Bulletin BULLETIN of the ILLINOIS NATURAL HISTORY SURVEY HARLOW B. MILLS, Chief Fusarium Disease of Gladiolus: Its Causal Agent JUNIUS L. FORSBERG Printed by Authority of the STATE OF ILLINOIS WILLIAM G. STRATTON, Governor DEPARTMENT OF REGISTRATION AND EDUCATION VERA M. BINKS, Director NAIuKAL HISTORY SURVEY STATE OF ILLINOIS William G. Stratton, Governor DEPARTMENT OF REGISTRATION AND EDUCATION Vera M. Binks, Director NATURAL HISTORY SURVEY DIVISION Harlow B. Mills, Chief Volume 26 BULLETIN Article 6 Fusarium Disease of Gladiolus: Its Causal Agent JUNIUS L. FORSBERG Printed by Authority of the State of Illinois URBANA, ILLINOIS Septetnht'r 1955 STATE OF ILLINOIS William G. Stratton, Governor DEPARTMENT OF REGISTRATION AND EDUCATION Vera M. Binks, Director BOARD OF NATURAL RESOURCES AND CONSERVATION Vera M. Bisks, Chairman A E Emerson, Ph.D., Biology Walter H. Newhouse, Ph.D., Geology l' H. Tiffany, Ph.D., Forestry Roger Adams, Ph.D., D.Sc, Chemistry Robert H. Anderson, B.S.C.E., Engineering David D. Henry, Ph.D., LL.D., HH.D., Litt.D., L.H.D., President of the University of Illinois Delyte W. Morris, Ph.D., President of Southern Illinois University NATURAL HISTORY SURVEY DIVISION Urbana, Illinois SCIENTIFIC AND TECHNICAL STAFF HARLOW B. MILLS, Ph.D., Chief Bessie B. East, M.S., Assistant to the Chief Section of Economic Entomology George C. Decker, Ph.D., Entomologist and Head J. H. Bigger, M.S., Entomologist L. L. English, Ph.D., Entomologist S. C. Chandler, B.S., Associate Entomologist Willis N. Bruce, Ph.D., Associate Entomologist Norman C. Gannon, Ph.D., Associate Ento- mologist John M. Wright, Ph.D., Associate Entomologist Paul Suranv, Ph.D., Associate Entomologist W. H. LuCKMANN, M.S., Associate Entomologist John D. Briggs, Ph.D., Associate Entomologist Ronald H. Meyer, B.S., Assistant Entomologist John W. Matteson, B.A., Field Assistant Robert Snetsinger, M.S., Field Assistant Sue E. Watkins, Technical Assistant H. B. Petty, Ph.D., Extension Specialist in En- tomolog\* Stevenson Moore, III, Ph.D., Extension Spe- cialist in Entomology* John Arthur Lowe, B.S., Research Assistant* Mohan Rao, M.S., Research Assistant* Clarence E. White, B.S., Research Assistant* Louise Zingrone, B.S., Research Assistant* Section of Faunlstlc Surveys and Insect Identification H. H. Ross, Ph.D., Systematic Entomologist and Head Milton W. Sanderson, Ph.D., Taxonomist Lewis J. Stannard, Jr., Ph.D., Associate Tax- onomist Philip W. S.mith, Ph.D., Associate Taxonomist Leonora K. Gloyd, M.S.. Assistant Taxonomist R. B. Selander, Ph.D., Assistant Taxonomist Thomas E. Moore, M.S., Technical Assistant Barbara Gutowsky, M.A., Technical Assistant Section of Aquatic Biology iologistGeorge W. Bennett, Ph.D., Aquatic Bic and Head William C. Starrett, Ph.D., Aquatic Biologist R. W. Larimore, Ph.D., Associate Aquatic Bi- ologist Donald F. Hansen, Ph.D., Assistant Aquatic Biologist Robert D. Crompton, Field Assistant William F. Childers, B.S., Technical Assistant* Section of Applied Botany and Plant Pathology J. Cedric Carter, Ph.D., Plant Pathologist and Head J. L. Forsberg, Ph.D., Plant Pathologist G. H. BoEVVE, M.S., Associate Botanist R. J. Campana, Ph.D., Assistant Plant Pathol- ogist I. R. Schneider, Ph.D., Assistant Plant Pa- thologist E. B. Himelick. M.S., Assistant Plant Pathologist A. W. Engelhard, Ph.D., Assistant Plant Pa- thologist Robert A. Evers, Ph.D., Assistant Botanist Rovenia F. Fitz-Gerald, B.A., Technical Assistant James D. Bilbruck, M.S., Research Assistant* Section of Game Research and Management T. G. Scott, Ph.D., Game Specialist and Head Ralph E. Yeatter, Ph.D., Came Specialist F. C. Bellrose, B.S., Game Specia'.isl H. C. Hanson, M.S., Assistant Came Specialist J. S. Jordan, Ph.D., Assistant Game Technician Ross J. Miller, M.S., Field Ecologist Frances D. Robbins, B.A., Technical Assistant Virginia A. Whipple, Technical Assistant John M. Schilling, Field Assistant William B. Robertson, Jr., Ph.D., Research Assistant* James Opsahl, M.S., Field Assistant* Jack A. Ellis, M.S., Field Assistant* Forrest D. Loomis, B.S., Field Assistant* Paul A. Vohs, Jr., B.S., Project Leader* Section of Publications and Public Relations James S. Avars, B.S., Technical Editor and Head Blanche P. Young, B.A., Assistant Technical Editor William E. Clark, Assistant Technical Photog- rapher Milan Dobrovic, B.A., Technical Assistant Technical Library Ruth R. Warrick, B.S., B.S.L.S., Technical Librarian Nell Miles, M.A., B.S.L.S., Assistant Technical Librarian Consultants: Herpetology, Hobart M. S.mith, Ph.D., Associate Professor of Zoology, University of Illinois; Parasitology, Norman D. Levine, Ph.D., Professor of Veterinary Parasitology and of Veterinary Re- search, University of Illinois. Employed on co-operative projects with one of several agencies: Illinois Agricultural Extension Service, Illinois Department of Conservation, United States Army Surgeon General's Office, United States Depart- ment of Agriculture, United States Fish and Wildlife Service, United States Public Health Service, and others. This paper is a contributionfrom the Section of Applied Botany and Plant Pathology. (17566—3M—6-55) ^ CONTENTS Acknowledgments 447 History of the Disease 447 Names of the Disease 448 Symptomatology 449 Etiology 45 1 Previous Accounts 45 1 Difficulties in Classifying Fus;iria 453 Purpose of Present Investigation 454 Methods 454 Physiological Studies 455 Influence of Temperature on Growth Rates 455 Reactions to Aniline Dyes 458 Reactions to Copper Salts 464 Reactions to Mercuric Chloride 467 Color Reactions on Steamed Rice 469 Variations in Culture Types and Pathogenicity 469 Morphology 479 Pathogenicity' Tests 48 1 Laboratory Tests 481 Greenhouse Tests 486 Discussion and Conclusions 496 Summary 500 Literature Cited 501 Examining gladiolus corms for disease symptoms preparatory to making laboratory cultures.^ The gladiolus Fusarium grows well on most of the common laboratory media. Fusarium Disease of Gladiolus: Its Causal Agent JUNIUS L. FORSBERG THE Fusarium disease of gladiolus is one of the most destructive ma'a- dies known to aftect flower crops. At a conference held in January, 1953, at Lleveland, Ohio, under the auspices of the Joint Research Committee of the North American Gladiolus Council and the North American Commercial Gladiolus Growers, the concensus of those present was that the Fusarium disease is the most important g'adiolus disease in the United States (Ryan 1953). The disease causes large losses to commercial gladiolus grow- ers in Illinois, Indiana, Michigan, Califor- nia, eastern Wa.^hington, and all of Ore- gon except the northern part. It causes an estimated loss of 11/4 '^o 2 million dol- lars per year in Florida alone. Because the Fusarium disease is corm-borne and be- cause many of the corms grown in Illinois are shipped to Florida and planted there for winter flower production, the fate of the crop in Florida is of importance to growers in Illinois as well as to growers in Florida. Only one commercial gladio- lus-growing area in the United States is not seriously troubled with the Fusarium disease. This is the cool region in western Washington where, according to Gould ( 1949) , Fusarium rot is uncommon except on recently introduced stocks. ACKNOWLEDGMENTS The writer wishes to express his sincere appreciation to all who have assisted in any way during the course of this investiga- tion, the greater part of which was pre- sented as a thesis submitted to the Gradu- ate College, University of Illinois, Ur- bana, in partial fulfillment of the require- ments for the degree of Doctor of Philos- ophy in Plant Pathology. He is especially grateful to the late Dr. Leo R. Tehon, for manv vears head of the Section of Applied Botany and Plant Pathology at the Nat- ural History Survey, for his guidance and many helpful suggestions during the course of the investigation and the preparation of the manuscript. Helpful suggestions were made by Dr. Wayne M. Bever, Pro- fessor of Plant Pathology, University of ll.inois, and Agent, United States Depart- ment of Agriculture. The photographic work was done by Mr. W^illiam E. v lark. Assistant Technical Photographer of the Natural History Survey, and by Mr. Ray R. Hamm, Manager of the University of Illinois Photographic Laboratory. Several present or former members of the Natural History Survey staff made valuable con- tributions. Mr. James W. Curfman drew the graphs. Mrs. Rovenia Fitz-Gerald and Mrs. Virginia Lee Johnstone assisted in preparation of the manuscript. Mr. James S. Ayars and Mrs. Blanche P. Young read all or parts of the manuscript. Each offered valuable criticism. The author is especially grateful to his wife, Edith L. Forsberg, for her encouragement and help in many ways during the course of this work. HISTORY OF THE DISEASE The disease of gladiolus which is cal'ed herein the Fusarium disease is a much mis- understood malady which exirts in three forms. These forms have been designated as the vascular, the brown rot, and the basal dry rot types, each of which has been described as a distinct disease by various workers. The disease was first recognized in the early 1920's but it probably existed prior to that time. W. A. Pryal (1909), a California grower, published a rote in which he described an interior corm rot and leaf yellowing of gladiolu;., but no proof was presented that the disease was caused by a fungus of the genus Fusarium. [447] 448 Illinois Natlral History Survey Bulletin Vol. 26, Art. 6 McL'ulloch (1944) reported that in 1^23 she received from two localities in California a large number of gladiolus conns which, although normal in external appearance, showed, when cut, % per cent of the interior rottoil. The rot varied from a slight discoloration in the basal scar to browning of the entire core and radiating fibrovascular strands. From these conns was isolated a Fusariuin that proved capa- ble of causing the disease. McCulioch (1944) reported further that in 1025 and 1926 she received sim- ilar specimens from states as widely sep- arated as North Dakota, Mississippi, and New Jersey. The progress of the disease seemed definitely from west to east, with prevalence increasing each season. In 1926 and 1927, McCulloch found the disease in shipments of conns from Holland. The usual i Itsarin ni was isolated from all these specimens. According to McCulloch (1944), N. van Poeteren "reported the vascular disease as present in Holland as early as 1925." A dry rot of gladiolus caused by Fusar'tum was mentioned in an Annual Report of the Experimental and Research Station, Cheshunt. Hertfordshire, Eng- land (Anonymous 1927). Moore (1939) reported a vascular Fusarium disease from the same countrv. Bellard (1933), Dim- ock (1941. 