Bulletin 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 3 Natural Availability of Oak Wilt Inocula E. A. CURL Printed by Authority of the State of Illinois URBANA, ILLINOIS June 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. Binks, Chairman A. E. Emerson. Ph.D L. H. TiKFANV. Ph.D. , Biology Walter H. Newhouse, Ph.D., Geology Forestry Roger Adams, Ph.D., D.Sc, Chemistry Robert H. Anderson, B.S.C.E., Engineering Llovd Morev, B.A.. B.Mus., C.P.A., LL.D., D.Sc, 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 t. H. Bigger, M.S., Entomologist . 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 Pavl Suranyi, Ph.D., Assistant Entomologist W. H. LiXKMANN, M.S., Assistant Entomologist Ronald H. Meyer, B.S., Assistant Entomologist John W. Matteson, B.A., Field Assistant Robert Snetsinger, M.S., Field Assistant Sl'E E. Watkins, Technical Assistant H. B. Petty, Ph.D., Extension Specialist in En- tomology* Stevenson Moore, III, Ph.D., Extension Specialist 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 Faunistic Surveys and Insect Identification Systematic Entomologist and Ph.D., Associate Tax- Assistant Tax- H. H. Ross, Ph.D. Head Milton W. Sanderson, onomist Lewis J. Stannard, Jr., Ph.D. onomist Philip W. Smith, Ph.D., Assistant Taxonomisl Leonora K. Gloyd, M.S., Laboratory Assistant Thomas E. Moore, M.S., Technical Assistant Bariiara Gutowskv, M.A., Technical Assistant Section of Aquatic Biology George W. Bennett, Ph.D., Aquatic Biologist and Head William C. Starrett, Ph.D., Associate Aquatic Biologist R. W. Larimore, Ph.D., Associate Aquatic Bi- ologist Donald F. Hansen, Ph.D., Assistant Aquatic Biologist Robert D. Crompton, Field Assistant Leonard Durha.m, M.S., Research 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., Associate Plant Pa- thologist G. H. Boewe, M.S., Assistant Plant Pathologist R. J. Campana, Ph.D., Assistant Plant Pathol- ogist J. R. Schneider, Ph.D., Assistant Plant Pa- thologist E. B. Himelick, M.S., Assistant Plant Pathologist Robert A. Evers, Ph.D., Assistant Botanist Rovenia F. Fitz-Gerald, B.A., Technical Assistant Harry J. Krueger, B.S., Research 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., Game Specialist F. C. Bellrose, B.S., Associate Game Specialist H. C. Hanson, M.S., Assistant Game 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 Assislaril* James Opsahl, M.S., Field Assistant* Robert H. Brough, B.S., Field Assistant* Section of Publications and Public Relations James S. Ayars, 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. W'arrick, B.S., B.S.L.S., Technical Librarian Oi.ga E. Griminger, B.A., Assistant Technical Librarian Consultants: Herpetology, Hobart M. Smith. 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 paprr is a ronlrihiilinrt from the Section of Applied Botany and Plant Pathology. (13164—4M—3-55) CONTENTS ackxowlldgments 278 Review of Literature 278 Field Methods 280 Selection of Trees 280 Examination of Trees 281 Classification of Mycelial Mats 281 Methods of Field Sampling 282 Wood and Bark on Forest Floor 284 Insect Collections 284 Weather Data 285 Laboratory Studies 285 Spore Germination 285 Sampling 287 Transporting Samples 289 Germination of Pad Cells 291 Production of Perithecia 292 Longevity of Conidia 292 Treatment of Samples From Nature 296 Conidia 296 Perithecia and Ascospores 296 ^Mycelial Pads 296 Further Treatment of ]\Lats 296 Insect Feeding Tests 297 Field Observatioxs 297 Development of Mats 297 Mats on Felled Trees ^qq Stimulation in Mat Production 3qj Fnvlronmental Conditions. Wounds Mats on Bark on Forest Floor. Decline of Mats 301 302 303 304 Insects Associated With Mats ^q^ Other Agents Associated With Mats 3 jq Ikocula IX Nature 3J2 Sources of Data 311 Availability of Conidia 3 j j Availability of Fertile Perithecia 3J5 Mycelial Pads 3J7 Discussiox 3 1 y Summary 310 Literature Cited 391 i^^ ^^•jhJ^'. Wilt-killed oak trees in a northern Illinois forest area. Dying of trees In "pockets," as illustrated here, indicates that a principal means of spread of the oak wilt fungus from infected to healthy trees is through naturally occurring root grafts. Natural Availability of Oak Wilt Inocula E. A. CURL* IN the past 10 years, oak wilt, caused by Endoconidiophora fagacearum Bretz, has become increasingly important as a destroyer of oak trees in the eastern half of the United States. It was first described about 13 years ago as a fungus disease in Wisconsin (Anonymous 1942), but ear- lier reports of dying oaks indicate that it probably has been present there for the past 20 years. The American oaks, which number about 300 species, are the most important group of hardwoods in North America, furnishing more native timber than any other related group of broadleaved trees (Finlay 1950). In 1948 the net volume of saw timber in Illinois totaled 10.3 billion board feet (King & Winters 1952). The oak species total was 56 per cent of the net board-foot volume. White oak accounted for 21 per cent of the total, and black oak and northern red oak each made up about 10 per cent. The esthetic as well as the commercial value of oaks must be consid- ered in evaluating the economic import- ance of oak wilt. Oaks are prized highly as both shade and ornamental trees. No species of oak yet tested has shown immunity to oak wilt (Kuntz & Riker 1950Z>). Other susceptible species of the family Fagaceae are Casianea mollissima Bl., Chinese chestnut; C. dentata Borkh., American chestnut ; C. sativa Mill., Euro- pean chestnut ; Lithocarpus densifiorus Rehd., tanbark oak; and Castanopsis sem- pervirens Dudley, bush chinquapin (Bretz & Long 1950, Bretz 1952«, Ernst & Bretz * Dr. E. A. Curl, now Assistant Plant Pathologist, Alabama Polytechnic Institute, Auburn, Alabanria, was a Special Research Assistant with the Illinois Natural His- tory Survey at the time he made the study reported here._ The study was made possible through assistance provided by the Forest Preserve District of Cook County, River_ Forest, Illinois. This article is based upon a thesis submitted by the writer to the Graduate College. Univer- sity of Illinois, Urbana, in partial fulfillment of the re- quirements for the degree of Doctor of Philosophy in Plant Pathology. 1953). Once infected, trees in the red oak group die rapidly, most of them within 6 weeks. Trees in the white oak group may die slowly over a period of 1 to 3 years. Since 1942 oak wilt has been reported from 18 states. The results of aerial sur- veys (Fowler 1951, 1952, 1953) con- ducted by the United States Department of Agriculture, Division of Forest Pathol- ogy, since 1951 and various individual re- ports (Bretz 1949, Carter \950b, 1952, Cummins 1949, Elmer et al. 1953, Fergus & Morris 1950, French & Christensen 1950, Strong 1951, Wysong 1949, Young & Bart 1951) indicate a considerable in- crease in wilt in most of these states. In Illinois the disease was first noticed in 1942 (Carter 1950fl). By the end of 1952 it had been found in 54 of the 102 counties in the state. Most of the coun- ties that are still free of the disease are located in areas where oak timber is not abundant. The threat of oak wilt was recognized early in Wisconsin (Anonymous 1942, Henry et al. 1944) and in Iowa (Dietz & Barrett 1946, Dietz & Young 1948), where some of the first research on the disease was conducted between 1942 and 1948. In 1950 the National Oak Wilt Research Committee (Anonymous 1950) was organized at Memphis, Tennessee, for the purpose of supporting research pro- grams in co-ordination with several uni- versities and with the United States De- partment of Agriculture, Division of For- est Pathology. Such programs have now been developed in most of the states in which oak wilt is found. The studies reported herein are in- tended mainly to supply information con- cerning the availability of oak wilt inocula and the relative importance of Illinois en- vironmental conditions at different times of the year on the longevity of the causal [277] 278 Illinois Natural History Survey Bulletin Vol. 26, Art. 3 fungus in nature. It seems that such infcr- mation would be applicable in findinji the means of spread of the disease beyond root graft distances and in selecting and de- veloping ettective control measures. ACKNOWLEDGMENTS The author grateful!) acknowledges the advice and guidance given him during the course of this study by the late Dr. Leo R. Tehon. While the study was being made, Ur. Tehon was Botanist and Head of the Section of Applied Botany and Plant Pathology, Illinois Natural History Sur- vey, and Professor of Plant Pathology, University of Illinois. The author wishes to express sincere appreciation to Dr. J. C. Carter, Plant Pathologist and present Head of the Section of Applied Botany and Plant Patholog)' of the Illinois Nat- ural History