Bulletin STATE OF ILLINOIS DwiGHT H. Green, Governor DEPARTMENT OF REGISTRATION AND EDUCATION Frank G. Thompson, Director NATURAL HISTORY SURVEY DIVISION Theodore H. Frison, Chief olume 23 BULLETIN Article 4 Wetwood of Elms J. CEDRIC CARTER Printed by Authority of tlic State of Illinois URBAN A, ILLINOIS August 1945 STATE OF ILLINOIS DwiGHT H. Green, Governor DEPARTMENT OF REGISTRATION AND EDUCATION Frank G. Thompson, Director BOARD OF NATURAL RESOURCES AND CONSERVATION Frank G. Thompson, Chairman William Trelease, D.Sc, LL.D., Biology* Arthur Cutts Willard, D.Eng., LL.D., Ezra J. Kraus, Ph.D., D.Sc, Forestry President of the University of Illinois L. R. HowsoN, B.S.C.E., C.E., Engineering Norman L. Bowen, Ph.D., Geology Roger Adams, Ph.D., D.Sc, Chemistry NATURAL HISTORY SURVEY DIVISION Urbana, Illinois Scientific and Technical Staff Theodore H. Prison, Ph.D., Chief Florence A. Nyberg, Assistant to the Chief Section of Economic Entomology G. C. Decker, Ph.D., Entomolgist M. D. Farrar, Ph.D., Research Entomolo- gist J. H. Bigger, M.S., Associate Entomologist S. C. Chandler, B.S., Southern Field Ento- mologist James W. Apple, M.S., Northern Field Entomologist B. G. Berger, M.A., Assistant Entomologist John M. Wright, B.A., Assistant Ento- mologist (on leave) H. B. Petty, Jr., M.A., Associate in Ento- mology Extension C. J. Weinman, Ph.D., Special Research Assistant Willis N. Bruce, B.S., Special Research Assistant Section of Insect Survey H. H. Ross, Ph.D., Systematic Entomologist Carl O. Mohr, Ph.D., Associate Entomolo- gist, Artist (on leave) B. D. Burks, Ph.D., Assistant Entomolo- gist (on leave) Milton W. Sanderson, Ph.D., Assistant Entomologist Kathryn M. Sommerman, M.S., Artist, Entomological Assistant Phyllis A. Beaver, Laboratory Assistant Section of Forestry James E. Davis, M.F., Extension Forester Section of Aquatic Biology George W. Bennett, Ph.D., Limnologist D. F. Hansen, Ph.D., Assistant Zoologist Paul G. Barnickol, M.A., Ichthyologist Bruno von Limbach, M.S., Special Re- search Assistant Section of Game Research and Management R. E. Yeatter, Ph.D., Game Specialist Section of Wildlife Experimental Areas Arthur S. Hawkins, M.S., Game Tech- nician (on leave) F. C. Bellrose, Jr., B.S., Assistant Game Technician Harold C. Hanson, M.S., Assistant Game Technician Section of Applied Botany and Plant Pathology L. R. Tehon, Ph.D., Botanist J. C. Carter, Ph.D., Assistant Botanist G. H. BoEWE, M.S., Field Botanist J. L. Forsberg, M.S., Research Pathologist Bessie B. Henderson, M.S., Research Assist- ant Section of Publications James S. Avars, B.S., Technical Editor Eleanor G. Wolff, B.Ed., Assistant Tech- nical Editor Technical Library Marguerite Simmons, M.A., M.S., Techni- cal Librarian Consultants in Herpetology: Howard K. Gloyd, Ph.D., Director of the Museum, Chicago Academy of Sciences ; Clifford H. Pope, B.S., Curator of Amphibians and Reptiles, Chicago Natural History Museum Deceased January 1, 1945. Thit paper ia a. contributionfrom the Section of Applied Botany and Plant Pathology. (81409—2M—4-45) PREFA CE THE results which Dr. J. Cedric Carter reports in this article of the Bulletin OF THE Illinois Natural History Survey are the outcome of six consecutive years, 1939 through 1944, of study in the field, greenhouse and laboratory of a complex of disease manifestations in the American elm. The elements of this complex, singly and in /arious combinations, have puzzled both practical and scientific tree experts for many .ears. Dr. Carter has demonstrated that there s a direct relationship between an obscure lisease of the heartwood of living elms, :ommonly called wetwood, and certain orms of twig and branch dieback, "bleed- ng" from crotches and wounds, and the wo conditions known to tree experts as nternal slime flux and external slime flux. !4e has isolated a species of bacterium, ipparently heretofore undescribed and un- lamed, which can produce elements of this lisease complex upon being inoculated into lealthy trees. Although supporting material