to be pathogenic on beans also. A culture of Chalaropsis thielavioides was isolated while attempting to isolate T. basicola from diseased poinsettias ex- hibiting symptoms like those of a Thie- laviopsis infection. Stock cultures were made by making single-spore isolations from the original cultures. They were maintained in culture by repeated transfer. Since C thielavioides has been re- ported to be a contaminant on pack- aged carrots (Boerema 1959; Lloyd & Lockwood 1962), the Chalaropsis may have come from the diseased poinsettia roots or from the carrot slices used as the isolating medium. A check was made on the isolation technique in an attempt to locate the source of the C. thielavioides. From every package of carrots used, several sterilized carrot slices were placed in petri dishes. These petri dishes were exposed to the same conditions as were the dishes in which pieces of diseased poinsettia roots had been placed on sterilized carrot slices. Chalaropsis was never found on any of the carrot slices that had not been brought into contact with diseased poinsettia root material. In every in- stance in which C. thielavioides grew on the carrot slices, a piece of root tissue was present. Consequently, the fungus must have come from the diseased poinsettia roots. Media Several media were used in an attempt to find the most suitable me- dium for the growth of the two fungi. Commercially prepared Difco media used were lima bean agar, prune agar, malt agar, cabbage infusion agar, com- meal agar, Noble agar, and potato- dextrose agar. Freshly prepared potato- dextrose agar and carrot-dextrose agar were also used in growth tests. Six media were prepared from living poin- settias. These poinsettia media consisted Aug., 1971 Perry: Two Components of Poinsettia Root Rot 423 of leaf-and-stem-dextrose agar, root- dextrose agar, and entire-plant-dextrose agar. The remaining three were of the same components without the dextrose. Six flowering poinsettias, each ap- proximately 30 cm tall, were cut be- tween the upper root and lower stem regions in preparing the root agar and the leaf-and-stem agar. The root sections were first thoroughly washed and then macerated in a Waring Blendor with 400 ml of distilled water for 5 minutes. The macerated material was added to 1,600 ml of distilled warm water in which 40 grams of agar had been dis- solved. This volume was then divided into two 1-liter portions. Twenty grams of dextrose were added to one 1-liter portion of the mixture. This medium was autoclaved at 121° C. for 45 min- utes and then poured into petri dishes. The leaf-and-stem agar was similarly prepared, except that the aboveground portions of six poinsettia plants were used. The entire-plant agar was pre- pared in the same way, except that the medium contained both the roots and the aboveground portions of the plants. Two broth cultures \\'ere used to test growth on a liquid medium. Potato- dextrose broth and V-8 juice broth were made in the same manner as the coun- terpart agars were made, except that the agar ingredient was omitted. Into 125-ml flasks were poured 100 ml of each broth. The flasks were plugged and sterilized. Seeding was done bv dropping fungus plugs into the broth. Poinsettias Nine varieties of poinsettias, Barbara Ecke Supreme, Elisabeth Ecke, Mikkel Pink, Mikkel Dawn, Paul Mikkelsen, Stop Light, Snow Flake, Snow Cap, and Ecke White, varying in flower color from dark red, to pink, to white, were used in establishing and maintain- ing the pathogenicity of Clialaropsis thielavioides and Thielaviopsis hasicola. Six of the varieties were varieties mar- keted commercially and three were ex- perimental varieties. METHODS Microscopic Examinations Samples of the fungi were obtained by cutting plugs from colonies on agar and by making needle scrapings of diseased host tissues. Microscopic ex- aminations of the fungi were made with a phase microscope. When samples con- taining only endoconidia were desired, a petri dish containing a fungus colony on agar was flooded with distilled water and slightly agitated. Drops of the liquid containing the spores were then removed from the dish and placed on microscope slides. Needle scrapings of host tissues were placed in drops of lactophenol on slides. The scrapings were usually composed of both my- celium and spores. Measurements of the fungus structures were made. pH Studies A pH range was prepared by alter- ing the components of fresh potato- dextrose agar and V-8 juice agar. Two solutions were prepared: a 25-percent lactic acid solution ( pH 2.0 ) and a sodium hvdroxide solution made by dissolving 4 grams of sodium hydroxide in 1 liter of water (pH 12.0). A re- corded number of drops of tliese solu- tions was then added to each of a group of sterilized petri dishes. Basic media were prepared by adding 5, 10, 15, or 20 drops of the sodium hydroxide solu- tion to petri dishes, each of which con- tained 20 ml of liquified potato-dex- trose agar or V-8 juice agar. The dishes were swirled to insure uniform distri- bution of the solution and the media. Readings of the pH values were taken with a Beckman pH meter before the seedings and after 10 days of growth. The media, after the addition of the sodium hvdroxide solution, varied from pH 5.7 to 9.2. The lactic acid solution was added in portions of 1, 3, 5, 8, 10, or 20 drops to dishes containing 20 ml of V-8 juice agar. These media, after the addition of tlie lactic acid, had a pH range of 7.1- 2.7. The agars were solidified and the 424 Illinois Natural History Survey Bulletin Vol. 30, Art. 7 dishes then inverted to prevent moisture condensation on the petri dish lids. Tests on the influence of pH on the growth of the isolates were made on samples of the two fungi grown on V-8 juice agar and potato-dextrose agar. These groups consisted of the various pH plates seeded and kept in constant darkness. The plates were maintained in an incubator at a constant 24° C. Di- ameters of the colonies were measured every 24 hours for 10 days. Each plate was seeded with a plug approximately 2 mm square taken from actively grow- ing areas in stock culture plates. Three replicate samples of each isolate were tested at each pH level. Temperature Studies Seeded plates of standard V-8 juice agar were placed in unlighted incuba- tors set at 5°, 10°, 15°, 20°, and 30° C. Linear growth was measured directly along a consistent diameter bisect every 24 hours for 13 days. Three plates of each of the three isolates at each tem- perature were measured. Light Studies Each of six V-8 juice agar plates was seeded with a plug of TBB isolate, six wdth a plug of TBT isolate, and six with a plug of Chdlaropsis. Three plates seeded with TBT, three with TBB, and three with Chalaropsis were placed in an unlighted incubator at 24° C. The remaining three plates of each isolate were placed at 24° C. in an incubator lighted by a constant light source of 95 footcandles. Daily measurements of