1945), and Nelson (1937rt, 1938fl, 1938/*, 1948) published brief ac- counts of the vascular form of the disease. McCulloch (1944) published an exten- sive account of this form. Massey (1922) published a brief note and, later (1926), a more extensive de- scription of a corm rot which he consid- ered primarily a disease of stored corms, although infections occurred in the field. McCulloch (1944) considered "the vascu- lar disease" entirely distinct from the conn rot described bv Massev. Nelson (1937/', 1948) described a Fu- sarium disease of gladiolus which he thought distinct from the diseases de- scribed by Massey and McCulloch. McClellan (1947) included as symp- tom expressions of one disease the symp- toms of the two diseases described by Mas- sey and McCulloch. McClellan recog- nized, however, that there are differences of opinion among those who have worked with Fusarium disease of gladiolus as to whether the two types are distinct dis- eases or merely forms of the same disease. NAMES OF THE DISEASE The use of various names in the litera- ture to designate the forms of the Fusar- ium disease of gladiolus has created much confusion in the minds of readers and re- search workers. McCulloch (1944) used the names yellows, wilt, and core rot for the vascular type of the disease. Massey (1922, 1926) designated the disease de- scribed b\ him mereh' as Fusarium rot. Creager (1944) used the name brown rot for the type of corm rot commonly as- sociated with the Picardy variety. He stated, "Fusarium brown rot is not the same as Fusarium yellows or core rot ; they are two distinct diseases." The symptoms commonly found on Picardy, however, seem to be the same as those described by Massey (1926) on other varieties. Pic- ardy was not introduced until 1931, 5 _\ears after Massey 's work was published. Dimock (1945) used only the name yel- lows and listed Picardy as one of the sus- ceptible varieties. Nelson (1948) used the names Fu- sarium dry rot and brown rot for the dis- ease originally described by Massey. He stated that this disease "has sometimes been confused with Fusarium yellows, an entirely different malady. The chief re- semblance between the diseases is that both cause a brown, dry rot of the conn. The best evidence of dissimilarity is demon- strated by the high resistance of the vari- ety Picardy to Fusarium yellows and its equally great susceptibility to Fusarium dry rot." The third tvpe of disease described bv Nelson (1937Z', 1948) was designated by him as basal drv rot. McClellan "(1947) stated, "At least two diseases of gladiolus have been de- scribed as being caused by fungi of the Fu- sarium group. One of these is a corm rot that is principally a storage disease ; the other is the yellows disease that occurs in the field. Yellows has been subdivided further into ( 1 ) a core rot type, a type confined to the water-conducting system ; and (2) a basal rot type." He then fol- September, 1955 Forsberg: Fusarium Disease of Gladiolus 449 lowed with a description of the several kinds of symptoms and considered them as expressions of the same disease. Alagie 5c Aliller (1948) referred to the Fusarium corm rot and yellows disease. In a later article the same authors (1949) used the name Fusarium hrown rot. Alagie (1950) used the names Fusarium yellows and Fusarium corm rot but referred to them as designating a single disease. He applied the term "yellows s\mptoms" to the leaf symptoms and vascular discolora- ton but not to the corm rot phase of the disease. SYMPTOMATOLOGY Symptoms of the Fusarium disease are produced on foliage, corms, and roots. De- tailed descriptions of symptoms associated Fig. 1.—Above: lengthwise sections of gladiolus corms showing, A, how the brown rot form of the Fusarium disease progresses from the mother corm to the daughter corm; B and C, how the vascular form progresses from the mother corms into the core and vascular tissues of the daughter corms. Below: sections of six older corms showing rotted cores and discolored vascular streaks associated with the vascular form of the disease. 450 Illinois Natural History Survhy Bulletin Vol.26, Art. 6 with the three forms of the disease, vascu- lar, brown rot, and basal dry rot, have been published by AlcCulloch (1^H4), Massey (1926), Nelson (1948), and Mc- Clellan (1947). The symptoms common to all three forms of the disease are a brownish to black dry rot of the corm tis- sues; yellowing, browning, and death of the foiiage; and browning and destruction of the roots. The three forms of the disease have been distinguished mainly by effects on the corms. In the vascular form of the dis- ease a sectioned corm will reveal a brown discoloration of the core and dark-colored vascular bundles extended laterally into the flesh, Hg. \B and C. In an advanced stage of the disease, the infected strands reach the surface of the corm at the nodes, and brown lesions develop at these points. In the brown rot form of the disease, tan, brown, or blackish lesions may occur anywhere on the corm but most commonly near the base, fig. 2. The rotted tissue is often quite thick and may extend all the way through the corm, fig. 2, bottom row. X^iscular discoloration is not associated with this form of the disease. The basal dry rot form of the disease differs from the brown rot form mainly in the thickness and position of the lesions. Basal dry rot lesions occur only on the bases of the corms and are usually re- stricted to the first and second internodes, fig. 3. The lesions are visible when the corms are dug and, under favorable curing conditions, they do not enlarge after har- vest. They rarely, if ever, extend deeper than 2 to -I- millimeters into the flesh. The diseased tissue is dark brown to black, Fig. 2.—Picardy gladiolus corms affected with the brown rot form of the Fusarium disease. Above: bottom views of six corms with lesions of various sizes. Below: sections of three corms showing thickness of rotted tissues. September, 1955 Forsberg: Fusarium Disease of Gladiolus 451 hard, rough, and usually somewhat scaly after the conns are dry. The affected area is sunken, and there is a sharp line of de- marcation between diseased and healthy tissues. While the majority of diseased corms in any given lot usually have symptoms characteristic of only one of the disease forms, it is not uncommon to find corms that have symptoms of two of the disease forms or symptoms intermediate between them, tig. 4. Bald (1953) stated, "In any large collection of gladiolus varieties in- fected with Fusarium diseases it ha:- not been found possible to maintain on a symp- tomological basis the division between Fu- sarium basal rot and Fusarium yellows. On different varieties a gradation was found between the 2 symptom types." ETIOLOGY The etiology of the Fusarium disease of gladiolus is quite typical for that of plant diseases caused by fungi of the genus Fti- sarium. The occurrence of the disease in more than one form and the great varia- bility commonly found in species of Fu- sariutii have contributed to the confusion regarding the cause of this disease. Previous Accounts A report by Massey (1922) was the first published account of a gladiolus dis- ease in which a fungus of the genus Fu- sarium was established as the cause. A more extensive description of this disease and its causal agent was later published by Fig. 3.—Corms of three gladiolus varieties affected with the basal dry rot form of the Fusarium disease: top row, variety CJold Eagle; middle row, Lake Placid; bottom row, Spot- light. The two sectioned Spotlight corms show the extreme thinness of the rotted tissue. 452 Illinois Natural History Survey Bulletin Vol. 26, Art. 6 the same worker (1926). Massey classi- fied the ortianism as iusariitm oxysporum Schlecht. emend. VVr. var. yladioH n. var. McCulloch (1944) considered the /•';/- sarium she found associated with the vas- cular form of the diease to be sufficiently distinct from iiisorhim oxysporum var. (jlaiUoli, described by Massey, to warrant putting it in another species. She classified it as I'usarium orthoceras App. et Wr. var. gladioli. In comparing the two organisms she stated. "In culture the bulb-rot organ- ism has. in most tests and examinations, shown less abundant aerial growth, less pigment, and wider macrospores than the yellows organism. . . . The most distinctive characteristics of these two Fusaria of gladiolus are the effects on the host." Other workers have been inconsistent in their use of names for the causal agents of the different forms of the disease. Mc- Clellan (1945) used the name lusaritini orthoceras App. & Wr. var. gladioli Mc- Culloch for the vascular Fusarium of glad- iolus. In a later article the same writer (1947) listed F. oxysporum f. gladioli Sny. & Hans, as the causal agent for yel- lows and rot. He described the other forms of the disease but did not name the causal agents. McCIellan &: Stuart (1947) used the name /•'. oxysporum f. gladioli (Massey) Sny. & Hans, for the causal agent of gladiolus "vellows, or corm rot." McCIellan (1948)' used both names. /•'. oxysporum var. gladioli Massey and F. or- thoceras var. gladioli McCulloch. Nelson (1948) listed F. orthoceras WoU. var. gladioli McCull. as the cause of yellows, Fig. 4.—Sectioned corms of gladiolus variety Dieppe, in upper two rows, and Golden Arrow, in lower two rows, showing symptoms of all three forms of the Fusarium disease. The second corm from the left in the top row shows core rot and vascular discoloration. The first and third corms in the second row and the fourth corm in the top row show symptoms interme- diate between basal dry rot and core rot. The first corms in the third and fourth rows have basal dry rot. The other corms show the brown rot form of the Fusarium disease. September, 1955 FORSBERC. : FUSARIUM DiSEASE OF GlADIOLUS 453 F. oxysporum Schlecht. \ar. yladioli Alas- sey as the cause of brown rot, and merely Fusarium sp. as the cauL^e of ba^al dry rot. Gould (1949) called F. oxysporum f. gladioli the cause of Fusarium rot. Miller & Alagie (1950) listed F. oxysporum f. gladioli as the cause of Fusarium storage rot of gladiolus corms. Magie (1950) re- ferred to the causal agent of Fusarium yel- lows and Fusarium corm rot as /'. oxy- sporum f. gladioli. Bald (1953) stated, "Typical single spore cultures from basal rot, yellows, and intermediate infections were submitted to Dr. W. C. Snyder for identification. He placed all the cultures in the species Fu- sarium oxysporum Schl. As these Fusaria were obtained from active lesions, and some similar isolates were shown by inocu- lation tests to be pathogenic on gladiolus corms, the strains causing basal rot, yel- lows, and intermediate symptoms on glad- ioli in southern California have been pro- visionally grouped under the name /•'. oxy- sporum f. gladioli (Alasse}) Sny. and Hans." Difficulties in Classifying Fusaria A review of the literature indicates that the difficulties encountered by plant pathol- ogists in classifying strains of the gladiolus Fusarium have been great and are similar to the experiences of many other workers faced with the task of determining the re- lationship and specific name to be applied to a pathogenic Fusarium. Some of the confusion is a result of the use of two different s^'stems of nomencla- ture and taxonomy now available for nam- ing and classifying isolates of Fusarium. These are the detailed system of ^\ollen- weber & Reinking (1935) and the simpli- fied svstem of Snvder & Hansen (1940, 1941, 1945). In the system of Wollenweber & Rein- king (1935), the genus Fusarium is di- vided into 16 named sections in which a total of 65 species, 55 varieties, and 22 forms are differentiated. This system is based largely on recommendations made at a conference held in Madison, Wisconsin, in 1924 (Wollenweber et al. 1925). Ac- cording to these recommendations, species and varieties must be distinguished by morphological characters only. Each spe- cies includes groups of individuals that can be distinguished by morphological charac- ters which must be "of such a nature as to be applicable and usable by mycologists in general and which will be most serviceable for practical purposes." Each variety is distinguished by morphological characters of less importance than those used for spe- cific segregation. Groups of individuals differing from the species and the variety only in certain physiological characters are separated as forms. This system failed to meet with unqualified approval because of difficulties that still were encountered h\ workers in attempting to classify specific isolates of Fusarium. The simplified system of nomenclature and taxonomy proposed bv Snyder & Han- sen ( 1940, 1941, 1945) was based on their extensive investigations of the variability shown in culture by species, varieties, and forms of Fusarium. Section Elegans was the first to be revised according to their concept of species. Simplification was achieved by emending the description of one species, Fusarium oxysporum Schl., to agree with the description of section Ele- gans given by Wollenweber (1913). The 10 species, 18 varieties, and 12 forms com- prising section Elegans of Wollenweber k Reinking (1935) were placed in one spe- cies, Fusarium oxysporum, on the sole basis of morphology. Twenty-five para- sites of the section were made forms of this common species on the basis of pathoge- nicity alone. Revision of the other sec- tions followed. As a result of the complete revision of the genus, the 16 sections, 65 species, 55 varieties, and 22 forms of Fu- sarium of Wollenweber & Reinking were reduced to 8 species and 34 forms. 