and ob- ervations were gathered in many places in Uinois, the major part of the field work onnected with this investigation was done n the Village of Hinsdale, in Du Page "ounty, Illinois. Much of the equipment lecessary for some of the studies and certain 'materials, such as fertilizers, were furnished y the Village, along with labor and other ;elp, as occasion demanded. Many of the lustrations in this paper show phases of 'lese Hinsdale studies. Mr. Robert S. Hopkins, Commissioner of Public Works for the V'illage, furthered the work in all possible ways. With his approval, Mr. William Ellsworth Rose, as Forester, took an enthusiastic interest in the work from 1939 through 1941, and Mr. Joseph F. Shafer, Assistant Commissioner of Public Works, showed an equal interest from 1942 through 1944. These men gave generously of their time, assisting personally and assigning men employed under them to various tasks as need arose. Among such tasks may be mentioned the collecting of wood, gas and sap samples for laboratory testing, the felling and cutting up of certain trees, the periodical recording through 2 years of the readings of gauges installed in trees, the feeding of several trees and the setting up of a number of experiments. Also, through their reports to the Village President and Board of Trustees, Mr. Rose and Mr. Shafer kept the officials and citizens of Hinsdale informed regarding the progress of the investigation. To acknowledge all of this help is a pleasure. The elements of personal and public interest did much to make Dr. Carter's study complete and exact in many details. Moreover, the cooperation between officials, of the Village of Hinsdale and scientists of the Illinois Natural History Survey, cordially maintained throughout, provides an example showing how com- munities in Illinois can proceed toward the solution of tree problems requiring the services of highly specialized scientists. T. H. J'RisoN, Chief Illinois Natural History Survey. CONTENTS Page Acknowledgments 407 Review of Literature 407 Hosts and Distribution 408 Trunk Pathology 409 Wood Discoloration 409 Gas 410 Sap 410 Root Pathology 413 Branch Pathology 414 Foliage Pathology 416 Dissected Elm 417 Isolations 418 Histology 419 The Wetwood Organism 420 Morphology 420 Cultural Characters 420 Biochemical Reaction 422 Taxonomy 422 Erwin'ta nimipressuralis New Species 423 Inoculations 423 ' American Elm, Greenhouse Inoculations 423 I Siberian Elm, Greenhouse Inoculations 424 Toxicity Tests for Wilt 425 Laboratory Tests on Cut Shoots 425 Field Tests on 2-Year-Old Elms 426 Greenhouse Tests on 2-Year-Old Elms 427 Greenhouse Tests on 7- to 12-Foot Elms 429 Pressures in Affected Elms 430 Field Studies 430 Greenhouse Studies 433 3as Analysis 436 Sap Analysis 437 The pH of Sap 438 Control Studies 439 Helione Injections and Feeding in 1940 439 Urea Feeding in 1941-42 439 Drains Installed in 1942 440 Injections in 1941 and 1942 440 VllSCELLANEOUS EXPERIMENTS 441 Injections of Toxic and Stimulatory Materials 441 Distribution of Malachite Green in Elms 441 Soil Tests 443 Elm Seeds 444 Growth Associations 444 onclusions 444 nummary 445 iterature Cited 446 American elm on the University of Illinois campus. The American elm, Ulmus amerkana L., unsurpassed ij beauty and grace, is the most popular and widely plante| shade tree in the Middle West. Wetwood of Elms J. CEDRIC CARTER THE investigation of wetwood reported in this paper is the out- growth of studies of an unusual wilt observed on a large number of American elms in the Village of Hinsdale, Assistant Professor of Bacteriology at the University of Illinois, made many helpful suggestions in connection with the identi- fication of the bacterium associated with wetwood. Preliminary analyses of sap Illinois, in July and August, 1939. As from wetwood-affected elms were run by studies of the wilt progressed, it became Dr. F. F. Weinard, Associate Professor apparent that this wilt was a secondary of Floricultural Physiology at the Univer- manifestation of