Survey, for many helpful suggestions. Others to whom the author wishes to give special recognition for contributions to the progress of the research and the preparation of the data are Mr. Noel B. W'ysong, Chief Forester of the Forest Pre- serve District of Cook County; Mr. H. W. Fox, Forester of the Sinnissippi Forest, Oregon, Illinois; Mr. R. E. Owens, Director of Parks, Peoria, Illi- nois; Mr. Ray R. Hamm, University of Illinois Photographic Laboratory; Dr. M. W. Sanderson, Mr. James S. Avars, Mr. W. E. Clark, Mr. J. W. Curfman, and Mrs. Rovenia M. Fitz-Gerald, all at the time of the study members of the Illinois Natura' History Survey stall. REVIEW OF LITERATURE A short time after the cause of oak wilt was established as a fungus (Anonymous 1942), the conidial stage was named Chalara quercina by Henry (1944), Later Bretz (1951, 1952Z') succeeded in pro- ducing perithecia of the fungus in labora- tory cultures and named this stage Endo- con'.diophora fagacearum. Symptoms of the disease have been adequately described by various workers (Henry & Moses 1943, Henrv et al. 1944, Henrv & Riker 1947, Riker 1948. Young 1949). In 1949 Kuntz & Riker (1950^) dem- onstrated local spread of the disease from diseased to healthy trees through natural root grafts. The means by which the pathogen is transmitted beyond root graft distances has not been determined. While transmission through root grafts is highly important in parts of the Midwest where the disease has become well established, it does not appear to be the primary method of spread in states such as Pennsylvania (Fergus 1953) and Ohio (Young et al. 1953). There many isolated single-tree in- fections occur, indicating long-distance transmission by a spore-carrying vector. For a decade following the identifica- tion of the causal fungus, the form of its fructification in nature was not known. In the summer and fall of 1951 Curl et al. (1952) discovered mycelial mats of En- doconidiophora fagacearutn under the loose bark of diseased oak trees in Illinois. Endoconidia were present in large num- bers on these mats. A little later Stessel & Zuckerman (1953) discovered the ascig- erous stage on mats in nature. Reports of endoconidium- and perithecium-bearing mats in other states (Barnett et al. 1952. Campbell & French 1953, Morris Si. Fer- gus 1952, Staley & True 1952) indicate that this type of growth is common on wilt-killed oak trees. The implication of the possible importance of mats in serving as reservoirs of inoculum from which the unknown vector or vectors might spread the disease is readily understood. C url et aK (1953) described the thick, sclerotium- like pad that usually occupies the center of the mycelial mat, and they succeeded in producing a similar structure in labora- tory cultures. Zixkerman & Curl (1953) presented proof that the pad is a growth form of E. fagacearum and showed that single cells from laboratory-grown pad> were capable of continuing growth. There are many physiological agents that affect the ability of fungus spores to germinate (Gottlieb 1950, Hawker 1950, Lilly & Barnett 1951, Wolf & Wolf 1947). Temperature and moisture have received more attention than other factors since they influence both the germinability of spores and the infection of the host. Many workers (Anderson et al. 1948, Heal'd & Gardner 1914, HeaM & Stud- halter 1915, Ling 1945. McCrea 1931, Rosen & Weetman 1940) have shown that spores of various fungi usually remain I June, 1955 Curl: Oak Wilt Ixocula 279 viable longer under comparatively dry conditions than when kept moist, and the resistance of the spores to extremes of tem- perature is greater under dry conditions. Heat may aftect reproduction by hasten- ing the fungus to maturitv (Lillv i!S: Bar- nett 1951). Weather conditions are known to influ- ence the incidence and relative prevalence of some plant diseases by influencing the availability of inoculum in the field. Ling (1945), working with stripe rust of wheat in China, found that the amount and dis- tribution of rainfall in late winter and spring are most important in determining rust epidemics. On the other hand, An- derson &: Rankin (1914) found that win- ter conditions have little effect on the via- bilit}' of pycnospores of Endothia para- sitica. Wilkins (1938) stated that the age of the spore is probably the most important single factor influencing germination of ascospores of Ustulina vulgaris. By mak- ing collections of ascospores and conidia of L'. vulgaris at intervals during the autumn and winter, he showed that the spores soon lose the power to germinate. Henry (1944) found that the oak wilt fungus grew best within a range of 24 to 28 degrees C. Young (1949) found the optimum range to be 22 to 26 degrees C. The optimum pH range for growth was found by Barnett & Lilly (1952) and "\'oung (1949) to lie between pH 5 and pH 7, and to have limits at pH 3 and pH 9. ^ oung obtained maximum germination of conidia on agar of low dextrose content at 25 to 30 degrees C. Henry obtained best germination of conidia in 1.25 per cent malt solution at 24 degrees C. Little previous work has been reported dealing with germination requirements of asco- spores of the fungus. Bretz (1952^) found that the ascospores germinated rap- idly in 2 per cent dextrose solution at 25 degrees C. Stessel & Zuckerman (1953), using ascospores taken in October from naturally occurring perithecia, obtained 30 per cent germination in 2 per cent dex- trose solution at 25 degrees C. The availability of oak wilt irocula un- der natural conditions and the effects of environmental conditions on longevity of the fungus have received some attention. McLaughlin (Sc True ( 1952) reported sur- vival of conidia of the oak wilt fungus for 173 days on a glass surface when kept at 10 degrees C. in controlled low relative humidities. The survival period was much shorter at temperatures above 25 degrees C. Jewell (1953) found that low tem- peratures and low relative humidity fa- vored the longevity of ascospores of the oak wilt fungus /// vitro. Curl (1953) reported that the greatest concentration of viable, naturally occurring conidia and ascospores of the oak wilt fungus in Illi- nois was found during ALirch, April, and May. Young (1949) demonstrated in Iowa that the fungus lives over winter in trees that become infected late in the sum- mer. Young & Spilker (1952) in Ohio failed to obtain the fungus from lumber that had been cut from wilt-killed oaks and piled during the summer, but isolated the fungus from large twigs, slabs, and stump wedges for a period of 3 weeks. Bretz & Morison (1953) found that the survival of the oak wilt fungus in small- diameter infected twag samples is relatively short at temperatures of 20 to 25 degrees C. and above. Fergus (1953) reported the presence of mycelial mats of the fungus in nature in Pennsyhania from INIarch through November, except in April and July. Morris & Fergus (1952) noted the appearance of mats in early March and found them to be viable a month later. They also observed that two mats collected in May continued to produce new peri- thecia and ascospores for approximately 2 months when kept at 8 degrees C. in a moist chamber. Campbell & French ( 1953) found mycelial mats of the fungus in Minnesota in November after 2yo months of drought. The possible significance of mycelial mats of Endoconidiophora fagacearum in relation to transmission of the disease has been discussed by various workers. Curl et al. (1953) noted the presence of insects, w^hich belonged to three families, beneath the bark of all mat- bearing trees exam- ined in Illinois. Some larvae and adults were seen in direct contact with the fun- gus. True et ah (1952) pointed out that mycelial mats might serve as natural res- ervoirs of oak wilt inoculum. Craighead z 40 FUNGUS MATS I I TEMPERATURE F^ PRECIPITATION DEVIATIONS FROM THE ANNUAL MEAN OCT. NOV. DEC. JAN. FEB. MAR. JUNE JULY Fig. 14.