5so va- rieties were recognized in the system of Snyder & Hansen. iVIassey (1926). lAIcCulloch (1944), and Nelson (1948) used the system of \Vollenweber & Reinking to designate spe- cies names for the gladiolus Fusarium. Gould (1949), Miller c^' Magie (1950), Magie (1950), and Bald (1953) used the svstem of Snvder (!^ Hansen. McClellan (1945. 1947.^ 1948) used both systems. Wollenweber's classification was the only one in existence at the time Massey pub- lished the results of his in\estigations. 454 Illinois Natural History Survey Bulletin Vol. 26, Art. 6 Both systems were available to workers after r940. PURPOSE OF PRESENT INVESTIGATION strains could be fitted into well-defined groups on the basis of their pathogenicity and physiological characters. METHODS This study was undertaken to rectify the confusion appearing in the literature regarding the relationship between the causal agent or agents of the different forms of the Fusarium disease on gladiolus and the symptoms produced. The main object of the investigation was to deter- mine if different strains of fusariu/n pro- duced the different s\mptoms and if these Several hundred isolates of Fusarium were cultured by the writer from diseased gladiolus corms in the years 1945 through 1950. Thirty-three of these isolates to- gether with six isolates received from Ray Nelson of Michigan State College and one isolate from Robert O. Magie of the Uni- versity of Florida Gulf Coast Experi- ment Station (a total of 40 isolates) Tuble 1.—Sources of the 40 isolates of the gladiolus Fusarium used in the infection ex- periments and physiological studies reported in this study. Isolate Date of Isolation Dec. Dec. Dec. Dec. Dec. Nov. Jan. Feb. Jan. Jan. Jan. Jan. Jan. Feb. April Nov. April Feb. Dec. Dec. Dec. Dec. Dec. Jan. Jan. Feb. Feb. Sept, Jan. Jan. Jan. Feb. Sept Jan. Jan. Jan. Jan. Nov Dec. 10, 1945 10, 1945 13, 1946 14. 1946 17, 1946 1, 1946 24, 1947 5, 1947 14. 1949 19, 1949 21, 1949 21, 1949 21, 1949 15, 1950 2, 1945 4, 1949 14. 1950 5, 1945 14, 1945 14, 1945 10, 1945 14, 1945 16. 1946 21, 1947 24, 1947 5, 1947 1.3, 1947 27, 1947 14, 1949 19, 1949 19, 1949 15, 1950 1950 3, 1948 23. 1947 24, 1947 14, 1949 19, 1949 25, 1936 23, 1948 Disease Form Vascular Vascular Vascular Vascular Vascular Vascular V'ascular Vascular Vascular Vascular Vascular Vascular Vascular Vascular Vascular Vascular Vascular Brown rot Brown rot Brown rot Brown rot Brown rot Basal dry rot Brown rot Basal dry rot Brown rot Brown rot Brown rot Brown rot Brown rot Brown rot Brown rot Brown rot Brown rot Basal dry rot Basal dry rot Brown rot Basal dry rot Basal dry rot Basal dry rot Gladiolis Variety Dr. F. E. Bennett Dr. F. E. Bennett Phyllis McQuiston Phyllis McQuiston Bit o' Heaven Unknown Beacon Dr. F. E. Bennett Margaret Beaton Lantana Myrna Dream of Beauty Mother Kadel Yellow Herald Unknown Corona Unknown Picardy Picardy Picardy Dr. F. E. Bennett Phyllis McQuiston Aladdin Picardy Beacon Corona Picardy Unknown Margaret Beaton •Abu Hassan Wings of Song Ohio Nonpareil Spic and Span Picardy Picardy Picardy Valeria Wings of Song Souvenir Souvenir Locality Wichert, III. Wichert, 111. Wichert, III. Wichert, III. Wichert, III. Cairo, III. Wichert, III. Wichert, III. Wichert, III. Champaign, III. Wichert, III. Wichert, III. Wichert, III. Wichert, III. East Lansing, Mich. Oregon California Wichert, III. Wichert, 111. Wichert, III. Wichert, 111. Wichert, III. Wichert, 111. Wichert, III. Wichert, III. Wichert, III. Fort Collins, Colo. Cocoa, Fla. Wichert, III. Wichert, 111. Wichert, 111. Champaign, III. Bradenton, Fla. East Lansing, Mich. Wichert, 111. WMchert, 111. _ Wichert, III. M Wichert, III. 1 East Lansing, Mich. East Lansing, Mich. •Isolated by Ray Nelson. Michigan State College, East Lansing. Ilsolated by Robert O. Magie, University of Florida Gulf Coast Experiment Station, Bradenton. September, 1955 Forsberg: Fusarium Disease of Gladiolus 455 Table 2.—Growth of six isolates of the gladiolus Fusarium on Coons's agar at various tem- peratures. Incubation period 160 hours. Temperature, Degrees C. 1-3. 5.... 20-22 24... 26... 27-28 30-31 32... 36... 37... 39... 40... Mean of Diameters (Millimeters) of Two Colonies Brown Rot Isolates 45-75 0.0 0.0^ 52.0 67.0 78.0 74.0 43.5 39.5 5.5 2.5 0.0 0.0 50-22 0.0 0.0 63.0 78.0 85.0 87.0 49.0 48.0 8.5 3.5 0.0 0.0 Vascular Isolates 45-73 0.0 0.0 66.0 83.0 90.0 90.0 57.0 54.0 7.0 3.0 0.0* 0.0 50-24 0.0 0.0* 50.0 63.0 68.0 71.0 50.0 47.0 11.5 5.5 0.0 0.0 Basal Dry Rot Isolates 47-3 0.0 0.0 58.0 64.0 73.0 75.0 49.0 48.0 6.0 4.0 0.0 0.0 50-23 0.0 0.0 51.5 63.0 74.0 75.0 49.0 47.0 6.0 3.0 0.0* 0.0 "A trace of growth insufficient for measurement appeared in thefe platei. were selected for comparison in pathoge- nicity tests and physiological studies. Sev- enteen of these isolates were obtained from conns with the vascular, 16 from corms with the brown rot, and 7 from corms with the basal dry rot form of the disease. They will be referred to hereafter as vas- cular, brown rot, and basal dry rot or basal rot isolates. The original isolations were made on Difco potato dextrose agar. Single-spore isolates were obtained from spore suspen- sions in sterile water blanks; each suspen- sion was diluted until a desired spore con- centration was reached, and then the di- luted suspension was poured over the sur- face of a thin film of 2 per cent water agar in a Petri dish. After a few seconds the excess suspension was poured off and the plate was allowed to stand for 15 to 16 hours. The Petri dish was then placed on the stage of a dissecting microscope, and germinated spores were picked off singly on the tip of a needle and transferred to potato dextrose agar slants. Progenies from these single-spore isolates were used in the infection experiments and physio- logical studies. The sources of all isolates are listed in table 1. Comparisons of isolates from the three forms of the disease were made on the fol- lowing bases: reactions to temperature, reactions to aniline dyes, reactions to cop- per salts, reactions to mercuric chloride, color reactions on steamed rice, growth types on Wellman's differential medium, pH changes produced in liquid media, spore measurements, inoculation tests in the laboratory, and inoculation tests in the greenhouse. PHYSIOLOGICAL STUDIES Physiological studies of the gladiolus Fusarium were made by the writer in an effort to determine if isolates from the three forms of the disease could be distin- guished by their physiological characters. Influence of Temperature on Growth Rates Massey (1926) reported that the glad- iolus Fusarium studied by him grew in cul- ture at temperatures ranging from 5 de- grees to 35 degrees C. ; the optimum was 27.5 degrees. McCulloch (1944) reported that the Fusarium she studied grew at tem- peratures ranging from less than 3 de- grees to about 34-36 degrees C. ; the op- timum temperature range was 23-26 de- grees C. In the present investigation, the influ- ence of temperature on the growth rates of the gladiolus Fusarium was studied on two isolates grown from each disease type on Coons's agar in Petri dishes through a series of 12 temperatures. The inoculum for the Petri dishes was prepared in the following manner to obviate erratic be- 456 Ii.i.iNOis Natural History Survey Bulletin Vol. 26, Art. 6 havior traceable to size or character of in- oculum : coarse white cotton thread was cut in pieces about H-j inches lonjr and autoclaved in distilled water. These pieces were laid on an agar slant, which was then inoculated from the isolate to be tested. About a week after fungus growth had overrun the cotton threads, the threads were lifted from the culture, scraped clean of adhering agar, and cut with sterile scis- sors into sections, each about 2 milli- meters long. One piece of Fusarium-'in- fested thread was placed in the center of each dish. Immediate'.v after inoculation the dishes were placed in electrically con- trolled incubators kept at temperatures shown in table 2 and left for 160 hours. At the end of the incubation period the diameter.? of the colonies were measured, table 2. Two dishes of each isolate were used for each temperature. Relative sizes of the colonies grown at different temper- atures are shown in figs. 5, 6, 7, and 8. None of the isolates grew at the 1-3 de- gree C. temperature range, table 2, and only brown rot isolate 45-75 and vascular isolate 50-24 showed traces of growth at 5 degrees C Since the next higher tempera- nmmm€(y Fig. 5.—Six isolates of Fusarium grown on Coons's agar for 160 hours at the following tem- peratures: 20-22, 24, 27-28 degrees C. (top row in each set of six dishes, left to right) ; 30, 32, and 36 degrees C. (bottom row in each set, left to right). Isolates 45-73 and 50-24 are vascular isolates, 45-75 and 50-22 are brown rot isolates, 47-3 and 50-23 are basal dry rot isolates. September, 1955 Forsberg: Fusarium Disease of Gladiolus 457 ture range used wai 20-22 degrees C, the cular isolate 45-73 and basal dry rot iso- minimum temperature for growth of these late 50-23 showed traces of growth at 39 isolates was not determined. The range degrees C. None of the isolates grew at of temperatures for optimum growth of all 40 degrees C. After the test period, all six isolates was 26-28 degrees C. All iso- Petri dishes that had been incubated at 40 lates grew at 37 degrees C, but only vas- degrees were kept at room temperature for tn 90 458 Illinois Natural History Survey Bulletin Vol. 26. Art. b (T 60 UJ UJ S 50 < o > 40 O 30o UJ ^ 20 q: u ^ 10 Isolate 47-3 10 15 20 25 30 TEMPERATURE, DEGREES CENTIGRADE Fig. 8.—Effect of temperature on the colony size of two basal dry rot isolates of Fusarium grown 160 hours on Coons's agar. several days. Five of the six isolates grew when moved to the lower temperature. Basal dry rot isolate 47-3 apparently had been killed by the 160-hour exposure to 40 degrees C. In these studies the general shape of the curves of growth responses of all isolates to various temperatures was of the same general pattern, figs. 6, 7, and 8. The basal dry rot isolates agreed much more closely in their responses to temperatures than did the isolates from the other disease forms. The vascular isolates showed the greatest divergence in responses to temper- atures. Isolates from the three disease forms could not be distinguished by their growth responses to various temperatures. Reactions to Aniline Dyes The 40 isolates of the gladiolus Fusar- ium listed in table 1 were grown on Coons's agar containing various concentra- tions of the aniline d\es malachite green, brilliant green, and crystal violet, accord- ing to the method developed bv Coons &; Strong (1931). The malachite green and brilliant green media were made from preparations of stock agar containing 10 milliliters of 0.5 per cent dje solution per liter of Coons's agar. For each medium, four concentra- tions, tables 3 and 5, were prepared from the stock agar diluted with appropriate amounts of sterile, melted Coons's agar. The crystal violet stock agar was pre- pared from 100 milliliters of 0.5 per cent dye solution added to a liter of Coons's agar, and the four concentrations, table 4, were made from this stock. Inoculum was prepared in the manner described for the temperature studies. Four isolates were grown in each dish. The center of each quadrant of the dish was seeded with a 2-millimeter piece of h u- sarium-\nie'>ted thread. The cultures were run in duplicate and were incubated for 10 days at 25 degrees C. Readings of the Petri dish cultures were made in accordance with the decimal num- bering scheme used by Coons & Strong (1931). This scheme is as follows: Color of mycelium White . ' 10. Red 20. Breadth of mycelial mat No growth Very slight growth tr. .5 cm. to 1 cm 1. 