disease intimately related to slime flux, a chronic bleeding at crotches and wounds, to abnormally high sap pressure in the trunk, and to wetwood, a water-soaked, darkly discolored condition of the heartwood. The investigation has shown that all of these conditions—wilt- sity of Illinois. Mr. O. T. Lay, in charge of the city office of the United States Department of Commerce Weather Bu- reau at Chicago, furnished the monthly meteorological summaries for Chicago. Photographic illustrations for the frontis- piece and for figs. 19, 20, 21 and 22 were ing, high sap pressure and water-soaked, prepared by Mr. Ray R. Hamm, Manager discolored wood—are phases of the wet- wood disease, and that a bacterium, described as a new species, can cause wetwood in elms. Acknowledgments The author wishes to express his sincere appreciation to all those who have assisted in any way during the course of this in- I'estigation. Special recognition is given to the Village of Hinsdale foresters — Mr. W. E. Rose through 1941 and Mr. J. F. Shafer since 1942—for their assist- ance with the portion of the investigation that was carried out at Hinsdale. Elms affected with wetwood on Hinsdale park- ways were used in parts of this investiga- tion. Permission to use these trees was arranged with the government of the V^illage of Hinsdale through Mr. Robert S. Hopkins, Commissioner of Public Works, and much of the labor, equipment and material used in treating and examin- ing them was furnished by the Village. Dr. O. W. Rees and Dr. G. C. Finger, chemists of the Illinois State Geological Survey, gave many suggestions in regard to methods of analyzing sap and gas taken from affected elms. Some gas samples were analyzed by Mr. C. D. Lewis, Assistant Chemist of the Illinois State Geological Survey. Dr. F. M, Clark, of the University of Illinois photographic and blueprinting laboratory. Mrs. Lucile Rogers Carter assisted in many ways in the preparation of this manuscript. The author is indebted to Dr. L. R. Tehon, head of the Section of Applied Botany and Plant Pathology of the Illinois Natural History Survey, for suggestions made during the course of this investigation. Review of Literature A bacterial vascular disease of Lom- bardy poplar, Populus nigra italica Muenchh., was described by Hartley & Crandall (1935). Crandall, Hartley & Davidson (1937) called this disease wet- wood and described it as "a water-soaked condition of the central wood." They reported wetwood in species of Abies, Morus. Platanus, Populus, Prosopis, Pru- nus, Quercus, Salix, Tsuga and JJlmus, and also Elaeagnus cuttings. Crandall, Hartley & Davidson (1937) mentioned that slime flux, in several species, appeared to be a pathological phe- nomenon of wetwood. Dodge (1937), in a discussion of slime flux, stated, "A tree should not be diagnosed as suffering from slime flux unless there is a 'wet wood' condition of the heartwood and unless the bleeding of moisture from the tree issues from this heartwood." He pointed out [407] 408 Illinois Natural History Survey Bulletin Vol. 23, Art. 4 that, in general, moisture is under pressure throughout the heartwood of such an affected tree. However, in a few cases he found pressure to be present only in isolat- ed sections of the heartwood. May (1942) showed that bleeding or fluxing from the heartwood of elms affected with wetwood was independent of sap flow in the sap- wood. He considered slime flux to be one of the manifestations of wetwood. Cran- dall (1943), working with winter-injured Platanus acerifolia Willd. in 1934, found a bacterial infection to be present in wet- wood-affected trunk wood. He suggested that the frost cracks that were present in the trees affected with wetwood had developed during periods of low tempera- ture. The affected trees fluxed freely through these cracks. Large (1944) described a flux of tung tree as alcoholic flux or white slime flux and stated that the disease was confined to the cambial region. He found bacteria and an Actino- mycete-like fungus associated