the diameters of the growing colonies were made. Samples were taken from the fungal colonies on the 2nd, 7th, 10th, and 14th days of the testing period for microscopic observation. Placed in each of thirty-six 125-ml flasks were 100 ml of potato-dextrose broth. In another thirty-six 125-ml flasks were placed 100 ml of V-8 juice broth. For each isolate, 12 flasks of potato-dextrose broth and 12 flasks of V-8 juice broth were used. Eighteen flasks of each broth type were seeded and wrapped in a double layer of aluminum foil. Nine wrapped flasks of each broth were placed on a 144-revolu- tions-per-minute shaker. Nine flasks of each broth were seeded, placed on the shaker, and exposed to constant light. The remaining 18 flasks of each broth, 9 wrapped in foil and 9 not covered, were placed in the light but not on the shaker. This experiment was conducted at room temperature. Seedings were made by dropping a 2-mm-square plug from an active colony into the broth. Observations were made every 3 days for 15 days. Pathogenicity Tests Nine varieties of poinsettias were used in testing tlie pathogenicity of the two fungi. Cuttings were rooted in sand and then were potted in a soil com- posed of equal portions of sand, loam, and vermiculite. Poinsettias in the vegetative and flowering stages were inoculated. The vegetative poinsettias averaged 20 cm in height and were growing singly in standard 4-inch pots. The flowering poinsettias varied from 20 to 50 cm tall. These were grown three plants per three-fourth size, 6-inch diameter pot ( a pan ) . All poinsettias were maintained in a greenhouse at the University of Illinois Floriculture and Ornamental Horticul- ture Division. The air temperatures varied from 16.7° to 18.3° C. Soil tem- peratures varied from 15° to 18.7° C. In one brief experiment, the soil tem- peratures were raised to the 19° to 25° C. range by regulating the air tempera- ture. The plants were watered twice daily and fertilized once daily with a mixture of equal parts (.68 kg, or 1.5 pounds) of ammonium phosphate and potassium phosphate dissolved in 379 liters ( 100 gallons ) of water. Artificial lighting was not used. Shading was used on exceptionally hot days. Most of the pathogenicity tests were conducted during the winter months. Inocula of each of the three isolates Aug., 1971 Perby: Two Components of Poinsettia Root Rot 425 were prepared by the following method. Four petri dish colonies of the isolate (including the V-8 juice agar, mycelium, and spores) were added to 1 liter of sterile, distilled water and blended for 5 minutes in a Waring Blender. Three different methods of placing the fungi in contact with the poinsettias were used. In the soil-drench method, the suspensions were poured over the soil surface in portions of 50 ml per plant (or 150 ml per pan). After the suspension had soaked in, 100 ml of distilled water were poured over the soil. A second method is referred to as the soil probe. Prior to drenching the soil \\ith 50 ml of inoculum as in the soil-drench method, the root balls of the poinsettias were probed six times \\'ith a handled scoopula. This probing served to break some of the roots. The soil around the check plants was drenched with 50 ml of sterile distilled water in place of the inoculum. The third method of placing the fungi in contact with the poinsettias involved digging the soil away from the crown area of the poinsettias. The exposed upper root-lower stem region was washed with sterile water; the surface was disinfested with 75-percent ethanol; and it was rinsed with sterile, distilled water. A sterilized scalpel \\as used to make a 1-cm sht into the pith region of the stem. From an actively growing agar colony, a mass of fungus my- celium and spores was removed with a sterilized transfer needle and transferred into the slit. The slit was then covered with petroleum jelly and the soil was replaced. The lower stems of the check plants were disinfested with ethanol, slits were made, the slits were covered with petroleum jelly, and the soil was replaced. The success of the inoculations was determined from disease symptoms ex- hibited by the plants and recovery of the fungi from the plants. Comparisons with check plants with regard to gen- eral vigor, leaf fall, chlorosis, dwarfing. premature flowering, lesions, and re- duced root growth were considered in evaluating the intensity of the diseases. Isolations from diseased roots and lower stem lesions were made to re- cover the pathogens. Altliough potato and sweet potato slices and non- diseased poinsettia stems and roots \\'ere also tried, Yarwood's carrot-slice isolation technique (Yarvvood 1946) was the only method successfully used in reisolating the pathogens. Packaged carrots were peeled and sliced into discs 40-70 mm thick. Four of these discs were placed in each sterile petri dish. The dishes were stacked under a bell jar. On top of the stack was placed an open dish containing 10 ml of a gaseous sterilant, propylene oxide (Klarman & Craig 1960). The bell jar was sealed for 24 hours to maintain die concentration of the propylene oxide. The jar was then removed and the un- opened dishes were allo\\'ed to air for 24 hours to disperse the propylene o.xide. Root balls of poinsettias show- ing symptoms of disease were opened and infected root segments approxi- mately 1 cm long were removed. These segments were surface disinfested by placing them in a 5-percent Clorox solution for 2 minutes and then rinsing them in sterile distilled water. The root segments were placed on the sterilized carrot slices in petri dishes, 10 ml of water were added to each dish, and the dishes were kept on a laboratory table. Daily observations were made for the appearance of fungus growth. RESULTS Morphology of Tbielaviopsis basicola Thielaviopsis basicola produced both reproductive and vegetative stages abundantly in culture as well as on the host. Characteristically, there were two types of vegetative mycelium (Fig. IB). The most common type was hy- aline, thin-walled, and septate. The younger hyphae varied from 1.9 to 2.6 fjL wide. In culture under favorable 426 Illinois Natural History Survey Bulletin Vol. 30, Art. 7 conditions,' the hyaline mycelium con- stituted about 90 percent of the vegeta- tive growth and was relatively dense. A second type of mycehum was found in the older areas of the colony, espe- cially in the center, or on the agar surface below the aerial growth. The older hyphae were wider, 2.6-3.9 /i, with dark brown pigmentation. Both types of mycelia produced spores. The hyaline mycelium produced endoconidiophores in which endoconidia were produced (Fig. ID). During this investigation, growth under mildly un- favorable conditions resulted in an in- crease in the amount of thick-walled mycelium. A reduction in the growth of both types of mycelia was caused by intensely unfavorable conditions. This fungus produced chlamydo- spores and endoconidia in its