1 cm. to 2 cm 2. September, 1955 Forsberg: Fusarium Disease of Gladiolus 459 2 cm. to 3 cm. 3. 3 cm. to 4 cm. 4. 4 cm. to 5 cm 5. Changes in medium color No change . . .0 Halo 1 Strong decolorization .2 Color intensified in mycelium .3 Edge of colony Even ' 00 Ramose 01 Frondose 02 Growth form of mycelium Submerged .000 Cottony 001 Villous 002 Sericeous 003 Tufted 004 Submerged, cottony center .005 Submerged, sericeous center .006 Submerged, with cottony fringe at edge, atoll 007 Woolly 008 In the key to the species of t usmium as arranged by Coons & Strong (1931), /•'. orthoceras and F. oxysporurn are recorded as being moderately sensitive to malachite green, i.e., growth usually 1-2 centime- Table 3.—Reactions* of isolates of the gladiolus Fusarium to four concentrations of mala- chite green in Coons's agar. Isolate 460 Illinois Natural History Survey Bulletin Vol. 26, Art. 6 ters broad in the 1 :400,000 and 1 :200,000 concentrations. Growth on crystal violet extended to about 1 :5,000 or 1 :4,000. At this point in the key the two species are separated by their reactions to brilliant green, brilliant green not being decolorized by F. orthoceras but being decolorized by F. oxysporum. Also, a difference in growth form of the two species on crystal violet is noted, growth being submerged with seri- ceous center for F. orthoceras and cottony for F. oxysporum. Diameters of the mycelial mats pro- duced in different concentrations of dye were used by the writer to compare abili- ties of isolates to tolerate the dye in the culture medium. Considerable variation in diameters was shown by the 40 isolates of gladiolus Fusarium, tables 3, 4, and 5. Although of little diagnostic value, growth forms of the various isolates were probably the most striking characters, figs. 9 and 10. When these isolates were taken through Coons (Sc Strong's key, 26 keyed out to Fusarium orthoceras var. triseptatuni. Since no isolates decolorized brilliant green, they did not fall into F. oxysporum. Vascular isolates 49-4 and 49-23, brown Table 4.—Reactions* of isolates of the gladiolus Fusarium to four concentrations of crys- tal violet in Coons's agar.f Isolate September, 1955 Forsberg: Fusarium Disease of Gladiolus 461 rot isolates 47-1, 50-7, and 50-22, and basal dry rot isolate 47-8 keN'ed out to F. euoxy- sporum. Brown rot isolates 45-74, 45-75, 46-4, and 49-19 fell into F. orthoceras var. longius, and brown rot isolate 49-1 and basal dry rot isolate 47-2 keyed out to F. nioniliforitie. Basal dry rot isolates 46-12 and 49-20 keyed out to F. fili- feru/n, but according to Coons & Strong (1931) this species decolorizes crystal vio- let. None of the isolates of the gladiolus Fusarium decolorized crystal violet. Although, on the basis of their reactions to the three aniline dyes, the majority of the isolates of gladiolus Fusarium fell into the F. oxysparum-F. orthoceras group, re- actions of so many of the isolates varied from typical reactions of this group that it would be impossible to use the aniline dye method of classifying Fusaria from gladi- olus with any degree of certainty. Neither would it be possible to separate isolates of the three disease forms, vascular, brown rot, and basal dry rot, on the basis of their reactions to aniline dyes. The results of this phase of the investi- gation agree with the results of Moore & Chupp (1952), who found that certain Table 5.—Reactions* of isolates of the gladiolus Fusarium to four concentrations of bril- liant green in Coons's agar.f Isolate 462 Illinois Natural History Survey Bulletin Vol. 26, Art. 6 Fig. 9. — Isolates of Fnsarium grown 10 days on Coons's agar containing three concentra- tions of brilliant green: left to right 1:200,000, 1:100,000, 1:50,000. Vascular isolates 45-73, 45-80, 46-3, 46-5, 46-9, 46-14, 47-6, and 47-10 are in the two top rows of dishes; brown rot isolates 47-12, 47-19, 47-32, 49-1, 49-17, 49-19, 50-7, and 50-22 are in the two lower rows. Isolates in the dishes at right and left are in the same relative positions as those in the center dishes. Other isolates in this series are shown in fig. 10. September. 1955 Forshero: Fusarium Disease of Gladiolus 463 Fig. 10.—Isolates of Fusarium grown 10 days on Coons's agar containing the same concen- trations of brilliant green as those shown in fig. 9: top row, four brown rot isolates; second row, brown rot isolates 46-4 and 47-1 and basal dry rot isolates 46-12 and 47-8 ; third row, basal dry rot isolates 47-2, 47-3, and 49-20 and brown rot i-olate 49-8 ; bottom row, basal dry rot isolates 50-23 and 50-26, brown rot isolate 50-25, and vascular isolate 50-28. Isolates in the dishes at right and left are in the same relative positions as those in the center dishes. 464 Illinois Natural History Survey Bulletin Vol. 26, Art. 6 isolates of Fttsaritim causing wilts of to- mato, cabbage, and muskmelon did not re- act toward malachite green and crystal violet in the way described by Coons & Strong (1931) for those species. Reactions to Copper Salts Coons & Strong (1931) used copper sulfate in the culture medium in some pre- liminary tests and expressed the opinion that this salt might be useful in identifying some species of Fusarium. Nelson, Coons, & Cochran (1937) reported that two forms of Fusarium which cause two forms of the yellows disease of celery could be differentiated if isolates of them were grown on synthetic agar containing either copper sulfate or copper chloride. The 40 isolates of gladiolus Fusarium used in the investigation reported here were grown on Coons's agar to which amounts of copper sulfate were added to make a series of concentrations that ranged from 1/100 molar (M/lOO) to 1/7000 molar, table 6. The strongest concentra- tion, M/lOO, was prepared from 2.5 grams of copper sulfate (1 mole = 249.6 Table 6.—Reactions* of isolates of the gladiolus Fusarium to seven different molar (M) concentrations of copper sulfate in Coons's agar.f September, 1955 Forsberg: Fusarium Disease of Gladiolus 465 Fig. 11.—Isolates of Fusarium grown 10 days in plates of Coons's agar containing (upper rows, left to right: .U/900, 37/700, M/500, (lower rows, left to right) A//300, and M/lOO, cop- per sulfate. In each plate, clockwise from upper left, isolates are as follows: A, brown rot 45-74, 45-8, 45-78, 45-75; B, vascular 49-15, 49-4, 49-30, 49-23; C, vascular 50-6, 49-31 50-27 50-24; D, vascular 45-80, 45-73, 46-5. 46-3. IfiN ..^^ ..^m -^ mm .-; y VJ? 'K_y As for fig. 11, except A, brown rot 47-19, 47-12, 49-1, 47-32; B, basal dry rot 46-12, K basal dry rot 4- " ' .- . ^ 49-8; D, brown rot 49-19, 49-17, 50-22, 50-7. brown rot 46-4, basal dry rot 47-8, brown rot 47-1 ; C, basal dry rot 47-3, 47-2, 49-20, brown rot 00. n 1 _„^ in in in i -! en tt rn -7 466 Illinois Natural History Survey Bulletin Vol. 26, Art. 6 t^rams) dissolved in a liter of Coons's a^ar. The other concentrations were prepared from this stock diluted with Coons's ajjar. All dilutions were made immediately after the a<:ar had been autoclaved. and the Petri dishes were poured immediately after the dilutions had been made. This procedure was necessary because it was found that Coons's ajjar containing copper salts, if remelted after being allowed to solidify, would not resolidify. Inoculum was prepared and the Petri dishes were inoculated in the same manner as that described for the aniline dve tests. The reactions of the 40 isolates of glad- iolus Ftisarium to copper sulfate are very interesting because they show extreme va- riability in tolerance to the salt, figs. 1 1 and 12. One isolate, 45-74, produced measurable growth in the iW 100 concen- tration. Seven isolates produced only traces of growth in the M/5G00 concen- tration and two isolates produced measur- able growth in only the 71//7000 con- centration, table 6. Although the reactions of the various isolate.^ to copper sulfate failed to distinguish between isolates of Fitsarium from the three disease forms, Table 7.—Reactions* of isolates of the gladiolus Ftisarium to four different molar {M) concentrations of copper chloride in Coons's agar.f September, 1955 Forsberg: FusARiUM Disease of Gladiolus 467 the brown rot isolates were, in general, less sensitive to copper sulfate than were the vascular and basal dry rot isolates. Copper chloride was used in a way sim- ilar to that in which copper sulfate was used. The J//100 concentration was pre- pared from 1.7 grams of copper chloride dissolved in a liter of Coons's agar and the other concentrations were prepared from this stock diluted with Coons's agar. Only four concentrations, M/lOO, J// 300, A//500, and M 1000, were used, table 7. Further dilutions of copper chloride were not used because the reactions of the iso- lates of Fusariuiii appeared to be follow- ing the same pattern with copper chloride as they did with copper sulfate. Reactions to Mercuric Chloride The preplanting treatment of gladiolus corms in a solution of mercuric chloride has been one of the methods used by grow- ers to control the Fusarium disease. Be- cause seemingly erratic results have some- times been obtained from chemical treat- ment of corms, it was decided to test the 40 isolates of gladiolus I usariuni for sen- Table 8.—Reactions* of isolates of the gladiolus Fusarium to six concentrations of mer- curic chloride in Coons's agar.f Isolate 468 Illinois Natural History Survey Bulletin Vol.26, Art. 6 siti'vity to various concentrations of mer- curic chloride in the culture medium. A jjradcd series of concentrations of mercuric chloride of 1 : 1.000 to 1 :25,000 was prepared in a manner similar to that used in the tests with copper salts. Results of the test are shown in table 8. Because none of the i^-olates grew in concentrations of 1 rl.OOO to 1 :10,000, only the dilutions of 1 : 10.000 and above are shown in table 8. As in the tests with aniline dyes and copper salts, the isolates varied considera- bly in their sensitivity to mercuric chlo- ride. One noticeable difference between the reactions to mercuric chloride and the reactions to the other growth-inhibitinjr substances was observed. In the various concentrations of aniline dyes and copper salts, size of the colonies of lusarium in- creased as the dilution became greater. Al- though this reaction occurred to some ex- tent with mercuric chloride, it was not so pronounced. In most cases, if growth oc- curred at all it was very vigorous and cov- ered the maximum space available in the Petri dish. In tables 3, 4, 5, 6, 7, and 8, w^hite mycelium and maximum growth are designated by 15, as derived from the Table 9.—Color reactions of isolates of the gladiolus Fusarium on steamed rice. Colors are from the manual of Ridgway (1912). Isolate September, 1955 Forsberg: Fusarium Disease of Gladiolus 469 Coons pe, she made no attempt to as- sociate these characters with differences in pathogenicity. Several investigators have reported cases of physiologic specialization in cer- tain species of Fusarium. Broadfoot (1926) reported that at least nine physio- logic forms of /•'. ///// Bolley can be distin- guished by their parasitism on four varie- ties of flax. Armstrong & Armstrong (1950) wrote that there are definitely two Table 10.—Growth forms of isolates of the jjladiolus Fusarium when originally cultured on potato dextrose agar and when grown on Wellman's agar, August, 1953. September, 1955 Forsberg: Fusarium Disease of Gladiolus 471 and probably more biological races of F. oxysporum f. tracheiphilum Sny. & Hans, on the basis of pathogenicity on varieties of soybeans and cowpeas. Studies on the relation of culture types and pathogenicity of the 40 isolates of gladiolus Fusariutii listed in table 1 were undertaken by the writer with the hope of finding a possible relation between colony type, disease form, and degree of virulence. Methods of determining virulence are de- scribed under "Pathogenicity Tests." Culture Types of the Gladiolus Fusariuw.