with this type of flux. Most of the work on slime flux before 1935 was concerned mainly with the visible manifestations of the disease on the outside of trees. Ludwig (1886, 1888, 1890) described alcoholic flux or white slime flux of oak, birch, poplar and maple, and brown slime flux of apple, elm, birch, horse chestnut, poplar and oak. He stated that in brown slime flux the sap or slime formed in the wood and broke through the bark, and both the bark and wood soon decayed. He associated Endoniyces magnusii Ludw. with white slime flux, and Micrococcus dendroporthos Ludw. with brown slime flux. Following Ludwig's early work, slime flux was investigated by Hansen (1889) in Denmark, by Holtz (1901) and Stautz (1931) in Germany, by Massee (1897, 1907) and Ogilvie (1924) in England, and by Stone (1916) and Cook (1918) in the United States. These investigators, with the exception of Ogilvie (1924), be- lieved that slime flux was caused by para- sitic organisms. Massee (1897) produced slime flux of apple and plum trees by inoculating them with Micrococcus den- droporthos hudw. Stautz (1931) pointed out that no fungus hyphae were found in affected wood but that bacteria were pres- ent in drops of the sap. Tubeuf & Smith (1897), Ward (1901) and Ogilvie (1924) believed that slime flux was not a parasitic disease but an abnormal physio- logical bleeding associated with normal or abnormal pressures in the tree. Guba (1934, 1942), after reviewing the literature, concluded that slime flux was not a parasitic disease but that it developed because of artificial environ- ment and other mechanical or physiologi- cal conditions. Hosts and Distribution In Illinois, wetwood has been found in American elm, Ulnius americana L., in- cluding the varieties Moline and Little- ford, slippery elm, Ulnius fulva Michx., English elm, Ulnius procera Salisb., and Siberian elm, Ulrnus pmnila L. Trees affected with this disease have been found in 40 towns and 21 counties, as follows: Quincy in Adams County ; Champaign and Fig. 1.—The black dots indicate the location of towns in which wetwood-affected elms have been found. August, 1945 Carter: Wetwood of Elms 409 Urbana in Champaign County ; Martins- ville in Clark County; Mattoon in Coles County; Barrington, Brookfield, Chica- go, Des Plaines, Evanston, Glencoe, La Grange, Northbrook, Oak Park, Park Ridge, Techny and Western Springs in L?ook County ; Timothy in Cumberland County ; Clinton in De Witt County ; Tuscola in Douglas County ; Addison, Elmhurst, Glen Ellyn, Hinsdale, Lombard ind Villa Park in Du Page County; Paris ind Redmond in Edgar County ; \Villo\v Hill in Jasper County; Aurora and St. [^harles in Kane County; Lake Forest in ^ake County ; Ottawa in La Salle County ; Decatur in Macon County ; Bloomington n McLean County; Hamlet in Mercer County; Hillsboro in Montgomery Coun- :y ; Peoria in Peoria County ; Danville in ^'^ermilion County and Rockford in iVinnebago County. Distribution of the 1-0 towns is shown in fig. 1. Trunk Pathology Wood Discoloration.—Wetwood in ;lms is characterized by dark brown dis- coloration that may appear in current- season trunk wood as streaks or broken bands or in several annual rings as narrow to broad streaks or broken bands, fig. 2. The discoloration sometimes extends beyond the current-season wood into the cambial region and phloem, Hg. 3. There it is grayish brown and appears as short streaks or irregular, elongate patches. Discolored wood appears water-soaked. When it is cut, sap oozes out. Fig. 3.—Wetwood discoloration can some- times be found in tiie cambial region and phloem of elms, where it is grayish brown and shows as short streaks or elongate patches. Fig. 4.—Fluxing through a wound made in Fig. 2.—Section of elm trunk affected with the removal of a large branch. Toxicity of the /etwood, showing discoloration of inner wood flux from wetwood-affected elm prevents cal- nd brown streaks in current-season wood. lus formation at the base of the cut. 410 Illinois Natural History Survey Bulletin Vol. 23. Art. 4 Pig. 5.