repro- ductive phase. The endoconidia were produced abundantly both on the host and in culture. They were single celled and stoutly cylindrical to barrel shaped with rounded comers (Fig. ID). The cellular contents appeared to vary with conditions. Under favorable conditions they were composed of a thin, hyaline cell wall enclosing granular contents and two conspicuous vacuoles, one in each end of the cell. Endoconidial cells produced under unfavorable pH or temperature condi- tions appeared to be inconsistent in contents. In some cases the contents appeared to be entirely granular or entirely vacuolated, while in others the two vacuoles appeared to be the sole constituents of the cells. Aging of the endoconidia produced a slight change in shape. The ends of the individual cells transformed from flattened with rounded corners to semi- ovoid. During maturation these cells widened slighdy, whereas Chalaropsis 'Throughout this paper references to un- favorable conditions indicate pH, temperature, or light conditions, individually or combined, that were not conducive to the best growth of the fungi. Favorable conditions are the pH, temperature, and light requirements indicated by this study as being most conducive to the growth of the two fungi. endoconidia appeared to elongate slightly. Endoconidia of the T. basicola isolate from citrus averaged 7.59 x 2.37 /x ( Table 1 ) . The endoconidia of this iso- late varied from 4.56 to 13.11 /i in length and from 2.28 to 2.85 /x in width. Mean dimensions of the endoconidia of the isolate from tobacco were 7.96 x 2.85 IX, with a range of 5.20-11.70 fi long and 2.28-3.42 /x wide. Except for these slight deviations in measurements, the endoconidia from the two isolates were identical. Germ tubes developed from the ends of the cells. In this study germ tubes were not observed devel- oping from the middle region of the cell in either Chalaropsis or Thielaviopsis. Thielaoiopsis endoconidia were in- distinguishable from those of Chalarop- sis. Generally, the biguttulations of Thielaviopsis endoconidial cells were more prominent, but this characteristic was not sufficiently consistent to permit separation of the two fungi. Thielaviopsis endoconidia were pro- duced within endoconidiophores similar to those of Chalaropsis. They were com- posed of tapering terminal cells (phialides) and one to four basal cells. In the isolates of the fungi studied in this investigation, basal cells varied in length from 6.3 to 9.1 /x and averaged 2.6 /x wide. In the Chalaropsis isolate the basal cells of individual endoco- nidiophores frequently varied slightly in size in such a way that the largest cell was at the end opposite the spore- producing cell. There was a progressive reduction in size of the remaining cells toward the terminal cell. In the two isolates of Thielaviopsis this condition occasionally occurred, but usually all of the basal cells were of the same size. This condition made it difficult to distinguish the termini of the hyphae and the origins of the endoco- nidiophores. However, endoconidio- phores arising from short, lateral branches were easy to differentiate. The tapering terminal cells varied in length from 15.6 to 32.4 ^. Endoconidiophores Aug., 1971 Pehhy: Two Components of Poinsettia Root Rot 427 Fig. 1, — Thiehviopiii basicolo: A, 10-day-old colony on V-8 juice agar; B, colony components — two kinds of spores and two types of mycelia (X 130); C, chlomydospore cfioins (X 520); and D, endoconidiopfiores with endoconidio (X 520). were produced individually or in arise from a common point but from clusters on terminal tips of hyphae or various subbasal, hyphal cells, on stubby lateral branches. Endoco- Conidia were produced endogenously nidiophores produced in clusters were by the phialides of the conidiophore. of various lengths, because they did not Although these spore-producing cells 428 Illinois Natural History Survey Bulletin Vol. 30, Art. 7 ^^ !^ -S :s B i I 'i s s e E= I i S E :s g s E Tt; Aug., 1971 Perry: Two Components of Poinsettia Root Rot 429 strand. Mature chlamydospore chains were released from tlie parent mycelium by fragmentation. The chlamydospore chains were released intact with a short fragment of hypha still attached to the basal cell. Some controversy has developed among plant pathologists over the ger- mination of Thielaoiopsis chlamydo- spores. Christias & Baker ( 1967 ) re- ported that before germ tubes could develop, the individual spores com- prising the chain had to be separated. Each spore was then believed capable of germination. Their work appeared to demonstrate the function of chitinase in breaking the spores apart so that germination could take place. How- ever, Papavizas & Adams (1969) later demonstrated that intact chlamydospore chains, as well as individual chlamydo- spores, were capable of germination. They found that the breaking up of chlamydospore chains into individual spores by chitinase was not a necessary prelude to germination. I did not study the germination of chlamydospores. There is no record of a necessary maturation or resting period before germination of Thielaviopsis chlamydo- spores as has been noted in Clwlaropsis chlamydospores (Longree 1940). Colony Characteristics of T. bos/co/o Observations were made and data were recorded on the development of Thielaviopsis basicola colonies on V-8 juice agar and potato-dextrose agar. More extensive development of vegeta- tive and reproductive structures oc- curred on V-8 juice agar. Growth of T. basicola on V-8 juice agar was luxuriant but slow. A 15-16 day growth period under favorable conditions was re- quired for both of the isolates studied to completely cover the agar surface in a 90-mm diameter petri dish. However, measurable growth was recorded 2 days after seeding. The first growth appeared as hyaline, vegetative hyphae radiating outward from the point of seeding. Throughout the development of the colony, the outermost perimeter was composed of these hyaline, nonsporulating hyphae appressed to the agar surface. On the 4th day after seeding, aerial, hyaline mycelia began to be produced around the seeding point and to develop pro- gressively outward. By the 5th day after seeding, endoconidiophores and en- doconidia could be found in the aerial hyphae. \\'ithin the next 48 hours the hyphae around the seeding plug began to darken. Microscopic examinations of the fungus in this region revealed that the darkening was brought about by the production of darkly pigmented, thick- walled hyphae. These dark strands predominated the fungal mass but re- mained close to the agar surface. Pig- mentation appeared to vary between dark gray to black and golden brown. Pigmentation did not appear to be con- sistent with the isolate, as both isolates produced both color types but not with- in the same colony. The dark, thick-walled hyphae gave rise to the terminal chlamydospore chains. The chlamydospores were also thick-walled