—Original isolations from dis- eased gladiolus corms were made on po- tato dextrose agar. Growth forms of the original cultures were not recorded for all isolates. However, 23 of the isolates were classified as belonging to the raised, intermediate, or appressed types on the basis of their aerial mycelium, table 10. Later, all isolates were grown on the me- dium found by Wellman (1942) to give more distinctive reactions between cul- tural variants. This medium, referred to as Wellman's agar in the present investiga- tion, has the following composition: pro- teose peptone 5.0 grams, dihydrogen pc tassium phosphate 0.5 gram, magnesium sulfate 0.5 gram, maltose 15.0 grams, fer- rous sulfate 0.03 gram, agar 12.0 grams, water 1,000.0 milliliters. 49-31 Fig. 13.—Raised, appressed, and intermediate growth forms of vascular isolates of Fusarium grown on Wellman's agar. The extreme raised and extreme appressed forms are shown in cul- tures 45-73 and 49-15, respectively. Cultures are 20 days old. 472 Illinois Natural History Survey Bulletin Vol. 26, Art. 6 Growth forms, based on the classi- tication of WeUman 5: Blaisdell (1941), of the 40 isolates of the gladiolus Fu- sarium as these isolates appeared in Au- gust, 1953, are given in table 10. When cultures of like form were grouped to- gether. 11 were classified as raised, 17 as intermediate-raised, 6 as intermediate-ap- pressed, and 6 as appressed. Photographs of 36 of the cultures are shown in figs. 13 and 14. Table 10 shows that by 1953 some of the cultures were no longer of the same type as the original isolations. When these Fig. 14.—Raised, appressed, and intermediate growth forms of brown rot and basal dry rot isolates of Fusarium grown on Wellman's agar. Cultures 45-8 through 50-22 in the upper four rows are brown rot isolates; cultures 46-12 through 50-26 in the two bottom rows are basal dry rot isolates. Cultures are 20 days old. September, 1955 FORSBERC: FUSARIUM DiSEASE OF GlADIOLUS 473 Table 11.—Changes in growth form of iso- lates of the gladiolus Fusarium after being grown for various periods of time on labora- tory media. Direction of Change 474 Illinois Natural History Survey Bulletin Vol. 26, Art. 6 Table 12.—Readings in pH of Tochinai liquid inoculated with different isolates of the gladiolus Fusariiim; orijjinal pH 6.21. September, 1955 Forsberg: Fusarium Disease of Gladiolus 475 The pH curves for these two isolates are reached the alkaline range ahead of iso- quite similar. Isolate 45-73 remained in late 46-3. After 30 days, the pH curves of the low acid range a little longer but these two isolates were nearly identical. pH 9.01— 8.0 6.0 4.0 - - -4 /^ ^^^ A'^lsolate 49-23 9.0 - 8.0 6.0 4.0 10 solate 50-28 solate 50-24> B 20 30 40 TIME IN DAYS 50 60 Fig. 15.—Progressive changes in pH produced in Tochinai liquid by four vascular isolates of Fusarium. 476 Illinois Natural History Survey Bulletin Vol. 26, Art. 6 In Hj:. \7B, brown rot isolates 50-7 50-22 was intermediate-raised. Isolate and 50-22 are compared. Isolate 50-7 50-22 was the more virulent in laboratory was of the intermediate-appressed type ; and greenhouse tests. The pH curves for PH September, 1955 Forsberg: Fusarium Disease of Gladiolus 477 these two isolates are similar except for a Two sets of basal dry rot isolates are divergence that occurred between the compared in fig. 18. In fig. 18.:/. isolate twelfth and thirty-fourth days. 47-2 was of the intermediate-raised type PH 478 Illinois Natural History Survey Bulletin Vol, 26, Art. 6 and 47-3 was raised. Isolate 47-3 was the the laboratory. Isolate 47-2 produced the more virulent in jjreenhouse tests ; 47-2 greater amount of acid during early was slightlv more virulent than 47-3 in growth and its pH readings remained pH 9.0 8.0 6.0 5.0 4.0 /"^Isolate 47-2 ^, -^t-z.::.:^^,^ 9,0 6.0 4.0 10 20 30 TIME IN DAYS 40 50 60 Fig. 18.—Progressive changes in pH produced in Tochinai liquid by four basal dry rot isolates of Fusarium. September. 1955 FORSBERC, : FUSARIUM DiSEASE OF GlADIOLUS 479 lower than those for isolate 47-3 until the thirty-fourth day. In fig. 185, isolate 46-12 was inter- mediate-raised, and isolate 47-8 was in- termediate-appressed. Isolate 46-12 was more virulent in laboratory tests. In this case the pH readings for the more virulent isolate were lower than those for the milder isolate from the beginning until the fifty-eighth day. In these experiments no consistent rela- tions between culture types, pH changes, or degrees of virulence were observed. In this respect, the gladiolus Fusarium ap- pears to differ from the tomato Fusarium. MORPHOLOGY A complete morphological study of all the isolates from the vascular, brown rot, and basal dry rot forms of the gladiolus Fusarium disease was not attempted. Some of the isolates produced the typically 2- to 5-septate macrospores rather consistently, but many isolates produced only the single- celled type. In some isolates, septate spores were very abundant in the original cul- tures from diseased gladiolus corms, but, after one or two transfers, only single- celled spores could be found. Spores seemed to be produced more abundantly on Well- Table 13.—Measurements of conidia produced by six isolates of the gladiolus Fusarium on Wellman's agar. Disease Form Vascular Vascular Brown rot Brown rot Basal dry rot Basal dry rot Isolate and Type OF Conidia Isolate 49-15 0-septate 1-septate 2-septate 3-septate 4-septate 5-septate Isolate 50-27 0-septate 1-septate 2-septate 3-septate 4-septate 5-septate Isolate 45-74 0-septate 1-septate 2-septate 3-septate 4-septate 5-septate Isolate 50-25 0-septate 1-septate 2-septate 3-septate 4-septate 5-septate Isolate 49-20 0-septate 1-septate 2-septate 3-septate 4-septate 5-septate Isolate 50-26 0-septate 1-septate 2-septate 3-septate 4-septate 5-septate Mean, Microns 9.2 480 Illinois Natural History Survey Bulletin Vol. 26, Art. 6 man's agar than on potato dextrose agar. From several isolates which produced septate macrospores rather abundantly on Wellman's agar, two isolates from each disease form were selected for spore meas- urements. The only purpose of making spore measurements was to find out if iso- lates from the different disease forms Fig. 19.—Corms of 14 gladiolus varieties, each corm inoculated with four isolates of Fusariiim. The locations of the inoculations on each corm are as follows: top, isolate 49-8; right, 50-27; bottom, 50-22; left, 49-17. Photograph was taken 34 days after inoculation. 4 September, 1955 Forsberg: Fusarium Disease of Gladiolus 481 might be distinguished by differences in spore sizes. As pointed out by Harter (1939), several factors influence the size of Fusarium spores produced in laboratory culture. It is often diflficult, if not impos- sible, by any means of manipulation of the culture to obtain a morphological agree- ment with the description of a given spe- cies. It seemed reasonable to assume, how- ever, that a fair comparison could be made of spores from cultures which had been kept under the same conditions. Six isolates were selected for use in making spore measurements and were grown on Wellman's agar in Petri dishes for 14 days in a location where the dishes were exposed to diffused light during day- light hours. All measurements were made within 2 or 3 days after the cultures were 14 days old. Cultures from which meas- urements could not be made immediately were placed in a refrigerator at 3-5 de- grees C. until measurements could be made. The spores to be measured were mounted on 2 per cent plain agar on a microscope slide. Thirty spores of each septation type were measured from each culture in which this number of spores was available. The 4- and 5-septate types were so rare in most of the cultures that it was impossible to find 30 spores for measure- ment. Mean measurements and ranges of measurements of spores from the six se- lected isolates are shown in table 13. The differences in spore sizes are not great enough to place the isolates in distinct groups on the basis of spore size. PATHOGENICITY TESTS In this investigation, the pathogenicity of the isolates of Fusarium was tested both in the laboratory and in the greenhouse. All 40 isolates were tested in the labora- tory ; 27 of them were tested in the green- house. Laboratory Tests Large mature gladiolus conns, free from blemishes, were selected for the lab- oratory inoculation tests. After the husks had been removed, the corms were washed well with water and then left on a table until dry. Four small wounds, approxi- mately equal distances apart, were made on the basal area of each corm with a three-sixteenths-inch metal drill bit turned rapidly with thumb and index finger. Uni- form disks of inoculum were cut from agar plate cultures with a sterile metal tube three-sixteenths inch in diameter. One disk of inoculum was pressed gently into each wound. Each corm was inoculated with four isolates ; each isolate was used on four corms in each of the various tests. Inoculations were identified by paper tags pinned near the points of inocula- tion, fig. 19. Immediately after they had been inoculated, the corms were placed in moist chambers and left for 48 hours. They were then removed and placed on a table in the laboratory. The corms were examined frequently, and at the end of 4 to 6 weeks final disease readings were made. These inoculations resulted in the de- velopment of three general types of le- sions: severe, in which the rot progressed steadily from the point of inoculation un- til most of the corm was rotted, shown in the Bit o' Heaven corm in fig. 19; mild, in which the rot progressed very slowly and only a narrow brown band appeared around the point of inoculation, shown in the Acca Laurentia corm in fig. 19; healed, in which no rot developed and the inocu- lation wound corked over, shown in the Annamae corm in fig. 19. A basis on which to compare virulence of the isolates of Fusarium, as well as to compare susceptibilities of gladiolus vari- eties, was derived from a modification of McKinney's (1923) formula for disease evaluation. Class values of 0, 1, and 2, respectively, were assigned to the healed, mild, and severe types of lesions. Indexes of rot severity were calculated by use of the following formula : p . . A N,0 + Nol + N32 ^ ,__ Rot severity index = r X 100 where Ni, No, N3 = number of corms in disease classes 1, 2, and 3, respec- tively 0, 1, 2 = values assigned to disease classes 1, 2, and 3 respec- tively t = total number of corms used 482 Illinois Natural History Survey Bulletin Vol. 26, Art. 6 Table 14.—Severity indexes for the rots produced by four isolates of Fusariutn on 68 jjladiolus varieties in 1951. The indexes were derived from the formula on page 481. Variety Isolate Abu Hassan .'^cca Laurentia .Aladdin .'Vlgonquin .Annamae Badger Beauty Beacon Big Top Bingo Bit o' Heaven Black Opal Blue Beauty Buckeye Bronze Burma Colonial Maid Convoy Corona Crinkle Cream Dieppe Dr. F. E. Bennett Dr. Whiteley Dusty Miller Early Rose Elizabeth the Queen. . . . Ethel Cave Cole Fair Angel Genghis Khan Gloaming Gold Eagle Golden Dream High Finance Johan van Konynenburg King Lear King William Lady Jane Lantana Larime Leading Lady Legend Majuba Malta Margaret Beaton Marguerite Minuet Miss Bloomington Mother Kadel Mrs. Lulu Hunt New Europe Ogarita Ohio Nonpareil Oklahoma Oregon Gold Pandora Phyllis McQuiston Prairie Gold Purple Supreme Red Charm Rewi Fallu Rosa \' an Lima Rose Ruffles 49-8 100.0 37.5 75.0 50.0 25.0 100.0 62.5 75.0 75.0 62.5 87.5 62.5 37.5 50.0 37.5 75.0 100.0 87.5 50.0 100 50.0 100.0 25.0 75.0 50.0 50.0 100.0 62.5 50.0 100.0 62.5 100.0 75.0 50.0 12.5 50.0 50.0 50.0 50.0 50.0 62.5 50.0 62.5 62.5 50.0 50.0 50.0 100.0 50.0 50.0 50.0 75.0 75.0 50.0 50.0 100.0 75.0 75.0 25.0 62.5 49-17 100.0 50.0 100.0 100.0 25.0 100.0 87.5 100.0 100.0 100.0 75.0 50.0 75.0 12.5 75.0 75.0 100.0 100.0 100.0 100.0 50.0 62.5 50 100.0 75.0 75.0 100.0 100.0 37.5 100.0 100.0 100.0 100.0 100.0 75 100.0 100.0 100.0 75.0 62.5 100.0 50.0 75.0 37.5 50.0 100.0 50.0 100.0 75.0 87.5 75.0 75.0 100.0 50.0 100.0 100.0 100.0 100.0 75.0 75.0 50-22 100.0 50.0 100.0 100.0 25.0 100.0 100.0 100.0 100 100.0 100.0 87.5 87.5 50.0 100.0 87.5 100.0 100.0 100 100.0 62.5 100.0 75.0 100 100.0 87.5 100.0 100 62,5 100.0 100.0 100,0 100.0 100.0 87.5 100.0 100.0 100.0 87.5 100 100,0 50,0 100 100 50 100 50.0 100.0 87.5 100.0 87.5 100 100.0 50.0 100.0 100,0 100,0 100,0 62.5 100.0 50-27 100.0 25.0 87.5 100 37.5 100.0 100.0 100.0 100 100. 