—Fluxing through branch crotches is common in elms affected with wetwood. The flux on this tree, invaded by air-borne organ- isms, has coated the bark below the crotch with slime. In most elms, wetwood is confined to the inner sapwood and heartwood, and there is very little or no streaking in the outer sapwood and no discoloration in the cambial region or phloem. However, in those affected elms that exhibit wilting of twigs and branches, grayish brown dis- coloration can be found in the outer sapwood and occasionally in the cambial region and phloem as well* as in older wood. Gas.—In wetwood-affected tissues, gas is produced in large amounts by the action of the fermenting bacteria on carbohy- drates and other materials in the trunk. This gas, confined in the trunk, causes abnormally high sap pressures to develop. Pressures up to 60 pounds per square inch have been recorded. Sap.—Sap accumulates in abnormally large amounts in affected wood. Because of the abnormally high pressure caused by the gas produced by fermentation, sap frequently is forced out of the trunk Fig. 6.—This elm is fluxing through a crack in the trunk, which originated as a frost crack during the preceding winter. Fluxing through the crack in the bark became apparent by June. August, 1945 Carter: Wetwood of Elms 411 through wounds made b\' the removal of branches, fig. 4, through cracks in crotch- es, fig. 5, and trunks, fig. 6, and through other trunk injuries. This exuding of sap is commonly called fluxing. The sap or flux as it oozes out of diseased wood is colorless to tan but turns dark upon ex- posure to air. When abundant bleeding occurs, the flux flows down the trunk, wetting and soaking large areas of bark, figs. 5 and 6. When it dries, it leaves a light gray to white incrustation on the bark, fig. 7. Ogilvie (1924), who worked J\ Fig. 8.—Crack in elm trunk wood around which a pocket formed between bark and wood. Such pockets develop when sap and gas seep out through cracks in the wood. in England on elm and other trees, suggest- ed that fluxing sap contains calcium car- bonate and forms a white incrustation on the bark upon drying. In some cases, especially when fluxing is prolific or long k-^^K '•'•Wm continued, air-borne bacteria, veasts and ''''•'"^^^"' J^aH fungi contaminate the oozing sap, ferment ^Bjfl/ ^ Gas Analysis Gas produced in wetwood-affected elms was analyzed in the field for carbon dioxide and in the laboratory for carbon dioxide, oxygen, hydrogen, methane, carbon mon- oxide, illuminants and nitrogen. The gas samples analyzed were from trees ranging from 5 to 12 inches d. b. h. Field analyses for the per cent of carbon dioxide in the gas were made by collecting approximate- ly 24 ml. of the gas in Smith fermentation tubes over water and by absorbing the carbon dioxide with N/5 sodium hydrox- ide. The gas samples analyzed in the field were collected in August, October and November, 1942, and in June, July and August, 1943. The analyses tended to show that in August the gas contained more carbon dioxide than it did in pre- ceding or succeeding months. The per cent of carbon dioxide in different trees varied considerably during any one day. There was no correlation between the diameter of the trunk and the per cent of carbon dioxide in the gas. For instance, each of two gas samples collected on August 19, 1943, one from a tree 12 inches d. b. h. and one from a tree 5 inches d. b. h., contained 19 per cent carbon dioxide. Gas was not present in sufficient quantities to be collect- ed for analyzing from December, 1942, to May, 1943. Samples of gas were analyzed in an Orsat apparatus, July 2, 1943, and found to consist of approximately 46.4 per cent methane, 33.8 per cent nitrogen, 14.3 per cent carbon dioxide, 4.5 per cent oxygen and 1.0 per cent hydrogen. Carbon mon- oxide and illuminants were not present. The amount of hydrogen recorded (1.0 August, 1945 Carter: Wetwood of Elms 437 per cent) is very small—less than the probable error in a combustion