—Monthly mean temperature and total precipitation, both shown as deviations from the annual means, and the numbers of mycelial mats of Endoconidiophora fagacearum found on wilt-killed oak trees in five Illinois study areas, October, 1952, through July, 1953. The bars illustrate the possible influence of monthly temperatures and precipitation on the numbers of mycelial mats of Endoconidiophora fagacearum produced on wilt-killed oaks. For temperature in October and precipitation in May there were no deviations from the means. month on all trees. It can be seen from these figures that mat development was greatly accelerated during April, May, and June. Some of the mats that were found in July were small and dry ; many of them consisted only of pad material. They were found by removing bark from the trees. None of these undeveloped mats bore conidia of Endoconidiophora fagacearum. Fig. 14 suggests the influences of monthly temperature and precipitation on mat formation. The average tempera- tures for May and June, respectively 16 and 22 degrees, approached the optimum temperature (about 25 degrees) for growth of the fungus. The maximum temperatures were 32 degrees in May and 40 degrees in June ; each of these oc- curred on a day near the end of the re- spective month. Although the average temperature for July was only 24 degrees, a number of days during the month had maximum temperatures of 35 degrees. Thermometer readings showed that the temperature under the bark of wilt-killed oaks in unshaded areas was frequently as high as 40 degrees. During the winter months the highest average temperature for any month was 9 degrees, which is too low for rapid growth of the fungus. The total monthly precipitation for the five study areas during the winter months was generally below the annual mean, but during the spring and summer it was av- erage or above. The ripe fruit odor associated with En- doconidiophora fagacearum was most no- ticeable during May, probably because of the presence of a large number of mats on the trees. Only at this time of the year could the odor be detected clearly at a dis- tance of 10 feet from the trunks of mat- bearing trees. Wounds.—The development of mac- roscopic mycelial growth of the oak wilt fungus was sometimes stimulated by wounds made through the bark of trees that were in or nearing the mat-producing stage. This type of growth, noticeable only during March, April, and May in the study areas, was first seen late in March on tree R-7, which had been felled in December. Where small areas of bark had been cut from trees with an ax 2 weeks previously to check on the condition of the wood, dense masses of fresh myce- lium had developed from the edges of the June, 1955 Curl: Oak Wilt Inocula 303 cuts to distances up to 2 feet back under the intact bark. Such patches of mycelium sometimes measured as much as 8 inches in width. Wound-stimulated mycelial growth was most prevalent during April and May. On any tree that had the fungus odor beneath the bark, macroscopic growth could be induced to form merely by lift- ing a section of bark and immediately nailing it back in place. From the trunk of felled tree R-7, six pieces of bark, each measuring approximately 8 by 12 inches, were lifted. On two of the areas from which the bark had been lifted, the bare wood was covered with thin plastic, and the bark was replaced and nailed down securely. On two other areas, the inner bark surfaces were covered with plastic and nailed back over the wood. On two other areas the bark pieces were only lifted and immediately nailed back in place. On the areas where the wood was cov- ered with plastic, abundant growth of Endoconidiophora fagacearum appeared within 2 weeks on the under side of the bark only. Two weeks later this growth was old, but still no mycelium had formed on the plastic-covered wood beneath. On the areas which had the inner surface of the bark covered, growth appeared on both bark and wood, more abundantly on the covered surface of the bark than on the wood. On the areas where bark was simply lifted and replaced, good growth occurred on both bark and wood surfaces. Similar results were obtained on felled tree R-8 and on several standing trees. Wounding not only stimulated free mycelial growth, but it also often hastened the development of typical mats with pads. For typical mats to appear adjacent to previously made cuts was common during April and May. In many cases the pads pushed the bark out at the edges of the cuts, exposing the mats to the outside. Also, mats appeared first on some trees at points where large nails had been driven through the bark in the process of building ladders on the trunks. These observations indicate that mat formation is stimulated by additional air from the outside. Mats on Bark on Forest Floor From November, 1952, through July, 1953, 213 bark pieces and 128 wood pieces were taken from wilt-killed oaks and placed on the forest floor in four Fig. 15.—Mycelial mat of Endoconidiophora fagacearum formed on a piece of bark from a wilt-killed oak while the bark lay on the forest floor. 304 Illinois Natural History Survey Bulletin Vol. 26, Art. 3 study areas. Mycelial mats, fig. 15, ap- peared on only 17 of the bark pieces, table 11. The first such growth known to ap- pear was found on March 24; the last in the early part of April. No visible my- celium of the oak wilt fungus tormed on the wood pieces at any time. This ab- sence of growth on the wood is not ex- plained, as it was shown earlier by Curl et al. (1953) in Illinois that mats of En- doconidiophora fagacearum developed on both bark and wood on the forest floor. All pieces of bark that produced a visible growth of fungus had been placed on the ground 2 weeks before the fungus ap- peared. Most pieces on which the fungus was not visible after 2 weeks were very dry or were overrun by other fungi. Wood and bark placed on the ground after April dried too rapidly to allow the fungus to develop, at least in visible amounts. Decline of Mats The rate of progress of mycelial mats from the immature stage to a deterior- ated condition varied considerably accord- ing to the time of year when the mats ap- peared, table 12. Most immature mats found during November, December, and January required 70 to 84 days to reach a stage of deterioration. Mats that were found in the immature condition during February, March, and April became dete- riorated in about 42 days, and mats that formed in May, June, and July declined very rapidly, sometimes requiring less than 14 days for complete deterioration. Table 11.—The development of mycelial mats of Endoconidiophora fagacearum on bark and wood pieces that were taken from diseased oaks and placed on the forest floor. Month (1952-1953) Bark Number of Pieces Number With Mats Wood Number of Pieces Number With Mats November December. January. . February. March April May June Total. 11 20 22 20 40 27 21 21 213 4 13 17 20 14 14 9 22 13 14 11 128 Table 12.—The rates of development and decline of mycelial mats of Endoconidiophora fagacearum appearing on wilt-killed oaks; as indicated by the condition classes of the mats at 14-day intervals, the rate of development and decline of each mat was influenced by the month in which it first appeared. Month Mat Appeared Condition Class* of Mats on Designated Number of Days After First Appearance Days 14 Days 28 Days 42 Days 56 Days 70 Days 84 Days November. December. January. . . February. . March. . . . April May June July II II II III II II II V V III III III IV III IV V IV IV IV V V V IV IV IV IV V V •Condition class I, immaturf; II, mature; III, aging; IV, declining: V, deteriorating. June, 1955 Curl: Oak Wilt Inocula 305 As field trips to the oak wilt areas were made only every 2 weeks, many mats that were found during the summer were al- ready in the more advanced stages of de- cline when first seen. Mycelial mats that developed on trees in March, April, and May as a result of stimulation by wounds made through bark followed essentially the same rate of de- cline as typical mats produced in these months. Mats that formed on bark pieces on the forest floor dried rapidly, and some of them disappeared entirely within 2 weeks. Table 13.—Numbers of mycelial mats of Endoconidiophora fagacearum on which individual species of insects were found in the 10-month period October, 1952, through July, 1953. Figures within parentheses indicate the numbers of mats examined. Insect Group Number of Mats With Insects Family Anthocoridae . Blattidae. . . . Brenthidae. . Buprestidae. . Carabidae. . . Colydiidae. . . Cucujidae. Collembola (order) , Curculionidae Elateridae Histeridae. Mycetophagidae. Nitidulidae Orthoperidae . Ostomidae Rhizophagidae. Scolytldae Staphylinidae. Tenebrionidae. Trichoceridae . Genus and Species Lyctocoris stalii Parcoblatta sp Eupsalis minuta Agrilus bilineatus, larvae. . Pristodactyla impunctata. . . AnloniiDn parallelopipeduni . Bothrideres geminatus Synchita parvtila Silvanus bidentatus Uleiota dubia Pandeleteitis hilaris Ampedus nigricayis Elater sp Paromalus bistriatus Platysoma lecontei Litargus sexpunctatus Carpophiliis sayi Carpophiliis sp. (1) Carpophilus sp. (2) Carpophiliis sp., larvae Colopterus truncatus Coloptenis semitectus Epuraea iimbrosa Epiiraea terminalis Glischrochilus obtusus GUschrochiliis sangiiinolentus . Glischrochilus sp., larvae Other nitidulid larvae Molamba Jasciata Molamba ornata Tenebroides laticollis Rhizophagus bipunctatits Monarthrum fasciatum Monarthrum mali Xyloteriniis politus Atheta sp Boletobius quaesitor Coproporus ventriciilus Philonthus laetulus Staphylinid larvae Tachinus sp Cynaeus angustus Platydema ruficorne Trichocera sp., larvae Q 3 3 1 8 2 3 1 10 5 1 1 27 4 1 2 2 4 3 17 19 1 11 18 3 3 29 4 53 2 1 2 61 45 2 17 4 1 37 38 34 4 12 32 4 43 42 4 2 14 6 10 11 23 71 12 5 6 12 3 4 3 11 306 Illinois Natural History Survey Bulletin Vol. 26, Art. 3 Insects Associated With Mats The possibility that several insects which are often associated with the fruit- ing mats of Endoconidiophora fagacearuni might spread oak wilt inoculum to wounds of healthy trees was pointed out by Nor- ris (1953) in Iowa and by Dorsey et al. (1953) in West V^irginia. These workers obtained mechanical transmission of oak wilt under experimental conditions with several species of the Nitidulidae. Gris- wald & Neiswander (1953) in Ohio sug- gested that the pomace fly, Drosophila Table 14.