100.0 62.5 87.5 50.0 100.0 62.5 100.0 100.0 100.0 100.0 50.0 100.0 37.5 100,0 87.5 50.0 100.0 100.0 62.5 100.0 100.0 100.0 100.0 100.0 87.5 100.0 100.0 87.5 50.0 50.0 100 50 100 100.0 50.0 100.0 50.0 100.0 87.5 87.5 75.0 87.5 100.0 50.0 100 100,0 100.0 100 37.5 87.5 84.4 September, 1955 Forsrerg: Fusarium Disease of Gladiolus 483 484 Illinois Natur.al History Survey Bulletin Vol. 26, Art. 6 In l')51, tour isolates were tested for ability to produce rot on conns of 68 va- rieties. Results are shown in table 14. Iso- late 50-27 was a vascular isolate; the others were brown rot isolates. All tour isolates caused rot on all varieties used in this test, but the severity of rot varied with isolates as well as with varieties. The av- erage inde-\e> in table 14 can be consid- ered indicative of the relative susceptibil- ity of the different varieties to Fusarium rot. Since the test was conducted under conditions that favored development of rot, varieties with an averajje index of to 50 can be considered resistant and va- rieties with an index jjreater than 50 can be considered susceptible. In 1952, all 40 isolates were tested on seven gladiolus varieties. The indexes of rot severity obtained in this test are shown in table 15. The isolates varied greatly in their ability to produce rot on corms of different varieties. Seven of the isolates failed to produce rot on any of the seven varieties tested. Only ba.-^al dry rot iso- lates 47-2 and 47-3 produced rot on all seven varieties. The average rot severity index for the 7 basal dry rot isolates was 26.5, for the 16 brown rot isolates it was 17.8, and for the 17 vascular isolates it was 6.7. Apparently vascular isolates are less capable of causing conn rot than are the brown rot and basal dry rot iso- lates. These results are in general agree- ment with those of McCulloch (1944), who found that the vascular isolates with which she worked caused only mild rot on conns inoculated in the laboratory, while isolates from corms having the disease type described by Massey (1926) caused severe rot. In this test the average severity in- dex for rot on the variety Picardy was 59.4 while on Maid of Orleans and Rosa Van Lima it was only 2.5. The differ- ence in susceptibility indicated by these figures agrees well with what is observed in commercial stocks of these varieties. Fu- sarium rot is very rare in stocks of Maid of Orleans and Rosa Van Lima but it is extremely common in stocks of Picardy. Six of the varieties used in the 1951 Table 16.—Severity indexes for the rots produced in 2 successive years by four isolates of Fusarium on six gladiolus varieties.* September, 1955 Forsberg: Fusarium Dise.ase of Gladiolus 485 test were used also in the 1952 test. The severity indexes for the rots caused by the same four isolates on corms of these vari- eties in the 2 years are shown in table 16. In 17 cases the rot indexes were lower in 1952 than they had been in 1951, in 5 cases they remained the same, and in 2 cases only they were higher. A third test was made in 1953 when all 40 isolates were used on the varieties Pic- ardy and Spirit of St. Louis. The results obtained with these varieties in 1952 and 1953 are shown in table 17. In the tests on Picardy the rot indexes in 27 isolates were lower in 1953 than in 1952, in 4 tliey were higher, and in 9 they remained the same. With Spirit of St. Louis the rot indexes in 5 isolates were lower in 1953 than in 1952, in 7 they were higher, and in 28 they remained the same. The indexes remaining unchanged included 7 instances in Picardy and 25 in Spirit of St. Louis in which no rot was produced in either year. Although no attempt was made to maintain the same conditions for all tests, it is not likely that laboratory conditions varied greatly during the three tests. Dif- ferences in corm lots used in different years may have been partly responsible for Table 17.—Severity indexes for the rots produced in 2 or 3 successive years by 40 isolates of Fusarium on two gladiolus varieties. Indexes were derived from the formula on page 481. Isolate 486 Illinois Natural History Survey Bulletin Vol. 26, Art. 6 Table 18.—Results of greenhouse tests on f^.ladiolus variety Picardy inoculated with Fusarium, 1948. September, 1955 P'orsberg: Fusarium Disease of (jLADIOlus 487 The new conns were dug July 24 ; they at 5 degrees C. and left until November were cleaned and examined August 13. or December, when isolations from the af- They were then placed in a refrigerator fected corms were attempted. Fig. 20.— Differences in reaction of, left to right in each picture, gladiolus varieties Dr. F. E. Bennett, Margaret Fulton, Picardv, and Variation to basal dry rot isolate 49-20 and vascular isolates 49-4 and 50-6 of Fusarium. 'Photographs were taken 52 days after planting date. 488 Ii.i.iNois Natural History Survey Bum.etix Vol. 26, Art. 6 Results obtained in these tests are shown conclusive, because disease symptoms de- in tables IQ, 20, 21, and 22. In the cases veloped in some corms of the checks, fu- of the Dr. F. E. Bennett and Picardy va- sarin/n was isolated from two corms of the rieties, the results cannot be considered Dr. F. E. Bennett checks. This finding Fig. 21.— Ditlereiices in reaction of, left to right in each picture, gladiolus varieties Dr. F. E. Bennett, Margaret Fulton, Picardy, and Variation to four brown rot isolates of Fusarium. Photographs were taken 52 days after planting date. September, 1955 Forsberg: Fusarium Disease of Gladiolus 489 indicated that the planting stock carried some Fusarium ; so all of the symptoms that developed in the new conns probably did not result from the inoculations. In the variety Picardy, five of the check plants died before reaching maturity and seven new corms had vascular discolora- tion. Fusarium was not isolated from any of these new corms. Curvularia sp. was isolated from four of them and Penicillium sp. was isolated from the other three. These two organisms appeared also in some isolations from corms in the inocu- lated series. In the variety Variation, vascular dis- coloration developed in one corm ; four corms of the 29 in the checks had slight basal rot lesions. Isolations from four of these affected corms yielded Penicillium ; no organism was obtained from the fifth corm. All plants from noninoculated Mar- garet Fulton corms remained healthy and produced healthy daughter corms. Fusar- ium was recovered from diseased corms in the inoculated series 36 times in 52 at- tempts. Tests in 1953.—Varieties Ur. F. E. Bennett, Margaret Fulton, Spotlight, and Elizabeth the Queen were inoculated late in 1952 with 13 isolates of Fusarium. Size No. 4 (three-fourths to 1 inch in diam- eter) corms of the variety Elizabeth the Queen and large cormels of the other three varieties were used in this test. Treating and planting was done December 29, 1952. New corms were harvested June 5, 1953; they were cleaned and ex- amined 2 weeks later. In this trial, because no disease was found in the new corms produced in the noninoculated checks, and because only one Dr. F. E. Bennett and one Spotlight plant failed to live and produce new corms, it was assumed that the planting stock was practically free of disease. Cause of death Table 19.—Results of greenhouse tests on gladiolus variety Dr. F. E. Bennett inoculated with Fusarium, February, 1952; new corms were examined in August of the same year. 490 Illinois Natural History Survey Bulletin Vol. 26. Art. 6 of the two nonsurvivinji check plants was not determined. As the results of this test are considered more conclusive than those of the previous test, they are given in de- tail hy isolates and varieties. Brown rot isolate 47-12 Dr. F. E. Bennett: Only 3 plants emerged ; none produced a new conn. Margaret Fulton : 8 plants emerged ; 1 survived and produced a new conn, which had thin basal rot. Spotlight: 5 plants emerged; 4 sur- vived and produced new corms, 3 of which had much of the bases rotted, but the rot- ted tissue was very thin. There was some vascular streaking, but it was not general. One corm showed no symptoms. Fusariutii was recovered from all 3 affected corms. Elizabeth the Queen: 10 plants emerged; all survived and produced new corms. Four of the new corms were badly decomposed ; 6 had typical brown rot le- sions. Of these 6 corms, 1 had numerous brown vascular strands; another had a single brown vascular strand in addition to the brown rot lesions. Fusariurn was re- covered from all 6 corms. Brown rot isolate 47-32 Dr. F. E. Bennett : 6 plants emerged ; 5 survived and produced new corms, all of which had core rot and brown vascular streaks. Fusariuni was recovered from all 5 corms. Margaret Fulton: 10 plants emerged; all survived and developed new corms. Two corms had thin basal rot le- sions ; 8 showed no symptoms. Fusariuni was not recovered from the affected corms. Spotlight : 5 plants emerged ; 7 sur- vived and produced new corms, all of which had diffused basal lesions. There was no vascular discoloration. Cultures were made from 3 corms, and Fusariurn was recovered from each corm. Table 20.—Results of greenhouse tests on gladiolus variety Margaret Fulton inoculated with Fusariurn, February, 1952; new corms were examined in August of the same year. September, 1955 Forsberg: Fus.arium Dise.a.se of Gladiolus 491 Elizabeth the Queex: 10 plants emerged; all survived and produced new corms, 7 of which had very thin small le- sions at the sides of the core bases. Fu- sariuni was recovered from 2 of 4 corms cultured. Brown rot isolate 50-7 Dr. F. E. Bennett: 8 plants emerged; all survived and produced new corms. All new corms had thin basal rot lesions. Fu- sarium was recovered from 2 of 5 corms cultured. Margaret Fulton: 10 plants emerged; all survived and produced new corms. No disease symptoms developed. Spotlight: 10 plants emerged; all survived and produced new corms. Nine of the new corms appeared to have thin basal rot lesions developing when the corms were cleaned, but the symptoms were not distinct. The corms were placed in an incubator at 26 degrees C. and left for 15 weeks. The lesions did not develop further, and attempts to recover the Fu- sariuni from these corms failed. Elizabeth the Queen: 10 plants emerged ; all survived and produced new corms, 9 of which had no disease symp- toms. The remaining corm had a diffused basal rot lesion spreading halfway up the side of the corm. Fusarium was recovered from this lesion. Brown rot isolate 50-22 Dr. F. E. Bennett: 6 plants emerged; 5 survived and produced new corms. Four of the new corms had typical thick brown rot lesions. One corm also had brown vas- cular strands. Fusarium was recovered from all 4 affected corms. One corm had no disease symptoms. Margaret Fulton: 10 plants emerged; 8 survived and produced new corms. Seven of the new corms had brown rot lesions on the bases and sides. Four Table 21.—Results of greenhouse tests on gladiolus variety Picardy inoculated with Fusarium, February, 1952; new corms were examined in August of the same year. 49: Illinois Natural History Survey Bulletin Vol. 26, Art. 6 conns were cultured, and Fusariittii was reco\erc'd from each of them, (^ne corm remained healthy. Spotlight: 10 plants emerged; 6 sur- vived and produced new corms. Three of the new corms were completely mummified at cleaninji: time. One corm was three- fourths rotted ; another contained a dis- colored vascular strand but had no exter- nal symptoms. Itisarium was recovered from the rotted corm but not from the dis- colored vascular strand. One corm re- mained healthy. Kl!