analysis. MacDougal (1932) reported that marsh gas, composed mostly of methane, or any other inflammable gas had not been found in normal trees. No tests were made in the present in- vestigation to determine whether the gas from the wetwood-affected trees was toxic to foliage of elms. Crocker ( 1931 ), Crock- er, Zimmerman & Hitchcock (1932) and Krone (1937) pointed out that natural gas, which contains about 80 per cent methane, is not highly toxic to trees and other plants. Gas was produced in abundance when the wetwood organism was grown in nutrient broth plus dextrose. The carbon dioxide content of six samples of this gas was determined by absorption with N/5 sodium hydroxide and the hydrogen content by passage of the gas through a palladium tube. The samples contained approximately 44.7 per cent carbon dioxide and 2.4 per cent hydrogen. How- ever, it was found that the amount of carbon dioxide contained in the gas varied with the age of the culture and with the rate of fermentation. More carbon dioxide was produced in young cultures, when the rate of fermentation was most rapid, than in old cultures. Sap Analysis Sap from the trunk wood of elms affect- ed with wetwood was analyzed for cal- cium, chlorides, copper, iron, magnesium, manganese, phosphate, potassium, sulfates, zinc, nitrates, nitrites, ammonia, starch, reducing sugars, indole and erythrodex- trin. Also, different quantities of distilled water in which healthy tissue and wet- wood tissue had been soaked were analyzed for the materials listed above. The meth- ods used in analyzing for these different materials were those given in publications of the Society of American Bacteriologists, Committee on Bacteriological Technic (1939), Merck and Compan\', Inc. (1940) and Connors & Tiedjens '(1941). Many of these methods were supplement- ed by recommendations of Dr. O. W. Rees, Chemist, Illinois State Geological Survey. Sap that accumulates in large amounts in elms affected with wetwood can be collected by tapping the trunks of affected trees. In all healthy elms tapped during this experiment, sap was not present in the wood in sufficient quantities to be collected. Analyses were made of distilled water in which wood samples from healthy trees and from wetwood-affected trees had been soaked. Sapwood and heartwood samples of both healthy and diseased trees were tested separately'. The water in which the wood had been soaked, and which contained the water-soluble materi- als of the wood, is referred to below as leach. All analyses of sap and leach were run in duplicate and most of them were re- peated two or more times. Ash of the sap of diseased elms was obtained by evaporat- ing the liquid and burning out the organic material over low heat. This ash was re- suspended in distilled water and used as a test material. Also, filtrate of the ash suspension was used as a test material. The sap—untreated, filtered, and fil- tered and autoclaved—and the ash suspen- sion filtrate contained a moderate amount of phosphate. In the ash suspension, phos- phate was abundant. Only small amounts of phosphate were present in the leaches from wetwood and healthy heartwood. Potassium was abundant in the sap—un- treated, filtered, and filtered and auto- claved—and in the ash suspension. There was possibly a trace of potassium in the leach from wetwood tissue but none in the leaches from healthy sapwood and heartwood. Magnesium was not present in any materials tested except for possibly a trace in the ash suspension. No nitrites were found in sap from wetwood tissues or in the leach from healthy heartwood. There appeared to be traces of nitrites in the leaches from healthy sapwood and from wetwood tissues. In our analyses, no calcium, chlorides, copper, iron, man- ganese, zinc, nitrates, ammonia, starch, indole or erythrodextrin were found in any of the materials tested. The ash suspension contained carbonates but it did not contain bicarbonates or hydroxides. It would seem from this fact that potassium and phos- phorus