—Numbers of mycelial mats of Endoconidiophora jagaceartim in five condition classes* on which individual species of insects were found in the 10-month period October, 1952, through July, 1953. Figures within parentheses indicate the numbers of mats examined. Family Anthocoridae. Blattidae. . . . Brenthidae. . Buprestidae. . Carabidae. . . Colvdiidae. . . Cucujidae. Collembola (order) , Curculionidae Elateridae Histeridae. Mycetophagidae. Nitidulidae Orthoperidae. Ostomidae Rhizophagidae . Scolvtidae Staphylinidae. Tenebrionidae. Trichoceridae . Insect Group Genus and Species Lyctocoris stalii Parcoblatta sp Eupsalis minuta Agrilus bilineatus, larvae . . . Pristodactyla impitnctata. . . Aulonium parallelopipedum . Bothrideres geminatus Synchita parvula Sihanus bidentatus Uleiota dubia Pandeleteius hilaris Ampedus nigricans Elater sp Paromaliis bistriatus Platysoma lecontei Litargus sexpunctatus Carpophilus sayi Carpophilus sp. (1) Carpophilus sp. (2) Carpophilus sp., larvae Colopterus truncatus Colopterus semitectus Epuraea mnbrosa Epuraea terminalis Glischrochilus obtusus Glischrochilus sanguinolentus . Glischrochilus sp., larvae Other nitidulid larvae Molambafasciata Molamba ornata Tenebroides laticollis Rhizophagus bipunctatus Monarthrum fasciatum Monarthrum mali Xyloterinus politus Atheta sp Boletobius quaesitor Coproporus ventriculus Philonthus laetulus Staphylinid larvae Tachinus sp Cynaeus angustus Platydema ruficorne Trichocera sp., larvae Number of Mats With Insects U U 3 11 7 3 11 1 1 12 2 25 2 1 2 13 12 2 6 1 18 8 13 1 5 1 12 18 3 2 9 3 3 2 9 28 5 3 2 2 1 2 U 4 12 5 3 2 11 2 15 38 26 1 1 13 1 2 7 24 15 1 2 2 1 1 13 2 2 2 11 1 7 2-*; 3 17 19 1 11 18 3 3 29 4 53 2 1 2 61 45 2 17 1 4 37 38 34 4 12 32 4 43 42 4 2 14 6 10 11 23 71 12 5 6 12 3 4 3 11 •Condition class I, immature; II, mature; III, aging; IV, declining; V, deteriorating. June, 1955 Curl: Oak Wilt Ixocula 307 melanogaster, which, like the Nitidulidae, is attracted to mycelial mats of the oak wilt fungus and to bleeding wounds on healthy oak trees, might be a vector of oak wilt. In the study reported here, many other insects were found to be asso- ciated with naturally occurring fungus mats. From October, 1952, through July, 1953, at least 40 species of insects belong- ing to at least ZZ genera of 19 families (exclusive of Collembola) were collected from mycelial mats on wilt-killed oak trees in Illinois, tables 13 and 14. Insects associated with mats are not only potential vectors of oak wilt, but they play other roles that affect the life Fig. 16.—Insect activity on naturally occurring mycelial mats. A. Adults of Gliscfirocfiilus ohtusus and larvae of Carpophilus sp. have completely destroyed this mat. B. Bark beetle (Scolytidae) and freshly made hole in fungus mat. C. and D. Holes made through perithecium- bearing mat and pad on wood and bark by adults of the Scolytidae. 308 Illinois Natural History Survey Bulletin Vol.26, Art. 3 Fig. 17.—Damage inflicted by squirrel or squirrels to the bark of an oak tree and the underlying mycelial pad of Endoconidiophora fagaccarum. cycle of the fungus. The destructive feeding habits of some of these insects, particularly the NitiduHdae, on labora- tory cultures of Endoconidiophora foga- cearum were mentioned earlier, page 297. Also, the role of some insects in the de- struction of mats in the field was ob- served, fig. XbA. During the spring three immature mats, which had no insects on them when examined, were covered with clean plastic covers so that no insects could reach the fungus; then the bark, which had been removed for the examination, was re- placed over the mats and nailed securely to the trees. Three other immature mats, which had several individuals of both Glischrochilus obtusus and Colopterus truncatus present, were covered in the same manner. The mats on which no insects had been seen remained in good condition for be- tween 4 and 6 weeks, after which con- tamination by bacteria and fungi other than hndoconidiophora fagacearum was evident. The mats with insects present declined rapidly, reaching a stage of com- plete deterioration in 2 weeks. It was observed that, throughout the spring and summer, mats infested by large numbers of NitiduHdae deteriorated rapidly. Dur- ing the winter months, when insect activ- ity was low, mats lasted much longer. Another role in which insects may be important is that of spermatizing mats with conidia of opposite compatibility groups, as shown experimentally by Leach et al. (1952) with two species of Niti- duHdae and one of Orthropidae. Any of the species of insects listed in table 13, or even mites, might conceivably perform this role. At least 3 of the 40 or more species of insects that were found on mats were present during each of the winter months as well as in the spring and summer, table 13. Several species were prevalent in October and November. During the cold months of January and February only 3 species w^ere constantly associated with the mats ; these were adults of the order Collembola and Glischrochilus obtusus and larvae of Carpophilus sp., which were usually present in large numbers through- out the 10-month period. Although Car- pophilus larvae were often found embed- ded in ice on the mats, many of them sur- vived and, on warm days, fed on the fun- June, 1955 Curl: Oak Wilt Inocula 309 gus. In winter months, Glischrochilus obtusus was present usually only in small numbers of 1 to 10 per mat. In April, large numbers of adult Scoly- tidae were seen running over the bark sur- face or making holes through the bark of diseased oaks that were near the mat-pro- ducing stage. Later, in May and June, many holes made by these beetles were seen in the wood and bark of most of the mat-bearing trees. It was common to find several holes extending through mycelial mats beneath the bark, fig. 165, C. Many of these holes had been made directly Fig. 18.—Mats of Endoconidiopliora fagacearum on which unidentified fungi hav all but the central pads. e overrun 310 Illinois Natural History Survey Bulletin Vol. 26, Art. 3 through the central pads, fig. 16D, and some beetles were found embedded there. Insect activity on mats was greatest during April, May, and June, when more species were found than in any other months. Very few fresh mats were found in July, and insects were seldom found on them. Few attempts were made to determine exact numbers of insects on mats. Mem- bers of the families Nitidulidae, Staphy- linidae. and Histeridae, and of the order Collembola, obviously were much more abundant than any of the others. Ap- proximately 75 to 100 adult Nitidulidae and as many larvae were commonly seen on a single mat. Members of the Staph- ylinidae and of the Collembola, which are much smaller than the Nitidulidae, were even more numerous. Adult insects were found most frequently on class III and class IV mats and larv^ae on class IV and class V mats, table 14. Insects were seldom present on immature mats, as the cracks in the bark over such mats usually were very narrow, barely perceptible openings that could admit only the small- est insects. Small individuals of the Niti- dulidae were often seen making unsuccess- ful attempts to squeeze through these openings. The fact that immature (class I) mats are not so strongly odoriferous as are mats of the mature and aging classes also may account for the presence of fewer insects on the younger mats. Other Agents Associated With Mats Agents other than insects may have been responsible for hastening the decline and deterioration of natural reservoirs of inoculum. Feeding by rodents on mycelial mats of Endoconidiophora fagacearum in Pennsylvania was reported by Morris & Fergus (1952). Squirrels caused consid- erable damage to mycelial mats in Illi- nois (Himelick et al. 1953) during the winter of 1952-53, and new damage of this kind, fig. 17, was seen in May and June of 1953. The rodents seemed to have been interested in only the central pads of young mats, but, to reach the pads, they had torn