and darkly pigmented in colors corresponding to those of the hyjDhae from which they had developed. As chlamydospore production continued to increase, endoconidial production decreased. A fully developed colony consisted of a series of concentric circles of visible differences in growth type (Fig. lA). The innermost region around the point of seeding was a mass of compacted, semi-aerial, dark-walled hyphae. Thick- walled, dark hyphae and chlamydospore chains composed approximately 95 per- cent of the fungus material in this region, but occasional hyaline hyphae were found. This center region, about 10 percent of the entire colony area, had a black, matted appearance. The next growth ring constituted about 40 percent of the colony area and was com- posed of approximately 75 percent dark hyphae and chlamydospore chains 430 Illinois Natural History Survey Bulletin Vol. 30, Art. 7 and 25 percent hyaline hyphae and endoconidia. Because of the presence of fewer dark hyphae in relation to the mass of hyaline hyphae and the more aerial nature of the hyaline hyphae, this region had a gray, wool-like appearance. In the remaining concentric growth patterns, the percentages of dark hyphae and chlamydospore chains com- prising the fungal colony decreased progressively as the distance from the point of seeding increased. The propor- tion of hyaline hyphae and endoconidia increased progressively as the distance from the seeding point increased. Apparently no standard number of con- centric growth rings made up a colony. Common borders between the growth rings were not sharp and well defined but were transition zones gradually in- tegrating one ring with the next. Addi- tional characteristics of typical T. basic- oJa colonies observed during this study were consistent with those described by Johnson & Valleau (1935). T. basicola on Poinsettias Thielaviopsis endoconidia were found on almost all external surfaces of diseased areas on poinsettias. Abundant endoconidia were found on decaying root segments, in lesions on lower stems, and on the inner walls of the lower pith cavities of the stem. These spores were always found on the external or internal surfaces but never embedded within the host tissue. As did those of Chal- aropsis, the thin-walled, hyaline hyphae ramified within the infected host tissue. Endoconidiophores developed from the hyaline hyphae that were either on the surface of the host or embedded in the diseased tissues. Endoconidial produc- tion seemed to be influenced little by environmental factors or host conditions. Endoconidia were found either on or within the host on internal surfaces at any time when symptoms of Thielaviop- sis infection were present. Thielaviopsis chlamydospores were produced much less frequently on the host than were the endoconidia. How- ever, the Thielaviopsis chlamydospores were found more frequently on the host tlian were Chalaropsis chlamydospores. Chlamydospore production of Thiela- viopsis seemed to be related to environ- mental conditions and the development of the disease. The development of longitudinal lesions on the lower stems of the poinsettia host was a symptom of an advanced stage of Thielaviopsis in- fection. Chlamydospores were found in these lesions. During my investigation, chlamydospores were detected only within the stem lesions, probably be- cause chlamydospore production re- quired light. Masses of thick-walled, darkly pigmented hyphae with multi- spored, terminal chlamydospore chains developed within these lesions. Morphology of Chalaropsis thielavioides The vegetative stage of Chalaropsis thielavioides consisted of septate hy- phae that varied in width from 1.2 to 2.6 /i. In culture most of the hyphae were thin and hyaline. As they aged, the cells became slightly wider, and the walls darkened. The reproductive struc- tures were produced on this type of mycelium. Even under favorable condi- tions the mycelia were sparse. Most of the growth consisted of endoconidio- phores arising from the older hyphae. Under unfavorable conditions the my- celia became even more sparse than that produced under normal conditions. The darkening of the mycelium as it aged varied inversely with the intensity of the unfavorable conditions. Under highly unfavorable conditions tlie mycelium failed to darken and remained thin walled, and vacuoles predominated the segmental contents. The hyphal cells sometimes enlarged to produce bead- like strands of mycelium. This fungus produced two types of asexual spores, endoconidia and chla- mydospores ( Fig. 2B ) . The endoconidia were produced within endoconidio- phores (Fig. 2D), each composed of two or three basal cells and a spore-pro- ducing cell (phialide). The basal cells varied from 6.5 to 9.1 /t in length and were about 2.6 ju wide. The spore-pro- Aug., 1971 Perry: Two Components of Poinsettia Root Rot 431 A B Fig. 2. — Chahropsis thielavioides: A, 10-day-old colony on V-8 juice agar; B, components of a typical colony (X 520); C, chlamydospores |X 1235); D, endoconidiophores with endoconidio |X 520). ducing terminal cell varied from 11.7 to 27.3 fi in length. The tapered phialides produced endoconidia singly. Although several endoconidia in various stages of formation were seen within a terminal cell, the mature endoconidia were ex- truded from the apex individually. A stubby side branch occasionally devel- oped from a mature strand of mycelium. One to five endoconidiophores devel- oped from this branch. It was not un- common to see several conidiophores on one lateral branch (Fig. 2D). The endoconidia of C. thielavioides 432 Ilunois Natural History Survey Bulletin Vol. 30, Art. 7 S 6 ._, ^ -* o> "O c4 c-i 1^ c< i> 00 00 2 O CD o S-S •i Tj -a o ccD Sio =in Jio H^as cG^ ecu c<^ 2io c o CcD Ph ' * s s cS to Aug., 1971 Perey: Two Components of Poinsettia Root Rot 433 were produced in great abundance on the host and in culture. These cqIIs were found clinging together in chains of a dozen cells or more. Endoconidia ger- minated at any time after they were released from the conidiophores. Germ tubes were produced from the ends or the sides of the spores. Individual en- doconidia were hyaline and biguttulate. They were barrel-shaped to cylindrical with rounded comers. As these cells aged, the ends became slightly more roimded. The endoconidia varied from 3.42 to 13.68 fi long and 1.14-2.28 ;li wide, with mean dimensions of 7.50 .x 1.67 /x. The endoconidia of minimal dimensions were almost always found in chains. Spore measurements of the isolate used in this work and the spore measurements of isolates used by other \\orkers are compared in Table 2. A second type of asexual spore, the chlamydospore (Fig. 2C), was pro- duced in fe\\'er numbers than were the endoconidia. Longree ( 1940 ) referred to these as macroconidia. These cells were produced only in the older parts of a colonv. Short lateral branches devel- oped from cells of die older, pigmented mycelium. The tips of