/.abeth the Queen: 10 plants emerged ; all survived and produced new corms. Two of the new corms were mum- mified at cleaning time, the other 8 had no disease symptoms. No attempt was made to recover the Fusarium. Vascular isolate 4?i'73 Dr. F. E. Bennett: 7 plants emerged; 5 survived and produced new corms. Two of the new corms had brown vascular strands; 3 had no symptoms. Fusarium was recovered from both of the affected corms. Margaret Fulton: 10 plants emerged ; all survived and produced new corms. One corm had brown vascular strands; 9 had no symptoms. Fusarium was recovered from the affected corm. Sf'OTLight: 10 plants emerged; all sur- vived and produced new corms. Three of the new corms had brown vascular strands ; 7 remained healthy. Fusarium was recovered from 2 of the affected corms. Elizabeth the Queen: 10 plants emerged ; all survived and produced new corms. Eight of the new corm^ had exten- sive discolored vascular strands ; 2 of them had thick brown rot lesions also. No connections between the brown rot lesions and the vascular streaks were found in any of the corms. Fusarium was recovered from all 8 of the afiFected corms. Two of Tiible 22.—Results of greenhouse tests on gladiolus variety Variation inoculated with Fusarium, February, 1952; new corms were examined in August of the same year. September, 1955 FoRsnERc: Kusarium Disease of Gladiolus 493 the 10 corms had no visible symptoms of disease. Vascular isolate 46-3 Dr. F. E. Bennett : 7 plants emerged ; all survived and produced new corms. One of the new corms was mummified ; 2 had basal core rot but no extensive vascular discoloration. Four of the corms remained healthy. Fusarium was recovered from both corms with core rot. Isolations were not attempted from the mummified corms. Margaret Fulton : 9 plants emerged ; all survived and produced new corms. One had a thin basal rot lesion ; 8 remained healthy. Fusarium was not recovered from the affected corm. Spotlight: 10 plants emerged; all survived and produced new corms. None had disease symptoms. Elizabeth the Queen: 10 plants emerged; all survived and produced new corms. All of the new corms remained healthy. Vascular isolate 47-10 Dr. F. E. Bennett: 7 plants emerged; all survived and produced new corms. Three corms had thin basal core rot but no vascular discoloration. Four corms had no disease symptoms. Fusarium was re- covered from 2 of the 3 affected corms. Margaret Fulton : 9 plants emerged ; 8 survived and produced new corms. One had extensive discolored vascular strands; 7 remained healthy. Fusarium was recov- ered from the affected corm. Spotlight: 10 plants emerged; all survived and produced new corms. All of the new corms remained healthy. Elizabeth the Queen: 10 plants emerged ; all survived and produced new corms. All of the new corms remained healthy. Vascular isolate 49-4 Dr. F. E. Bennett: 8 plants emerged; 7 survived and produced new corms. Two corms were mummified ; 1 had a deep core rot extending to a thin lesion at the side of the core base. Three corms had deep core rot but no extensive vascular discol- oration. One corm remained healthy. Fusarium was recovered from 3 of the af- fected corms. Margaret Fulton : 9 plants emerged ; all survived and produced new corms. Eight corms had thin basal rot. There was no vascular discoloration. Three corms were cultured, and Fusarium was recov- ered from all of them. One corm had no disease symptoms. Spotlight: 10 plants emerged; 9 sur- vived and produced new corms. At clean- ing time 2 of the corms were mummified ; 6 had thin basal rot. Fusarium was re- covered from 5 of the 6 corms affected with basal rot. Elizabeth the Queen: 10 plants emerged ; 9 survived and produced new corms. Four of the new corms had thin basal rot. In 1 corm, the brown discol- oration extended from the base into one vascular strand. Fusarium was recovered from 1 of the 4 affected corms. Vascular isolate 50-24 Eight Dr. F. E. Bennett, 9 Mar- garet Fulton, 10 Spotlight, and 10 Elizabeth the Queen plants emerged; all survived and produced new corms. No disease symptoms developed. Basal rot isolate 47-2 Dr. F. E. Bennett: 10 plants emerged ; all survived and produced new corms. One of the new corms had one brown vascular strand ; another had vet}- short discolored vascular streaks around the core. Fusarium was not recovered from the affected corms. Eight corms had no disease symptoms. Margaret Fulton: 10 plants emerged ; 9 survived and produced new corms. All new corms remained healthy. Spotlight : 8 plants emerged ; all sur- vived and produced new corms. Six corms had thin basal rot lesions, most of which chipped out when the corms were cleaned. One corm also had pronounced vascular discoloration. Fusarium was recovered from 3 of the affected corms. Two corms remained healthy. Elizabeth the Queen: 10 p'ants emerged ; all survived and produced new corms. All new corms remained healthy. Basal rot isolate 47-3 Dr. F. E. Bennett : 7 plants emerged ; all survived and produced new corms. All 494 Illinois Natural History Survey Bulletin Vol. 26, Art. 6 7 corms had brown core bases; 5 of these conns had brown vascular strands also. Fusarium was recovered from all 7 corms. Margaret Fultox: 10 plants emerged ; all survived and produced new corms. Each of the corms had a thin, light brown discoloration around the core base. Only 1 corm had a discolored vascu- lar strand. Three of the corms were cul- tured ; Fusarium was recovered from all of them. Spotlight: 10 plants emerged; all survived and produced new corms. One corm had a hard core base and pronounced vascular discoloration. Two other corms had lighter vascular discoloration. Fu- sarium was not recovered from these corms. Seven corms showed no symptoms of disease. Elizabeth the Queen: 10 plants emerged ; all survived and produced new corms. Four corms had typical thin basal rot lesions; 2 had discolored vascular strands but no basal rot lesions. Fusarium was recovered from 5 of the 6 affected corms. Four corms had no symptoms of disease. Basal rot isolate 50-23 Dr. F. E. Bennett: 6 plants emerged; all survived and produced new corms. One corm had a thin basal rot lesion ; 1 had a brown vascular strand in the top half of the corm. Fusarium was recovered from both affected corms. Four corms remained healthy. Margaret Fulton: 10 plants emerged ; 9 survived and produced new^ corms. Eight corms had thin brown dis- colorations over the core bases. In 1 corm the discoloration extended slightly beyond the core. One corm had no symptoms. Attempts to recover Fusarium failed with all 8 affected corms. Spotlight: 10 plants emerged; all survived and produced new corms. Nine corms had typical thin diffused basal rot lesions. There was no vascular discolora- tion. Fusarium was recovered from 8 of the 9 affected corms. One corm remained healthy. Elizabeth the Queen: 10 plants emerged ; all survived and produced new corms. No disease symptoms developed in any of the plants or new corms. Basal rot isolate 50-26 Dr. F. E. Bennett: 8 plants emerged; all survived and produced new corms. Seven corms had brown core bases. One corm also had a thick brown rot lesion on its side ; 1 corm had a thin brown rot le- sion on its side, and 1 corm had a brown vascular strand. One corm had no disease symptoms. Fusarium was recovered from 5 of the 7 affected corms. Margaret Fulton : 9 plants emerged ; all survived and produced new corms. Six corms had brown core bases ; 3 had no symptoms. Fusarium was recovered from only 1 of the affected corms. Spotlight: 9 plants emerged; 8 sur- vived and produced new corms. Two of the corms were mummies; 6 had typical thin basal rot lesions. Fusarium was re- covered from all 6 of these corms. Elizabeth the Queen: 10 plants emerged ; all survived and produced new corms. Two corms had very small, thin basal rot lesions. Fusarium was recovered from both corms. Eight corms had no dis- ease symptoms. Noninoculated checks: Three sets of checks consisting of 10 corms of each vari- ety in each set were planted. Dr. F. E. Bennett: 24 plants emerged ; 23 survived and produced new corms. No disease symptoms developed. Margaret Fulton: 29 plants emerged ; all survived and produced new corms. No disease symptoms developed. Spotlight: 29 plants emerged; 28 sur- vived and produced new corms. No dis- ease symptoms developed. Elizabeth the Queen: 30 plants emerged ; all survived and produced new corms. No disease symptoms developed. Fusarium reisolates: The cultures that were recovered from the new corms agreed closely in growth type on Wellman's agar with the growth types of the respective isolates used for inoculation. Variation in Virulence of Isolates. —Relative virulences of the isolates used in the greenhouse tests were rated numer- ically by means of disease indexes com- puted as follows : Values were assigned to the disease symptoms in the new corms and these values were applied in a modifi- September, 1955 Forsberg: Fus.arium Disease of Gladiolus 495 cation of the formula used in determina- tion of rot severity indexes in the labora- tory tests. Complete decomposition of the corms was considered the most severe re- sult of the disease and was assigned a value of 3. Brown rot and vascular symptoms were considered equal in severity and were assigned a value of 2. Thin basal dry rot was considered less severe and was given a value of 1. Absence of symptoms rated Table 23.—Severity indexes (obtained by use of formula on pajje 496) for the disease pro- duced by 20 isolates of Fiisariiim on four gladiolus varieties in greenhouse tests, 1952.* 496 Illinois Natural History Survey Bulletin Vol. 26, Art. 6 a value of 0. The forimila used was as follows: Disease index = ^^— r X 100 where N'l, No, \:i, N4 = number of corms in each severity class 0, 1, 2, 3 = values assit^ned to severity classes t = total number o\ corms counted The disease indexes obtained from these calculations are shown in tables 23 and 24. The isolates varied in their effects on the test varieties, but the variations did not follow any definite pattern. The results obtained in the inoculation tests show that an isolate does not always produce the same form of the disease. For example, in the 1953 tests, all three forms of the disease appeared in plants inocu- lated with isolates 47-32 and 50-26. In most cases, more than one disease form was obtained from inoculations with a single isolate. A summary of the disease forms produced in plants inocu'ated with the va- rious isolates is given in table 25. Although the same methods were not employed, these results are in general agreement with those obtained by Mc- Clellan (1948), who used mass culture isolates of Fusnrium for inoculations on the Picardy and Dr. F. E. Bennett varie- ties. Some isolates caused a vascular rot, some a surface rot, and others caused a combination of symptoms. McClellan mentioned that the pathogenicity of iso- lates varied from year to year but he did not describe the nature of the variation. DISCUSSION AND CONCLUSIONS The occurrence of different forms of a single fungus in laboratory cultures has been reported by many workers in many parts of the world. The terms "varia- tion," "saltation," "dissociation," "muta- tion," and "sectoring" have been used to designate this phenomenon. Most of the studies of variations in the genus Fusarium have been made on the relation of pathogenicity to forms of growth. Most of the workers have re- ported that cultures of Fusarium vary be- tween a type with abundant aerial growth and a type with all the mycelium appressed in the nutrient substrate. The type of cul- ture having aerial mycelium