large holes in the bark, exposing the fungus to other de- structive elements, such as insects, wind, rain, and other fungi. During the summer months, the mat- bearing trees were often exposed to the direct rays of the sun, and temperatures became very high under the bark. The temperatures for one tree at 3:00 p.m. on July 23, 1953, w^ere 32.5 degrees out- side and 41.0 degrees under the bark on the sunny side of the trunk, as determined by actual thermometer readings. On the shaded side, the temperatures were 29.5 degrees outside and 30.5 degrees beneath the bark. No further mat production oc- curred on this tree, and the fungus could not be isolated from the wood. During the winter months, mats were subjected to alternate freezing and thawing. At this time the mats often were continu- ously wet for several days and finally be- came slimy with bacteria and other micro- scopic forms of life. Many mycelial mats were found to be overrun by wood-rotting fungi, fig. 18, ex- cept for the central pad of each, which seemed to repel invasion of these fungi. However, the pads were readily attacked by other fungi. Graphium, in particular, throve well in aging pads, where it en- tangled the pad cells in a thick mass of hyphal strands and produced abundant coremia and slimy masses of spores. Other agents that were commonly asso- ciated with the mats of the oak wilt fun- gus but that were less destructive were mites, nematodes, and Crustacea. Mites were usually abundant during all months on mats in all stages of decline. Nema- todes of the genus Diplogaster* were very abundant on old perithecium-bearing mats. They infested the masses of exuded asco- spores of Endoconidiophora fagacearum, in some cases hundreds in a single mass. These nematodes had oral openings that measured about 5.6 microns, large enough to admit conidia or ascospores of the oak w^ilt fungus. However, attempts to en- tice the nematodes to feed upon the spores of the fungus on the surface of agar were unsuccessful. Crustaceans of the species Porcellio rathkei were sometimes found on mycelial mats that had developed on bark pieces on the forest floor, but they were not observed feeding on the fungus of the oak wilt disease. * Nematode; were identified by Dr. M. B. Linford, De-^q partment of Horticulture, University of Illinois. June, 1955 Curl: Oak Wilt Inocula 311 INOCULA IN NATURE Sources of Data A total of 629 mycelial mats of Endo- conidiophora fagacearum were found be- neath the bark of 27 of the 30 wilt-killed oaks that were thoroughly studied over a 10-month period, table 10. The remain- ing 3 trees, 1 standing white oak, 1 stand- ing bur oak, and 1 felled black oak, did not produce mats. Twenty additional mats were taken from 4 red oaks that were examined only once and that were not included among the thoroughly stud- ied trees. These 20 mats brought the total number of mats found on 31 mat-bearing trees to 649. The figure for mats includes only typical mats with central pads accom- panied by cracks in the bark. The detailed data presented on the fol- lowing pages were obtained from 365, or 56 per cent, of the total number of mats found. Forty-three of these mats were left on the trees, where each was resam- pled at 2-week intervals until it reached a deteriorated condition. The total number of additional samplings* that were made of the 43 mats on the trees was 100. Thus, the data came from 465 samplings of 365 mats, table 15. As each additional sampling of a mat was made at a later * A sampling involved taking three mycelial disks each 7 mm. in diameter from a mat, as described on page 284. time than the one preceding and when the mat was more advanced in its develop- ment or decline, the data obtained from this sampling were treated as if taken from a separate mat. The figures in table 15 represent mats sampled in all five study areas. More samples were taken during the spring and summer than at other pe- riods of the year, because more mats were produced at that time. Also, more mats of classes IV and V than of other classes were sampled, as they were found more often than mats of the other classes. Availability of Conidia The data regarding numbers and ger- minability of conidia obtained from sam- ples of naturally occurring mycelial mats of the oak wilt fungus in their different stages of development and decline during 10 months are presented in table 16. The method of counting and germinating conidia is described in another section, "Treatment of Samples From Nature," page 296. The average number of conidia per mat, as determined from mats of all classes, increased from October to Decem- ber, 1952, at which time the highest con- centration of the 10-month period was reached, fig. 19. The concentration of conidia then decreased steadily during a period of low winter and spring tempera- Table 15.—Numbers of samplings and average sizes (cm.) of mycelial mats of Endoconidio- phora fagacearum in five condition classes* sampled in the 10-month period October, 1952, through July, 1953. Month Class I 312 Illinois Natural History Survey Bulletin Vol. 26, Art. 3 turcs until April, 1^53, when a sharp rise occurred with a rise in monthly mean tem- perature. After April, the number of conidia decreased again, as the monthly mean temperatures increased, and no conidia were found on mats that were sampled in July. It is interesting to note in fig. 19 that the peak in conidium con- centration in December and a rise in April were preceded by months in which precipitation was above the annual mean. The time of the highest average per 14r ^1 CONIOIA I I TEMPERATURE VTA PRECIPITATION DEVIATIONS FROM THE ANNUAL MEAN PRECIP. (INCHES) OCT. NOV. DEC. JAN. FEB APR. JUNE JULY TEMP. CF.) +4--+40 +3--+30 +2--+20 + 10 2.5--48 -10 -2 20 -3 30 Fig. 19.—Monthly mean temperature and total precipitation, both shown as deviations from the annual means, and average numbers of conidia (per ml. of spore suspension prepared from three-disk sampling of each mat) obtained from samples of mycelial mats of Endoconidiophora fagacearum, October, 1952, through July, 1953. For temperature in October and for precipita- tion in May there were no deviations from the means. Table 16.—Average number per mat sample* and per cent germination of conidia taken from naturally occurring mats of Endoconidiophora fagacearum in five condition classesf in the 10-month period October, 1952, through July, 1953. June, 1955 Curl: Oak Wilt Inocula 313 Table 17.—Average number per mat sample* and per cent germination of conidia taken from mycelial mats of Endoconidiopkora jagacearum in five condition classesf in five study areas in the 10-month period October, 1952, through July, 1953. 314 Im-inois Natural History Survey Bulletin Vol.26, Art. 3 cent gerniiiiability of conidia taken from mycelial mats during the lU-month period did not coincide with the time of highest concentration of conidia. The best aver- age germination rate (39 per cent) was obtained from conidia that were collected in April, tig. 20. The next best germina- tion rate was obtained from conidia col- lected in November. A germination rate as high as 80 per cent was not uncommon. The number and germinability of conid- ia that were taken from mats of the same condition class did not vary unexpectedly frt)m one study area to another, table 17, All areas included in the study were in the northern half of the state, and the dis- tance between any two areas was not greater than 165 miles. Pronounced differences were found, in the number and germinability of conidia, between the mats belonging to different condition classes, fig. 21. The highest average numbers of conidia were obtained from mature, or class II, mats. The high- est average per cent of germination oc- curred in conidia from immature, or class I, mats. Both the number and germina- bility of spores changed as the condition of the mats advanced from class I toward class V or deterioration. The data dealing with number of co- nidia on a mat were based upon esti- mates of the average number of spores in 1 ml. of suspension prepared from a 3-disk sampling of the mat. A rough approxi- mation of the number of conidia on an entire mat could be made by using figures obtained from the samples. The number of conidia in a mature mat which meas- ured 12 by 5 cm. with a central pad which measured 4 by 2 cm. was estimated as fol- lows. The average number of conidia from one disk of mat surface 7 mm. in diameter (38 sq. mm.) contained in 1 ml. of a 10-ml. water blank was 350,000 spores or 3,500,000 spores from the entire disk. The area of the mat, after the area of the central pad (which had few or no spores) had been deducted, was 4,100 square mm. The number of conidia on 5 — < o z (rt 3 O 3 o o UJ cc ffi Q 5 z 2 Z I CONIDIA GERMINATION - 50 40 t 30 z 20 10 CLASS I CLASS n CLASS m class cr CLASS 3C Fig. 21.