these lateral branches and the tenninal tips of the hyphae became bulbous. Longree ( 1940 ) reported that macroconidia and niacroconidiophores were frequently found clustered at the base of an en- doconidiophore. In my investigations I did not find this condition. Macroconidia from the C. thielavioides used in this work were alwavs produced individ- ually and not at the bases of endo- conidiophores. Maturing chlamydospores were thin walled and hyaline for a short while, but soon became thick walled and darkly pigmented. The mature chlamyd- ospores were subglobose to ovoid with small flattened bases. Mature chlamyd- ospores were freed from the colony without any portions of the conidio- phores still attached to them. Spores freed shortly before maturity often still had a short fragment of conidiophore attached. Freed, hyaline chlamydo- spores sometimes had a fragment of conidiophore equal in length to several times their diameters. The diameter of the chlamydospore varied with the isolate. Mature chla- mydospores from the isolate used in this investigation were spherical, with a mean diameter of 7.64 fi. They varied from 6.27 to 8.55 fi in diameter. As indicated by the data in Table 2, other reports of this fungus show the chla- mydospores to be more ovoid than spherical. These spores did not germinate readily. None of the chlamydospores observed showed any evidence of germination. However, the cultures were not maintained for more than a month. Longree (1940) stated that the macroconidia either require a resting period or mature slowly. She found tliat 2-nionth-old macroconidia ger- minated poorly, whereas the percentage of germination was greater in 3-month- old spores. Chlamydospores were pro- duced only after the mycelium had reached a certain stage of maturity or when the mycelium was subjected to unfavorable conditions. Colony Characteristics of C. thielavioides Chalaropsis thielavioides grows lux- uriantly on only a few agar media. Al- though it does grow on most common agars, the fungus sporulates abundantly and produces an extensive colony on only a few. Good growtli was produced on V-8 juice-dextrose-yeast extract agar (Fig. 2A), but even this growth was slow. On V-8 juice agar Chalaropsis covered die agar surface in a 90-mm diameter petri dish in 10-12 days under favorable conditions. If a plug of agar widi fungal spores and mycelium was placed on agar in a petri dish, measur- able growtli was observed after 2 days. This first growth appeared as non- sporulating hy-phal strands living on or slightly below the agar surface. The vanguard of hyaline mycelia continued outward, preceding the subsequent 434 Illinois Natural History Survey Bulletin Vol. 30, Art. 7 colonial development. If a single point of seeding was made on tlie agar, a circular colony was almost always produced. For the first few days the colony ap- peared hyaline, and then it began to whiten. The change to white was brought about by the production of endoconidiophores and endoconidia. These reproductive structures grew witli a concentric perimeter slightly (about 5 mm) within the enlarging hyaline perimeter. After 5-6 days of growth under favorable conditions, Chalawp- sis produced a colony that covered about two-tliirds of a 90-mm diameter petri dish. By this time a small, circular halo immediately surrounding the ini- tial plug had turned black. This black- ened area consisted of developing and matiue chlamydospores. The mycelium was almost entirely on the surface of tlie agar or slightly submerged, and it was pigmented a golden brown. The black color in the colony was due to the dark-brown-to-black pigmentation of the numerous chlamydospores. Al- though the mycelium in the chlamydo- spore area remained on the surface, the surrounding mycelium became more aerial and fluffy. This caused the center area to appear sunken in tlie white my- celium. After 8-10 days a light gray area constituting^ about one-half of the colony began to appear in the center of the colony. The gray color was due to the intermingling of maturing chla- mydospores and the accompanying abundant endoconidia. The fully developed colony appeared as a series of progressively lighter colored, concentric circles. The inner- most circle, about one-tenth of the total area, was black; sunken; and composed of many chlamydospores, few endo- conidia, and much thick-walled my- celium. The next zone was fluffy and whitish gray and accounted for about one-half of the colony area. This area consisted of abundant endoconidia, much thin- walled mycelium, and abundant chla- mydospores. When this area was scraped from the agar and the surface of the agar was examined, a dark surface growth of thick-walled mycelium was seen radiating outward from the center. This surface growth appeared to ter- minate after enlarging to about one- half of the area of the entire colony. Chlamydospores which grew up through the aerial endoconidia! mycelium and caused the graying of this region were produced from this growth. The next outermost circle was white and, although definitely aerial, not quite as fluffy as the adjacent, inner region. This white area constituted about one- third of the colony and was composed almost entirely of endoconidia, endo- conidophores, and thin-walled hyphae. The surface growth below this region frequently was grayish and contained few chlamydospores. The outermost concentric region made up about one-tenth of the total area. It consisted entirely of surface and subsurface, hyaline, thin-walled hyphae with no spores of either type. Under ideal conditions, the margin of the colony was regular. Abnormal or ad- verse conditions frequently produced lobed or irregular margins. A lO-day- old Chalaropsis colony on V-8 juice agar grovwi under favorable conditions is shown in Fig. 2A. C. fhielavioides on Poinsettias Signs of Clwlaropsis were more difficult to find on the poinsettia plant than were signs of Thielaviopsis. Chala- ropsis endoconidia were found in mod- erate numbers on the external surfaces of the diseased areas of the roots. These spores were also found externally in the areas of the stem where bark had been loosened or sloughed off near the ground line. All of the mycelium produced within the infected host tissues was thin walled and hyaline. This mycelium was difficult to locate in the decaying tissues. It appeared to produce endo- conidiophores that protruded through the epidermis of the host tissue and ex- truded the endoconidia externally. Chlamydospores of Chalaropsis were Aug., 1971 Perry: Two Components of Poinsettia Root Rot 435 not produced abundantly on poinset- tias. Environmental conditions or host influence may have been responsible for the lack of abundant chlamydospores. Chlamj'dospores from the isolate used in this experiment were found only in the pith cavities of the lower stem region of the host. These spores ap- peared to be restricted to the lowest pith chamber, since they were