generally has been found to be most pathogenic, the type having only appressed mycelium least pathogenic, but observers on this point have not agreed unanimously (Armstrong, MacLachlan, & Weindling 1940, Burk- holder 1925). Numerous attempts have been made to account for these variations, but none has been entirely satisfactory. Leonian (1929, 1932) considered different cultural forms merely as phases in an orbit of variation of a species. Hansen (1938) explained varia- bility in many of the Fungi Imperfecti as a "dual phenomenon" resulting from a segregation of genetically different nuclei in a multinucleate mycelium. Hansen Si Smith (1932) earlier concluded that vari- able forms of the Fungi Imperfecti may owe their instability to nuclear heteroge- neity, and that this condition can be brought about by nuclei of one strain en- tering the cells of another strain through anastomoses, and that reassortment of di- verse nuclei can be accomplished by such mechanisms as anastomosis and unequal cell division. Anastomoses, or fusions, between the hyphae and between the germ tubes of va- rious fungi have been described by other workers. Zeller (1926) found conjugat- ing macroconidia of Nectria sanffuinea (Sibth.) Fr. in sporodochia on apple bark. He observed some comparatively long con- jugating tubes connecting cells of one spore with those of another. In each case the nu- cleus had migrated from one cell to the other, producing a binucleate condition. Also, Zeller saw conjugation between two cells of the same spore. He did not discuss the significance of these conjugations. Dickinson (1932) studied hyphal fu- sions in Fusarium fructigenum (Fries) and F. vasinfectum (Atk. ). In no case did he observe fusion between segments (cells) of a conidium and only once did he find fusion between two segments of ad- jacent conidia. He concluded that in these species saltation was due to mutation rather than to heterocaryosis or cytoplas- mic inheritance. September, 1955 Forsberg: Fusarium Disease of Gladiolus 497 That germintitintr spores of the gladi- olus Fusariuin anastomose was observed in the studies reported here, fig. 22, but cyto- logical studies were not made and no spe- cific attempt was made to relate this phe- nomenon to subsequent variation of the isolates. If, however, the interpretation of Hansen (S: Smith (1932) is correct, this phenomenon could account for the \ariants both in form of growth and in pathogenicitv of the gladiolus Fusariu/n. Miller (i945rt, 1945^-, 1946rt, \94bb), in studies of the muskmelon-wilt Fusar- ium and other species, reported that when Table 25.—Disease forms produced by 27 isolates of Fusarium in experiments performeJ in 1, 2, or 3 years. Isolate 498 Illinois Natural History Survey Bulletin Vol.26, Art.6 i \CS^l?'?^!^ Fig. 22.—Anastomoses among germinating spores of the gladiolus Fiisanum: A, macro- spores ; B, microspores. September, 1955 Forsberg: Fusarium Disease of Gladiolus 499 monosporous isolations were made from an isolate derived from a diseased plant, all cultures were alike and of a form that pro- duced abundant aerial mycelium on which conidia, mostly nonseptate, were borne rather sparsely. He called this form the wild type. Cultural variants that were derived from the wild type were consid- ered to be laboratory mutants. Miller con- tended that the taxonomy of Fusariuni should be based only on w^ld types and not on laboratory mutants. The classification of WoUenweber & Reinking (1935) is based on the view that the macroconidia are the spore types on which species descriptions should be based and that those I usaria which do not produce such spores when isolated from nature may be induced to do so by fre- quent cultural transfers. Such transfer- ring gives rise to a cultural state char- acterized by abundant production of macroconidia, a condition that has been called "normal culture," "high culture," "Normkultur," and "Hochkultur." Miller contended that this method results in dis- placement of the wild type by mutants. According to Miller, mo^t of the species de-criptions appearing in the literature have been based on laboratory mutants rather than on natural tvpes. Hence, he considered the classification of WoPen- weber & Reinking unsatisfactorv. The Snyder & Hansen (1940. 1941,' 1945) revision disregards morphological criteria and places primary emphasis on the host relationship. Miller contended that this s'.stem also is unsatisfactory, stating that if proper cultural methods are employed the morphologv cf ihee organisms will be found sufficientlv constant to warrant an attempt at morphological classification. He based this statement on two facts: ( 1 ) his original isolates were aU of the ra'sed type and (2) he was able to main- tain this t\pe by a soil culture technique in which the fungus was kept in a dried, in- active condition for long periods of time. From these two facts he concluded that variants of the types found in laboratory cultures do not occur in nature, or at least that variants are very rare. These conclusions do not agree with the report of Orton (1935), who found that several strains of Fusariuni niveuni Smith may change ;is readily in soil as upon labora- tory media. While an extensive study of the occur- rence of changes in culture form of the gladiolus Fusarium was not made by the writer, such changes were observed, table 10. Since the raised, appressed, and in- termediate forms were obtained in orig- inal isolations of the gladiolus Fusarium, it must be assumed that all of these forms occur in nature and that the wild types of this fungus are not all of one form. The main object of this investigation was to determine if strains of Fusarium obtained from gladiolus corms having dif- ferent disease symptoms could be fitted into well-defined groups on the basis of their pathogenicity and physiological characters. The isolates used in these studies do not fall into we'l-defined groups. Also the pathogenicity tests show that a single isolate is capable of produc- ing more than one form of disease. Evi- dence obtained in the other tests in this investigation shows that the strains of the gladiolus Fusarium are extremely var- iable and some apparently are quite un- stable. No definite pattern for as:ocia- tion of the many variables could be deter- mined ; the variations seem to occur inde- pendently. It was not intended that this paper should enter the controversy on the rela- tive merits of the two available systems of classifying Fusaria. But, as both sys- tems have been used in previous studies of the Fusarium disease of gladiolus, and at least two specific names under one sys- tem have been used for strains of the fun- gus associated with different forms of the disease, it becomes necessary to take a definite stand regarding the nomenclature and taxonomy of the gladiolus Fusarium. Since this investigation failed to establish the existence of well-defined strains of a Fusarium associated with the different forms of the disease, it seems most logical to regard all variants as members of a single species. Because the system of Sny- der & Hansen is better suited for classi- fication of this type of organism, it is pro- posed that all forms of the gladiolus Fu- sarium be included under the name Fu- sarium oxysporum f. gladioli (Massey) Snvder & Hansen. 500 Illinois Natural History Survey Bulletin Vol. 26, Art. 6 SUMMARY Three forms of the Fusarium disease of {zladiolus, known as the vascular, brown rot, and basal dry rot forms, have been described by other workers. The ajient or agents which cause these disease forms have been assigned various specific names, with the result that the exact rela- tion of the different symptom types and their causal agent or agents has been in a state of confusion. The purpose of this investigation was to rectify the confusion by determining if strains of Fusariutn producing different symptoms could be fitted into w^ell-defined groups on the basis of their pathogenicity and physiological characters. From several hundred isolates of Fu- sarium that had been cultured from dis- eased gladiolus corms, 40 isolates were selected for comparison in pathogenicity tests and physiological studies. Compari- sons of isolates from the three disease forms were made by means of their reac- tions to temperature, reactions to aniline dyes, reactions to copper salts, reactions to mercuric chloride, color reactions on steamed rice, growth types on differential media, pH changes produced in liquid media, spore measurements, and tendency to reproduce the same or different disease forms in inoculated plants and corms. The isolates varied a great deal in their reactions in these tests, but no definite pat- tern for association of variables could be determined ; the variations seemed to occur independently. The isolates did not fall into well-defined groups; the isolates from the three disease forms could not be dis- tinguished by any of the tests used. The pathogenicity tests showed that a single isolate is capable of producing more than one form of the disease. The evidence obtained in these studies shows that strains of the gladiolus Fu- sarium are extremely variable and that some of them apparently are quite unsta- ble. It is proposed that all forms of the gladiolus Fusarium be included under the name Fusarium oxysporum f. gladioli (Massey) Snyder & Hansen. LITERATURE CITED Anonymous 1927. Mycological investigations. [Cheshunt] Expt. and Res. Sta. Ann. Rep. 12(1926) :26. [Cheshunt, Herts., England.] Armstrong, G. M., and Joanne K. Armstrong 1950. Biological races of the Fitsarinm causing wilt of cowpeas and soybeans. Phyto- pathology 40(2) :181-93. Armstrong, G. M., J. D. MacLachlan, and R. Weindling 1940. Variation in pathogenicity and cultural characteristics of the cotton-wilt organism, Fusarium vas'inffctum. Phytopathology 30( 6) : 515-20. Bald, J. G. 1953. Control of disease by heat-curing and dipping gladiolus corms. II. 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The species concept in Fusarium. Am. Jour. Bot. 27(2):64-7. 1941. The species concept in Fusarium with reference to section Martiella. Am. Jour. Bot 28(9) :738-42. 1945. The species concept in Fusarium with reference to Discolor and other sections. Am. Jour. Bot. 32(10) :657-66. Ullstrup, Arnold J. 1935. Studies on the variability of pathogenicitv and cultural characters of Gihherella sau- hinetii. Jour. Ag. Res. 51(2): 145-62. VVellman, Frederick L. 1942. Difference in pH relations of some pathogenicallv variable strains of tomato Fusarium. Phytopathology 32(4) :271-87. Wellman, Frederick L., and Dorothy J. Blaisdell 1941. Pathogenic and cultural variation among single-spore isolates from strains of the tomato-wUt Fusarium. Phytopathology 31 (2) :103-20. Wollenweber, H. W. 1913. Studies on the Fusar'um problem. Phytopathology 3(l):24-50. Wollenweber, H. W., and O. A. Reinking 1935. Die Fusarien, ihre Beschreibung, Schadwirkung und Bekampfung. P. Parey, Berlin, viii + 355 pp. Wollenweber, H. W., C. D. SherbakoflF. O. A. Reinking, Helen Jchann, and Alice A. Bailey 1925. Fundamentals for taxonomic studies of Fusarium. Jour. Ag. Res. 30( 9 ) :833—43. Zeller. S. M. 1926. Species of Nectria, Gihherella, Fusarium, CylinJrocurpou and Ramularia occurring on the bark of Pyrus spp. in Oregon. Phytopathology 16(9):623-7. SOME RECENT PUBLICATIONS A.—ILLINOIS NATURAL HISTORY SURVEY BULLETIN. Volume 25, Article 1.—Characteristics of Residual Insecticides Toxic to the House Fly. By Willis N. Bruce. July, 1949. 32 pp., frontis, + 14 figs., bibliog. Volume 25, Article 2.—Effect of Permanent Flooding in a River-Bottom Timber Area. By Lee E. Yeager. August, 1949. 34 pp., frontis. + 21 figs., bibliog. Volume 25, Article 3.—Canada Geese of the Mississippi Flyway, with special reference to an Illinois flock. By Harold C. Hanson and Robert H. Smith. March, 1950. 144 pp., frontis. + 82 figs., bibliog. 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