—Average number and per cent germination of conidia of Endoconidiophora fagarearum taken from mycelial mats in five stages of development and decline over the 10- month period October, 1952, through July, 1953; class I, immature; class II, mature; class III, aging; class IV, declining; class V, deteriorating. June, 1955 Curl: Oak Wilt Inocula 315 the entire mat was estimated as being ap- proximately 378,000,000. Another mat which measured 24 by 10 cm., the pad 6 by 2 cm., and which had the same average number of spores on a disk sample as the mat above, was estimated to have 1,650,- 000,000 conidia, more than four times as many as the other mat. From these figures one can appreciate the significance of mat size as well as numbers of mats in accounting for the concentration of conidia in an oak wilt area. It can be seen in table 15, which gives the average sizes of all mats sampled in each month, that the mats of May and June were considerably larger than those of other months. The sizes of the mats sampled in all months ranged from 1 by 1 cm. to 48 by 14 cm. As pointed out earlier, other possible mycelial sources of oak wilt inoculum might be afforded by the padless mycelial mats that form on bark pieces on the for- est floor and by wound-stimulated myce- lial growth on standing and felled trees. The conidial sporulation on such mats and the ability of the spores to germinate appear to be equal to those of typical mats wirh pads, table 18. Availability of Fertile Perithecia The total number of mats found with perithecia during the 10-month study pe- riod was 90, or 23 per cent of 393 mats studied in detail, table 19. Perithecia were never present on class I mats. The month with the highest percentage of perithecium-bearing mats was December and the next highest May. Table 18.—Average number and ^erminability of conidia of Endoconidiophora fagaceantm obtained from wound-stimulated mycelial mats and from mats on bark pieces on the forest floor during 3 months of 1953. 316 Illinois Natural History Survey Bulletin Vol.26, Art. 3 The averaj^c number of perithecia on a presented by months and by mat classes mm. square area of mat surface and the in table 20. Some mature mats had many average germinability of ascospores are young perithecia not exuding ascopores, Table 20.—Avera({e numbers of perithecia on 6-mm. square areas of surface on perithe- cium-bearinii mats of Endoconidiophora fagacearum in four condition classes* and per cent ger- mination of ascospores in the 9-month period October, 1952, throujjh June, 1953. Month October. . . November December. January. . February . March. . . . April May June Class II n 32 25 30 14 8 31 5 0,0 27 t t 36 t Class III 28 86 5 35 48 38 U ^^ 49 34 38 Class IV 1) u 50 16 42 5 3 42 16 Class V -C-C t t 27 16 7 4 25 ^S3 0,0 11 •Condition class II. mature: III, aging: IV, declining; V, deteriorating. tBecause the surface w.is infested with nem.itodes, bacteria, and Graphium sp., it could not be told with certainty whether the ascospores had germinated. OCTOBER NOVEMBER DECEMBER JANUARY FEBRUARY MARCH APRIL MAY JUNE 10 20 30 40 NUMBER OF PERITHECIA 50 10 20 30 40 50 GERMINATION OF ASCOSPORES (PER CENT) Fig. 22.—Average number of perithecia on a 6-mm. square area of mat surface and average per cent germination of ascospores taken from mycelial mats of Endoconidiophora fagacearum, October, 1952, through June, 1953. June, 1955 Curl: Oak Wilt Inocula 317 Table 21.—Per cent of mats of Endoconid- iophora fagacearum with perithecia, average number of perithecia on a 6-mm. square area of mat surface, and average per cent ger- mination of ascospores (465 mat samplings in five condition classes*). 318 Illinois Natural History Survey Bulletin Vol.26, Art. 3 wounds on mat-producing; trees and on the inner sides of pieces oif bark from dis- eased trees while these pieces lie on the moist forest floor. Perithecia were never found on such mats. It was well known before the present study was undertaken that the injection of either conidia or ascospores of Kiido- conidlopliorn f(u/accorum into healthy oaks would result in diseased trees. How- ever, it was not realized that the irregu- larly shaped cells that constitute the pseudoparenchymatous interiors of myce- lial pads are capable of rapid germination and abundant sporulation comparable to that of conidia and ascospores. Like co- nidia and ascospores, the pad cells ger- minate well on plain water agar, but whether they will germinate and produce oak wilt symptoms when injected into healthy oak trees has not been determined. It has been shown that cultures obtained from pad cells are pathogenic. Pad cells do not seem to be adapted for insect trans- mission, but the possibility of their spread by squirrels or woodpeckers is not remote. While woodpecker damage to mycelial mats has not been observed in Illinois, damage caused by the feeding of squirrels is common. The spread of fungus diseases by these and other unusual agents has been reported bv Gravatt & Marshall (1917), Heald & Studhalter (1914), and Talbot (1952). The decline of diseased oak trees and the development and decline of the result- ing mycelial mats seem to follow a pat- tern. The brown streaks that are nor- mally found in diseased trees of the red oak group first become more pronounced, usually on one side of the tree ; then they increase in width and length until a large area of wood is brown. During this proc- ess the bark loses its tight grip on the wood, which begins to emit the charac- teristic amyl acetate or ripe fruit odor of the fungus. Now having "room" to grow, the fungus appears in a macroscopic form, in most cases in a few days. The time re- quired for the conditioning process to occur before mats appear is dependent on the season of the year. In Illinois the first wilt symptoms of the year are seen on trees early in June. Trees wilting at this time decline rapidly and may produce mats late in August or in September. The decline of trees that wilt in the last part of July or in August is retarded by low winter temperatures, and these trees may not produce mats until the following spring or summer, unless the winter is unusually mild. Mycelial mats of Endo- ronidiopliora fagacearuni have not been reported on trees of the white oak group in Illinois. The bark on diseased white oaks is thin and adheres to the wood, even after the trees have reached an advanced stage of decline. No ripe fruit odor was detected in the white oaks studied. In the period of this studv, more mats were found during April, May, and June than in other months. These months were the ones during which large numbers of bark beetles (Scolytidae) w^ere boring hundreds of tiny holes through the bark and wood of wilt-killed trees. Such per- forating of the bark m.av have been in- strumental in bringing about a subcorti- cal aeration that resulted in a condition favorable for rapid mat development. The inducement of mat formation by in- tentional wounding of trees that were nearing the mat-producing condition indi- cated that a supply of air from outside may hasten mat initiation. When the wood surface of mat-producing trees was covered with plastic, the fungus did not grow, at least to a visible form, on the wood but grew abundantly on the uncov- ered inner bark surface and on other areas of uncovered wood. Several factors seemed to be influential in determining the occurrence of mycelial mats and the longevity of inocula in na- ture. Weather conditions affected both the development and decline of the fun- gus. Mat production on individual trees seemed to be accelerated following periods of cool, rainy weather. Macroscopic growth of the fungus continued over a longer period of time during the winter months than during the summer, and ap- parently low winter temperatures, along with a minimum of insect activity, in- creased the longevity of the fungus mats. That the occurrence of new mats in July was rare was due probably to high tem- peratures plus the fact that wilt-killed trees w-ere, by that time, nearing a state of deterioration which favored the growth of various wood-rotting fungi other than Etidoconidiophora fagacearuni. Observa- tions have shown that new mats are sel- dom found during August in Illinois. June, 1955 Curl: Oak Wilt Inocula 319 Apparently the freezing of mats during the winter does not in itself have a marked deteriorating effect on the fungus in na- ture. Laboratory tests showed that a tem- perature of degrees C. favored longevity of the fungus in both humid and dry at- mospheres ; a continuous dry atmosphere was more favorable to the fungus than a continuous humid atmosphere. In the field, the fluctuating temperature and moisture conditions, combined with the