not found in the upper chambers of the stem. Thick-walled, darkly pigmented hyphae developed on the walls and septations of the lowest chamber. Individual ter- minal chlam)'dospores were produced from the tliick-\\alled hyphae. No chlamydospores were found embedded in the host tissues, on diseased roots, or on any diseased surface exposed to light. Light may have been the critical factor that determined the location of Chalaropsis chlamydospore production. Comparison of Growth of Cha/oropsis thielavioides and Thielaviopsis basicola on Various Media Various media were tested to deter- mine the one most satisfactory for cul- turing the three isolates. Of the media tested, V-S juice agar was tlie most satisfactory'. Prepared as suggested by Miller (1955), this agar had a pH range of 7.0-7.25. Both fungi produced vegetative and reproductive stmctures abundantly on this medium. Both fungi grew and sponilated ecjually well on carrot-dextrose agar and V-8 juice agar, but the carrot-dex- trose agar was not used because of its more involved method of preparation. V-8 juice can be easily purchased, whereas carrot juice is not so readily available. A juice was easier to use in the media preparation than macerated carrot tissue. Potato-dextrose agar was not as satis- factory as the V-8 juice agar. Hyaline mycelia and endoconidia were produced on potato-dextrose agar, but chlamydo- spores and dark hyphae were produced infrequently and sporadically. Colonies of both fungi on potato-dextrose agar were sparse and usually occurred be- low the surface or appressed to the surface. Testing of the poinsettia agars proved interesting but not too beneficial. No growth was made by either fungus on agar derived from poinsettia tops only, either with or without dextrose. Growth of Thielaviopsis on agar derived from the entire poinsettia plants was poor. This growth consisted of light gray, random mycelia on the agar surface. Sporulation did not occur. Chalaropsis produced a sparse, aerial mycelium with some endoconidia. Again, the presence or absence of dextrose had little eS^ect. All three isolates grew well on the poinsettia-roots-only agar. Both types of vegetative and reproductive structures were produced abundantly. Gro^^'th of all three isolates was notice- ably better on the dextrose-supple- mented agar. Fungal growth on these b.\o media was comparable to growth on V-8 juice agar. The poinsettia-roots medium was not used, however, be- cause of tlie extra time and effort re- quired in its preparation. Growth on lima bean agar, prune agar, malt agar, cabbage infusion agar, cornmeal agar, and Noble agar was negligible. Of all the agars tested, only potato-dextrose agar and V-8 juice agar were used in subsequent experiments. Two liquid media were tested. Potato- dextrose broth and V-8 juice broth supported good growth of both fungi. Chalaropsis produced a complete sur- face mat in 7 days on V-8 juice broth. Thielaviopsis produced a complete mat in 10 davs. Both fungi failed to produce a complete mat on potato-dextrose broth. Neither fungus produced any growth in either of the tsvo broths in shake cultures. Comparison of Growth Rates Daily measurement of the colonial enlargement of the three isolates in- dicated that Chalaropsis was the fast- est growing. The colony increased at the rate of 8.0 mm in diameter per day. The colony produced was dense and 436 Illinois Natural History Survey Bulletin Vol. 30, Art. 7 contained both coarse and fine hyphae as well as both types of spores. The colonies of both isolates of Thie- laviopsis increased 5.9 mm in diameter daily. This fungus grows somewhat slower than Chalaropsis. The colonies produced were similar to those pro- duced by Chalaropsis. They were dense and contained both types of hyphae and both types of spores. The rate of growth of each isolate is shown graphically in Fig. 5. Effect of pH Although temperature influenced the amount of growth, pH influenced the type of growth of the fungi reported on here. Conspicuous variations in the morphology of the fungi accompanied variations in the pH of the media. Fig. 3 shows the effect of pH on colony growth. C. thielavioides had a narrower favorable pH range than either isolate of T. basicola. C. thielavioides produced some growth at pH 3.9 and fair growth at pH 9.2. In my opinion, based on the amount of growth and quality of the colony, the optimum pH range for Chalaropsis was 6.7-8.0 (Fig. 3A). Al- though colonies on agars with a pH in excess of 8.0 produced normal daily colonial enlargement, the hyaline hy- phae were distorted in shape and the endoconidia were nearly oval. An in- crease in pH to above 8.0 produced an intensification of the distortions. Chla- mydospores and dark hyphae were sparse or absent in all colonies on agar viath a pH greater than 8.0. Colonies on agars with a pH between 8.0 and 8.7 were appressed to the agar surface. Colonies on agars with a pH higher than 8.7 were almost entirely sub- merged and definitely hyaline. Chalaropsis grew better on basic agar than on acid agar. Although the growth of reproductive and vegetative struc- tures was retarded on agars in the lower pH range, the deforming of structures was not as extensive as it was on the basic agars. Growth was reduced on agars with a pH of less than 7.0. Colonies varied from dark gray at pH 6.8 to whitish gray at pH 3.8. On none of the acidic agars did the colonies appear hyaline. Their color was due to the dark, thick-walled hyphae inter- mingled with some hyaline hyphae and endoconidia. At the lower end of the pH range, there were fewer chlamyd- ospores. The colony produced at pH 3.8 contained occasional thick-walled hyphae but no chlamydospores. An abundance of endoconidiophores was responsible for the whitish color of this colony. The type of growth also varied as pH values varied. A decrease in pH pro- duced an increase in the mass of aerial hyphae. Colonies on highly acidic agars appeared fluffy in comparison with the wooly colonies grown at pH 7.0. Variations in pH influenced the growth of isolate TBT more than that of isolate TBB. Neither isolate grew at a pH lower than 3.2. Although the basic limit was not defined, a progressive decline in the growtli and formation of typical structures of the colonies at pH 9.0 indicated that the maximum was somewhere near that point. As Fig. 3C shows, after 10 days of growth the diameters of TBB colonies on agars with various pH values ranged from 18 to 62 mm. A variation of 27-74 mm in the diameters of the TBT isolate colo- nies occurred on these agars (Fig. 3B). The optimum for both isolates was be- tween pH 5.9 and 6.7. The colonies on agars outside of the optimum pH range produced more vegetative structures than reproductive structures. TBB and TBT colonies on agars with- in the optimum pH range were white for the first 2-3 days, due to the abun- dant production of thin-walled hyphae and endoconidiophores. After several days the colony, except for a narrow white perimeter, developed shades of gray. The gray color resulted from the production of chlamydospores and thick-walled hyphae. Daily diameter en- largement varied between 5.8 and 7.4 mm. Morphologically, the colonies on the Aug., 1971 Perry: Two Components of Poinsettia Root Rot 437 more basic media tended to be light gray to whitish. As the basic condition was intensified, chlamydospore produc- tion was lessened and endoconidial pro- duction became greater. At pH 8.0 the colonies of both isolates consisted almost entirely of masses of endoconidiophores with few other structures. These colo- nies were characterized by alternate rings of dense and sparse masses of en- doconidiophores. Occasionally chlamyd- ospores and some dark hyphae were CHALAROPSIS LIGHTED DARKENED n I—I—I I—I—I—I—I—I—I—I—I— r I 23 456 7 69 10 II 12 13 14 T. BASICOLA TBT ISOLATE -I—I — \ — \ — \ —1 — \ — \ — I —! — \ — \—r— I I 23456789 10 M 12 13 14 Fig. 3. — The effect of vari- ations in pH and medium type upon the growth of: A, Chalarop- sis thielavioides: 8, the TBT iso- late of 7hietaviop$i$ faosico/o; C, the TBB isolate of T. faosico/o. T. BASICOLA TBB ISOLATE 1—1 1 1 1 I I I— r I 23456789 10 11 12 13 14 438 Illinois Natural History Survey Bulletin Vol. 30, Art. 7 observed in random clumps, usually within the denser mycelial circles. Colonies of both isolates of Thiclaviop- sis on agar with a pH value of 8.6 were composed almost entirely of hyaline hyphae appressed to the agar surface. A few endoconidia were present. Each colony on agar more basic than pH 8.6 consisted only of thin, distorted, hyaline hyphae embedded in the agar. The daily diameter growth of the colonies growing on media with pH values above 6.7 varied from 3.6 to 6.5 mm. Acid media induced the formation of a difiEerent type of colony. Each colony on an acid agar was composed mainly of aerial mycelium. Concentric circles within the colony were produced on agars with pH values of 7.0-4.5. How- ever, the mycelium in each of these circles was black to intermediate gray. Growth on media with a pH as low as 4.5 resulted in an increase in the pro- duction of chlamydospores. This in- crease produced a corresponding dark- ening of the colonies to almost entirely black. Colonies of both isolates grown on agars in the pH range 4.5-4.1 were lighter in color than colonies on slightly less acidic agars. Chlamydospores and dark hyphae were produced in clumps. They were not produced on strongly acidic agars. No chlamydospores were produced on agars with pH 3.7 or below. Fluffiness (or the amount of the aerial myceHum) increased as the pH of the agar decreased down to pH 3.7. Colonies on the pH 3.7 agar were al- most entirely aerial and composed of elongated strands of hyaline hyphae. A few endoconidia and endoconidiophores were found. Colonies on these highly acidified agars were dense enough to form visible margins. These margins were characteristically reticulately lobed. Margins of colonies produced on the highly basic agars were characteris- tically smooth. Daily increases in di- ameters of the colonies on acid media varied from 1.8 to 6.2 mm. Both iso- lates of Thielaviopsis failed to grow on agars with a pH of 3.2 or lower. The effect of fungus growth on the pH of the media was not determined. Before the agar plates were seeded, the pH was adjusted by adding a pre- determined number of drops of acid or base solution to the liquified media. After the 10-day testing period, the media were remelted and tested for pH with a pH meter. The changes in pH of most media were so slight as to be negligible. Fluctuations that may have taken place during the 10-day period were not determined. Effect of Temperature Temperature exerted an effect on the rate of growth but not on the type of growth of the fungi reported on here. In all tests where temperature did not severely restrict growth, the three iso- lates produced colonies characterized by a dense composite of both types of hyphae and both types of spores. The only variation was in the diameters of the colonies. As shown in Fig. 4A, the greatest amount of growth of Chalaropsis oc- curred at 30° C. Daily enlargement of colonies growing at this temperature varied between 6.4 and 8.5 mm. An abrupt decrease in growth at 35° C. resulted from the thermal death of many mycelial cells. Although growth at 10° C. was slight, it was measurable and averaged about 1.2 mm per day. Growth was evident at 5°C.; however, it was restricted to the seed plug and too limited to be measured. The optimum temperature for growth of both isolates of Thielaviopsis was 25° C. (Fig. 4B and C). The TBB isolate grew slightly better at 30° C. than did the TBT isolate. Colonies maintained at 20° C. and 15° C. pro- duced both types of spores and both types of hyphae but were smaller than the colonies grown at 25° C. The TBT isolate produced no growth at either 5° C. or 10° C. TBB, however, pro- duced visible growth at 10° C, but it was confined to the seed plug and was too limited to be measured. This isolate produced no growth at 5° C. ( Aug., 1971 Perby: Two Components of Poinsettia Root Rot 439 GROWTH ON PDA GROWTH ON V-e JUICE AGAR jlW jg/^Asis ihiclavioidcs colony characteristics, 433, 434 component of poinsettia root rot complex, 420, 447 environmental factors, influence on growth, 420, 422, 435-442, 446-449 hosts, 421, 430, 446 i.soIates, 422 morphology, 422, 426, 429-431, 433, 446 on poinsettias, 420, 430, 434, 435, 442-444, 446, 447, 449 patliogenicitv, 420, 421, 423, 442, 446, 449 rate of growtli, 433-435, 438, 439, 446 surface contaminant on carrots, 422, 447 Colonics C. ihielacioides, 429, 433-436 T. haskolu, 425, 429, 430, 435-438, 440-442, 447 zonation, 429, 430, 434, 438 Comparisons of T. hasicola and C. thielavioides, 420, 422, 426, 434, 436, 442, 447, 449 Environmental factors light, 426, 446 pH, 421, 426, 446 temperature, 421, 422, 426, 446 Eiiplinrhia pulchcrrinm Willd., 419 Fungal structures chlamydosporcs, 421, 426, 428-430, 433-442, 445-447, 449 endoconidia, 423, 425-431, 433-436, 438-440, 442, 444-446 endoconidiophores, 425-431, 434, 436-438 mycelium ( hyi^ha ) , dark, 426, 428-430, 434-436, 438, 440-442, 446 mycelium (hypha), hyaline, 425, 428^30, 433-436, 438-440, 442. 446 Germination of spores, 426, 429, 433, 446 Gnift failure, 422 Host-parasite contact, methods of initiating, 425, 441, 442 Incidence of disease, 421, 441—447 Internal collar rot disease, 420 Light effect on rate of growth C. thielavioides, 424, 435, 439-442, 447-449 T. hasicola, 424, 430, 440-442, 447-449 influence on chlamydospore production, 430, 435, 439-442, 449 M Measincments of fungal structures Chalaropsis. 