effects of mat-invading microorganisms, may account in part for the fact that the germinability of spores collected during the winter months was lower than that of spores collected in spring and early sum- mer. Also, during the winter months, fewer new mats form than in spring and early summer, and it was found that spores from new or immature mats were more highly viable than those from older mats. Perithecia occurred most abun- dantly on aging mats at temperatures of about 16 degrees C. This was the aver- age temperature for the five study areas in May, 1953, the month during which the greatest number of perithecia occurred. The data obtained in this study indi- cate that the greatest inoculum potential of the oak wilt disease may be expected to occur in Illinois during April, May, and June. This indication is made clear by the following facts : ( 1 ) The greatest number of mats was found in May and June, (2) more viable conidia were pres- ent on mat samples taken in April than in other months, (3) the average size of mats that developed was greater in May and June than in other months, (4) the largest number of perithecia occurred on mats in May, (5) ascospores were most highly viable in April and May, (6) pad cells from immature mats were most highly viable and more mats of this class were found in March, April, and May than in other months. It is also interest- ing to note, from the standpoint of possi- ble transmission, that insect activity was greatest on mats during April, May, and June. Insects, particularly the Nitidulidae, in addition to being possible disease vectors and spermatizers of mycelial mats, are probably the most potent destroyers of naturally occurring oak wilt inocula. The feeding of squirrels on mycelial pads ap- pears to be of minor significance in reduc- ing the amount of inoculum on trees, but the possibility of their spreading conidia, ascospores, or pad cells to healthy trees is evident. The perpetuation of the oak wilt dis- ease seems to be dependent not on the abil- ity of the fruiting mats of the fungus to survive long periods of adverse conditions but rather on the continued existence of the fungus in an oak wilt area where new inocula are produced from time to time. As some trees cease to produce mycelial mats, other trees, more recently wilted, continue the process. Thus, an almost con- stant supply of fresh inoculum is present. To control the spread of such a disease beyond root-graft distances, it would seem necessary either to prevent the mac- roscopic fruiting of the fungus, particu- larly during March, April, May, and June, or to prevent the feeding of insects and other possible vectors on the mycelial mats on both standing and felled trees. The possibility of the extensive spread of the oak wilt disease from fungus develop- ment on bark or wood chips on the forest floor seems negligible and might be elim- inated altogether by placing the pieces so that they dry rapidly. SUMMARY Need for information that would be useful in explaining the spread of oak wilt prompted an intensive study of the avail- ability of oak wilt inocula. This study was made in five major wilt areas in Illi- nois and covered the 10-month period from October, 1952, through July, 1953. Results of preliminary laboratory tests showed that a 2 per cent water agar me- dium and an incubation temperature of 28 degrees C. for 36 hours were best for determining the germinability of both conidia and ascospores of the oak wilt fun- gus, Endoconidiophora fagacearum. Good germination was obtained with cells from the interiors of pads of Endo- conidiophora fagacearum mycelial mats that had not started to decline. Mycelial pad cells were found to have a slightly lower optimum temperature for germina- tion than have conidia and ascospores. Germinated pad cells sporulated abun- dantly on water agar. Formation of Endoconidiophora faga- cearum perithecia on a wheat bran-agar 320 Illinois Natural History Survey Bulletin Vol.26. Art. 3 medium was favored by temperatures of 16 and of 25 degrees C Tests in vitro showed that longevity of conidia of Endoconidiophora fagaceorum was favored by low temperatures and a dry atmosphere. Under conditions of high humidity, conidia on artificially grown mats remained viable for 102 days at and at 12 degrees C. Under conditions of low humidity, conidia on similar mats were still viable after 250 days at 0, at 12, and at 16 degrees. Conidia on a mat taken from nature and subjected to dry storage at 16 degrees were still viable after 330 days. Laboratory feeding tests revealed that insects of the family Nitidulidae were ef- fective destroyers of mats of the oak wilt fungus. The rate at which wilt-infected oaks declined and the length of time required for trees to reach a condition favorable for the development of mycelial mats were found to be closely related to the time of incipient wilt. The influence of monthly temperature and precipitation on formation of mats of the oak wilt fungus was suggested by ac- celerated mat development during April, May, and June. The average tempera- tures for May and June were near the optimum temperature for growth of the fungus. The development of macroscopic my- celial growth of Endoconidiophora faga- ceorum was readily induced during March, April, and May simply by mak- ing cuts through the bark of trees that were nearing the mat-producing stage. This experience suggests a strong air re- lationship in mat formation. In March and April, padless mats of the oak wilt fungus formed and grew on 17 of 213 pieces of bark that were taken from diseased oaks and placed on the for- est floor. No macroscopic growth formed on any of 128 wood pieces that were treated in the same manner. The time required for fungus mats on trees to progress from an immature stage to a deteriorated condition varied accord- ing to the season in which the mats first appeared. Mats that first appeared dur- ing the spring and summer declined rap- idly as compared with mats that first ap- peared during the autumn and winter. Insect activity on mats in nature was greatest during April, May, and June, when more species of insects were found than in other months. Members of the families Nitidulidae, Staphylinidae, and Histeridae, and of the order Collembola were more abundant than others. Among other agents commonly associated with mycelial mats were nematodes, mites, bac- teria, fungi of various kinds, and crusta- ceans. A total of 649 typical mycelial mats, each with a pad in the center, were found on 31 selected trees, some of the mats in each month of the 10-month study period. Spore counts from mat samples indicated that the highest concentration of conidia on mats was reached in December, after which there was a steady decrease during a period of low winter and spring tem- peratures until April, when a sharp rise occurred with the rise in temperature. After April the number of conidia de- crea•^ed rapidly until, in July, no conidia could be found on mats. In the laboratory, best germination was obtained from conid- ia that were collected in April and good germination from those collected in Octo- ber, November, and March. The highest average number of conidia was obtained from mature mats but the highest ger- mination rate of conidia was obtained from immature mats. The significance of mat size, as well as numbers of mats, in accounting for the concentration of conidia in an oak wilt area was demonstrated from sample data. A mat that measured 24 by 10 cm. was estimated to contain 1,650,000,000 co- nidia. Twenty-three per cent of 393 mats (which were presumed to be old enough to have perithecia) contained perithecia. Mats with perithecia were found in all months of the study except July, and the highest average number of perithecia per mat sample was found on aging mats in May. The best germination rate was ob- tained with ascospores collected from ag- ing mats in April. Mass transfers of cells from the inte- rior portions of 423 mycelial pads to po- tato dextrose agar showed that cells from immature, mature, and aging mats were viable ; 82 per cent of the pads from im- mature mats gave positive cultures. LITERATURE CITED Anderson, A. L., B. W. Henry, and T. L. Morgan 1948. The effect of temperature and relative humidity upon the viability of the conidia of Pirictilaria oryzae. (Abs.) Phytopathology 38(7) :574. Anderson, P. J., and W. H. Rankin 1914. Endothia canker of chestnut. Cornell Univ. Ag. Exp. Sta. Bui. 347:531-619. Anonymous 1942. Oak wilt a fungus disease. Wis. Ag. Exp. Sta. Bui. 455. Pt. II. 58th Ann. Rep. 75-6. 