430, 431, 433, 448 Thielaviopsis, 425, 426, 428, 448 Mecha agars acidic, 421, 423, 436, 438 basic, 421, 423, 436-438 cabbage infusion, 422, 435 carrot dextrose, 422, 435 corn meal, 422, 435 lima bean, 422, 435 malt, 422, 435 Noble, 422, 435 poinsettia, 422, 423, 435, 447 potato dextrose, 422-424, 429, 435 prune, 422, 435 \'-8 juice, 423-425, 427, 429, 431, 433-435, 439-441, 448 broths potato dextrose, 423, 424, 435, 442 V-8 juice dextrose, 423, 424, 435, 442 Methods hght studies, 424 microscopic examinations, 423 patliogenicitv tests, 424, 425 pH studies, 423, 424 temperatrne studies, 424 Patliogenicitv tests results of,' 442.-446, 449 techniques used, 425 effect on rate of growtli C. thielavioides, 435^37, 446, 448, 449 T. hasicola, 421, 435-438, 446, 448, 449 influence on disease development, 419, 421 growth of fimgi in culture, 421, 423, 424, 435-438, 446, 449 growth of fiuigi in soil, 419, 421, 446 452 Aug., 1971 Perhy: Two Components of Poinsettia Root Rot 453 Poinsettia agar component, 423 as host, 419, 442, 446, 447 maintenance, 424, 446 symptoms produced on C. thiclavioidcs, 442, 446 T. hasicola, 430, 443, 445 \'arieties, 423, 424, 447 Poinsettia a»ir, 422, 423, 435, 447 Propylene oxide, 425 Pythiiuii root rot, 419 Pythiiim ultinium, 419, 420, 447 technique, 425 Sterilization host tissues, 425 isolation material, 425 S\mptonis of disease uiused bv C. thiclavioidcs, 420, 442, 444-i47, 449 caused by P. ultimum, 419 caused by R. solani, 419 caused bv T. hasicola, 419, 420, 430, 443-447, 449 used to evaluate pathogenicity, 425, 442 Results light studies, 439-442, 447, 449 microscopic examinations, 425^35, 449 patliogenicity tests, 442-446, 449 pH studies, 436-438, 449 temperatiu-e studies, 438, 439, 449 Rhizoctonia root rot, 419 Rhizoctonia solani, 419, 420, 447 l^oot rot complex, 419, 420, 447 Signs of pathogens C. thiclavioidcs. 434, 435, 442, 445-447, 449 T. hasicola, 430, 444-447, 449 Soil-drcncli metliod results, 442, 446 teclmique, 425 Soil pH, 419, 421 Soil-probe method results, 442, 444-446 technique, 425 Stem-incision method results, 442, 443 Temperaturi' edect on rate of growth C. thiclavioidcs, 422, 438, 439, 446, 44H. 449 T. hasicola. 421, 438, 439, 446, 448, 449 influence on disease development, 419, 421, 446, 447 growtli of fungi in culture, 421, 422, 424, 436, 438, 439, 446, 448, 449 growtli of fungi in soil, 419, 421, 424, 446 Th ickiviopsis hasicola colony characteristics, 421, 429, 440, 441 component of poinsettia root rot complex, 419, 420, 447 environmental factors, influence on growth, 420, 421, 436^42, 446-149 hosts, 419-422, 426, 446 isolates, 422, 436--138, 440-443, 446 morphology, 425-430, 446 on poinsettias, 419, 420, 430, 443^47, 449 pathogenicitv, 423, 446, 449 rate of growtli, 429, 435, 436, 438, 440, 442, 446 Tliielaviopsis root rot, 419, 421. 443, 445 Some Publications of the ILLINOIS NATURAL HISTORY SURVEY BULLETIN Volume 29, Article 4.—The Thrips, or Thysa- noptera, of Illinois. By Lewis J. Stannard. May, 1968. 338 p., frontis., 310 fig., bib- liogr., index. Volume 30, Article 1.—Largemouth Bass and Other Fishes in Ridge Lake, Illinois, 1941- 1963. By George W. Bennett, H. Wick- liffe Adkins, and William F. Childers. Sep- tember, 1969. 67 p., 10 fig., bibliogr., in- dex. Volume 30, Article 2.—D>'namics of One- Species Populations of Fishes in Ponds Subjected to Cropping and Additional Stocking. By D. Homer Buck and Charles F. Thoits III. March, 1970. 97 p., 10 fig., bibliogr., index. Volume 30, Article 3.—Migrational Behavior of Mallards and Black Ducks as Deter- mined from Banding. By Frank C. Bell- rose and Robert D. Crompton. September, 1970. 68 p., frontis., 25 fig., bibliogr., index. Volume 30, Article 4.—Fertihzation of Es- tablished Trees: A Report of Field Stud- ies. By Dan Neely, E. B. Himelick, and Webster R. Crowley, Jr. September, 1970. 32 p., frontis., 8 fig., bibliogr., index. Volume 30, Article 5.—A Survey of the Mus- sels (Unionacca) of the Illinois River; a Polluted Stream. By William C. Starrett. February, 1971. 137 p., 17 fig., bibliogr., index. Volume 30, Article 6.—Comparative Uptake and Biodegradability of DDT and Methoxy- chlor by Aquatic Organisms. By Keturah A. Reinbold, Inder P. Kapoor, William F. Childers, Willis^ N. Bruce, and Robert L. Metcalf. June, 1971. 12 p., frontis., 5 fig., bibliogr., index. BIOLOGICAL NOTES 64.—The Value of In Vitro Fungicide Tests. By Dan Neely. January, 1969. 8 p., bibliogr. 65.—Trends in Pheasant Abundance in Illi- nois: 1958 to 1968. By Ronald F. Labisky, May, 1969. 8 p., 4 fig., bibliogr. 66.—Tree and Shrub Hosts of VerticiUium albo-atrum. By E. B. Himelick. July, 1969. 8 p., bibliogr. 67.—Concentrations of Chemical Elements in Phea.sant Tissues. By William L. Anderson and Peggy L. Stewart. April, 1970. 15 p., bibliogr. 68.-Illinois Birds: Mimidae. By Richard R. Graber, Jean W. Graber, and Ethelyn L. Kirk. September, 1970. 38 p., 32 fig., bibliogr. 69.-The Life History of the Dusky Darter, Percina sciera, in the Embarras River, Illi- nois. By Lawrence M. Page and Philip W. Smith. September, 1970. 15 p., 11 fig., bibliogr. 70.—An Ecological Study of Four Darters of the Genus Percina (Percidae) in the Kas- kaskia River, Illinois. By David L. Thomas. December, 1970. 18 p., 11 fig., bibliogr. 71.—A Synopsis of Common and Economic Illinois Ants, with Keys to the Genera ( Hymenoptera, Fomiicidae ) . By Herbert H. Ross, George L. Rotramel, and Wallace E. LaBerge. January, 1971. 22 p., 27 fig., bibliogr. 72.—The Use of Factor Analysis in Modeling Natural Communities of Plants and Animals. By Robert W. Poole. February, 1971. 14 p., 14 fig., bibliogr. 73.—A Distributional Atlas of Upper Mississippi River Fishes. By Philip W. Smith, Alvin C. Lopinot, and William L. Pflieger. May, 1971. 20 p., 2 fig., 107 maps, bibliogr. 74.—The Life History of the Slenderhead Dar- ter, Percina phoxocephah, in the Embarras River, Illinois. By Lawrence M. Page and Philip W. Smitli. July, 1971. 14 p., 10 fig., bibliogr. CIRCULAR 46.—Illinois Trees: Their Diseases. By J. Cedric Carter. June, 1964. (Third printing, with alterations.) 96 p., frontis., 89 fig. 49.—The Dunesland Heritage of Illinois. By Herbert H. Ross (in cooperation with Illi- nois Department of Conservation). August, 1963. 28 p., frontis., 16 fig., bibliogr. 51.—Illinois Trees: Selection, Planting, and Care. By J. Cedric Carter. August, 1966. 123 p., frontis., 108 fig. 52.—Fertihzing and Watering Trees. By Dan Neely and E. B. Himelick. December, 1968. (Second printing.) 20 p., 9 fig., bibliogr. 53.—Dutch Elm Disease in Illinois. By J. Cedric Carter. October, 1967. 19 p., fron- tis., 17 fig. List of available publications mailed on request No charge is made for publications of the Illinois N.^tural History Subvey. A single copy of most publications will be sent free to anyone requesting it until the supply becomes low. Costly publications, more than one copy of a publication, and pubhcations in short supply are subjects for special correspondence. Such correspondence should identify the writer and explain the use to be made of the publication or publications. Address orders and correspondence to the Ctiief, Illinois Natural History Survey Natural Resources Building, Urbono, Illinois 61801