1950. Industry fights oak wilt. Am. Forests 56(5) :39. Barnett, H. L., and V. G. Lilly 1952. Physiological factors affecting growth and sporulation of Chalara quercina in culture. (Abs.) Phytopathology 42(1) :2. Barnett, H. L., John M. Staley, and R. P. True 1952. Mycelial mats of Chalara quercina on killed oak trees as a potential source of perithe- cia in nature. Phytopathology 42( 10) :531-2. Bretz, T. W. 1949. The present known distribution of oak wilt in Missouri. U. S. Dept. Ag. Plant Dis. Reptr. 33(11) : 437-8. 1951. A preliminary report on the perithecial stage of Chalara quercina Henry. U. S. Dept. Ag. Plant Dis. Reptr. 35(7) :298-9. 1952a. New hosts for the oak wilt fungus, Chalara quercina Henry. (Abs.) Phytopathol- ogy 42(1) :3. \9S2b. The ascigerous stage of the oak wilt fungus. Phytopathology 42(8) :435-7. Bretz, T. W., and W. G. Long 1950. Oak wilt fungus isolated from Chinese chestnut. U. S. Dept. Ag. Plant Dis. Reptr. 34(10) :291. Bretz, T. W., and David W. Morison 1953. Effect of time and temperature on isolation of the oak wilt fungus from infected twig samples. U. S. Dept. Ag. Plant Dis. Reptr. 37(3) :162. Campbell, R. N., and D. W. French 1953. Mycelial mats of oak wilt found in Minnesota during dry weather. U. S. Dept. Ag. Plant Dis. Reptr. 37(4) :243. Carter, J. C. 1950a. Oak wilt in Illinois. U. S. Dept. Ag. Plant Dis. Reptr. 34(3) :81-2. 1950^. Status of oak wilt and elm phloem necrosis in the Midwest. Arborist's News 15(5):45-51. 1952. Distribution and spread of oak wilt in Illinois. U. S. Dept. Ag. Plant Dis. Reptr. 36(l):26-7. Craighead, Frank C, and Caleb L. Morris 1952. A progress report— : Possible importance of insects in transmission of oak wilt. Pa. Forests and Waters 4(6) : 126-9. Cummins, George B. 1949. Oak wilt in Indiana. U. S. Dept. Ag. Plant Dis. Reptr. 33(8) :332. Curl, E. A. 1953. Studies on the availability of oak wilt inoculum in Illinois. (Abs.) Phytopathology 43(9) :469. Curl, E. A., G. J. Stessel, and Bert M. Zuckerman 1952. Macroscopic growth of the oak wilt fungus in nature. (Abs.) Phytopathology 42(1):6. 1953. Subcortical mycelial mats and perithecia of the oak Avilt fungus in nature. Phytopa- thology 43(2) :61-4. Dietz, S. M., and J. W. Barrett 1946. Spread and control of oak wilt. (Abs.) Phytopathology 36(5) :397. Dietz, S. M., and Roy A. Young 1948. Oak wilt— a serious disease in Iowa. Iowa Ag. Exp. Sta. Bui. P91. 20 pp. Dorsey, C. K., F. F. Jewell, J. G. Leach, and R. P. True 1953. Experimental transmission of oak wilt by four species of Nitidulidae. U. S. Dept. Ag. Plant Dis. Reptr. 37(8) : 419-20. Elmer, O. H., I. J. Shields, and C. T. Rogerson 1953. Oak wilt in seven Kansas counties. U. S. Dept. Ag. Plant Dis. Reptr. 37(1) :44. Ernst, Raymond A., and T. W. Bretz 1953. American chestnut susceptible to oak wilt fungus. U. S. Dept. Ag. Plant Dis. Reptr. 37(3) :163. Fergus, Charles L. 1953. Mycelial mats of the oak wilt fungus. Pa. State Col. Ag. Exp. Sta. Prog. Rep. 100. 7 pp. [321] .122 Illinois Natural History Survey Bulletin V^ol. 26, Art. 3 Fergus, Charles L., and C. L. Morris 1950. Oak wilt in Pennsylvania. U. S. Dcpt. Ag. Plant Dis. Reptr. 34(10) :291. Finlay, .Margaret Curtin 1950. The might) oaks. Am. Forests 56(4) : 7-9. Fowler, .Marvin E. 1951. Survevs for oak wilt. V. S. Dept. Ag. Plant Dis. Reptr. 35(2): 112-8. 1952. Oak wilt surveys in 1951. U. S. Dept. Ag. Plant Dis. Reptr. 36(4) : 162-5. 1953. Oak wilt: its destruction and control. U. S. Dept. Ag. Plant Dis. Reptr. 37(2):104-9. French, David W., and Clyde M. Christensen 1950. Oak wilt in Minnesota. U. S. Dept. Ag. Plant Dis. Reptr. 34(3): 82. Gottlieb, David 1950. The physiologj- of spore germination in fungi. Bot. Rev. 16(5) :229-57. Gravatt. G. F., and R. P. Marshall 1917. Arthropods and gastropods as carriers of Cronartium ribicola in greenhouses. Phyto- pathology 7(5) : 368-73. Griswald, C. L., and R. B. Neiswander 1953. Possible insect vectors of oak wilt. Trees 13(4): 18, 22. Hawker, L. E. 1950. Phvsiology of fungi. Universitj' of London Press, Ltd., Bickley, Kent, England. 360 pp. Heald, F. D., and M. W. Gardner 1914. Longevity of pycnospores of the chestnut blight fungus in soil. Jour. Ag. Res. 2:67-75. Heald. F. D.. and R. A. Studhalter 1914. Birds as carriers of the chestnut blight fungus. Jour. Ag. Res. 2:405-22. 1915. Longevity of pycnospores and ascospores of Endotliia parasitica under artificial con- ditions. Phytopathology 5(1) :35-45. Henry. Berch W. 1944. Clialara qtiercina n. sp., the cause of oak wilt. Phytopathology 34(7):631— 5. Henry, Berch W., and C. S. Moses 1943. An undescribed disease causing rapid dying of oak trees. Arborist's News 8(6) :46. Henry, Berch W., C. S. Moses, C. Audrey Richards, and A. J. Riker 1944. Oak wilt: Its significance, symptoms, and cause. Phytopathology 34(7) :636—47. Henry, Berch W., and A. J. Riker 1947. Wound infection of oak trees with Clialara qtiercina and its distribution within the host. Phytopathology 37 ( 10) : 735-4-3. Himelick, Eugene B., Richard D. Schein, and E. A. Curl 1953. Rodent feeding on mycelial pads of the oak wilt fungus. U. S. Dept. Ag. Plant Dis. Reptr. 37(2): 101-3. Hoffman, Paul 1952. Early trials in oak wilt chemotherapy. (Abs.) Phytopathology 42(1) :11. Jewell, Frederick F. 1953. Ascospore longevity of the oak wilt fungus as affected by temperature and humidity. (Abs.) Phytopathology 43(9) :476. King. D. B., and R. K. Winters 1952. Forest resources and industries of Illinois. III. Ag. Exp. Sta. Bui. 562. 95 pp. Kuntz, J. E., and A. J. Riker 1950a. Root grafts as a possible means for local transmission of oak wilt. (Abs.) Phyto- patholog>- 40(1) :16-7. 1950Z'. Oak wilt in Wisconsin. Wis. Ag. Exp. Sta. Stencil Bui. 9:1-9. Leach. J. G., R. P. True, and C. K. Dorsey 1952. A mechanism for liberation of spores from beneath the bark and for diploidization of Clialara f/uercina. Phytopathology 42( 10) : 537-40. Lilly, Virgil Greene, and Horace L. Barnett 1951. Physiology of the fungi. McGraw-Hill Book Company, Inc., New York. 464 pp. Ling, Lee 1945. Epidemiology studies on stripe rust of wheat in Chengtu Plain, China. Phytopathology 35(10) : 885-94. McCrea, Adelia 1931. Longevity of conidia of common fungi under laboratory conditions. Mich. Acad. Sci., Arts, and Letters Papers 13:165-7. McLaughlin, W. D., and R. P. True 1952. The effects of temperature and humidity on the longevity of conidia of Clialara quer- cina. (Abs.) Phytopathology 42(9) :470. McNew, George L., and Roy A. Young 1948. The nature and control of oak wilt. Natl. Shade Tree Conf. Proc. 24:123-30. June, 1955 Curl: Oak Wilt Inocula 323 Morris, C. L., and C. L. Fergus 1952. Observations on the production of mycelial mats of the oak wilt fungus in Penn- sylvania. Phytopathology 42(12) : 68 1-2. Norris, Dale M. 1953. Insect transmission of oak wilt in Iowa. U. S. Dept. Ag. Plant. Dis. Reptr. 37(8) :417-8. Riker, A. J. 1948. The menace of oak wilt. Arborist's News 13(7):53-5. Rosen. H. R., and L. M. Weetman 1940. Longevity of urediospores of crown rust of oats. Ark. Ag. Exp. Sta. Bui. 391. 20 pp. Staler, J. M., and R. P. True 1952. The formation of perithecia of Chalara quercina in nature in West Virginia. Phyto- patholog>' 42(12) :691-3. Stessel, G. J., and Bert M. Zuckerman 1953. The perithecial stage of Chalara quercina in nature. Phytopathology 43(2):65—70. Strong, F. C. 1951. Oak wilt found in Michigan. U. S. Dept. Ag. Plant Dis. Reptr. 35(8) :383. Talbot, P. H. B. 1952. Dispersal of fungus spores by small animals inhabiting wood and bark. British Myco- logical Society Transactions 35:123. True, R. P., J. M. Staley, J. G. Leach, H. L. Barnett, and C. K. Dorsey 1952. Liberation of spores from natural reservoirs facilitates overland spread of oak wilt. (Abs.) Phvtopathology 42(9) :476. Wilkins, W. H. 1938. Studies in the genus Ustuliria with special reference to parasitism. III. Spores-germina- tion and infection. British Mycological Society Transactions 22:47-83. Wolf, Frederick A., and Frederick T. Wolf 1947. The fungi. Vol. II. John Wiley and Sons, Inc., New York. 538 pp. Wysong, Noel B. 1949. Rapid spread of oak wilt in the Midwest. Am. Nurseryman 90(10) :14, 17, 55-7. Young, Roy A. 1949. Studies on oak wilt, caused by Chalara quercina. Phytopathology 39(6) :425^1. Young. H. C., and G. J. Bart 1951. Oak wilt in Ohio. Aerial survey shows general infection in state's timber area. Ohio Farm and Home Res. 36(272) :67-8, 71. Young. H. C., and Oren Spilker 1952. Longevitv of disease organism getting attention in oak wilt project. Ohio Farm and Home Res. 37(279) : 97-8. Young, H. C., G. J. Bart, Oren Spilker, W. H. Brandt, and R. B. Redet 1953. Progress of oak wilt investigations in Ohio. U. S. Dept. Ag. Plant Dis. Reptr. 37(4) :244. Zuckerman, Bert M., and E. A. Curl 1953. Proof that the fungus pads on oak-wilt killed trees are a growth form of Endoconidio- phora fagacearum. Phytopathology 43(5) :287—8. 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. 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