were present as carbonates in the materials tested. No reducing sugars were detected in sap or in the leaches from wetwood tissues, healthy sapwood or healthy heartwood. There appeared to be traces of reducing 438 Illinois Natural History Survey Bulletin Vol. 23, Art. 4 sugars in the leaches produced by auto- claving healthy sapwood and healthy heartwood. Ogilvie (1924) found no sugar, or only traces of it, in the clear fluid from "water-soaked wood" of elm. The pH of Sap To' compare the pH of sap from wet- wood-affected elms with the pH of water- soluble materials in the wood and bark of healthy trees and in the wood of trees affected with wetwood, each of the tissues named was soaked in distilled water and the resulting leach, containing water- soluble materials, was tested for its pH. Ten samples of sap and 10 samples of wetwood leach were alkaline, pH 7.9, when tested with indicators in the field. Five samples of leach from healthy wood and five samples of leach from healthy bark were acid, pH 6.3. The distilled water used in these tests was neutral to slightly acid. The pH values of wetwood sap and of wetwood leaches, and of healthy wood and healthy bark leaches, were determined with the Youden apparatus in the labora- tory on several occasions and are given in table 4. Both sap and leach from wetwood- affected elms were alkaline, averaging pH 7.67 and 7.39, respectively. The leaches from healthy wood and bark were acid, averaging pH 6.35 and 5.89, respectively. These data indicate that sap in healthy elm wood and bark is acid, while sap in wood affected with wetwood and also the water- soluble materials from wood affected with wetwood are alkaline. This is in general agreement with the findings of Ogilvie (1924), who reported the brown and red flux from elm to have a pH of 9 or above, and of Crandell, Hartley & Davidson (1937), who found wetwood-affected tissues of elm and other trees to have a higher pH value than either live sapwood or true heartwood. Additional pH tests were made on fil- Table 4.—The pH of wetwood sap and of leaches of wetwood, healthy wood and healthy bark of elms. The readings were obtained with the Youden apparatus, with platinum electrodes. Date Collected July 23, 1941. August 15, 1941. November 12, 1941*. August 11, 1942. Average pH. Tree No. 1 2 3 4 5 6 7 10 11 12 13 14 15 16 17 18 Wetwood Sap 7.43 7.36 7.80 8.34 7.56 7.11 7.87 7.31 7.62 7.36 7.70 8.55 8.00 7.40 7.67 pH Value Wetwood Leach 7 August, 1945 Carter: Wetwood of Elms 439 tered and autoclaved sap from wetwood- affected elms. For these tests a sample of sap was divided into four portions. One portion was untreated, one was autoclaved at 15 pounds for 20 minutes, one was passed through a Berkefeld filter and one was filtered and then autoclaved. The un- treated sap had a pH of 7.40, the auto- claved sap a pH of 8.09, the filtered sap a pH of 6.26 and the filtered and auto- claved sap a pH of 6.33. Autoclaving resulted in an increase in the pH of both the unfiltered sap and the filtered sap. When samples of unfiltered and filtered sap were autoclaved, a brown, fluffy pre- cipitate was formed, \\hich settled to the bottom of the container upon standing. The formation of this precipitate may have accounted for the differences in the pH readings obtained before and after autoclaving. Sap collected from the diseased tis- sues of a wetwood-affected tree is alkaline. Supposedly it is, or contains, the products of the long-continued fermentation of fer- mentable materials present in the tree. In the laboratory, nutrient broth plus dex- trose eventually becomes strongly acid (p. 422) as the result of fermentation by Erivinia nimipressuralis. The difference in pH between fermented sap and fermented medium appears inconsistent. However, Conner, Peterson & Riker (1937) have shown that the crown gall bacterium, Phytomonas tumefaciciis, brings about an acid condition when it is grown with glu- cose as the main source of energy but an alkaline condition when grown in a medi- um that does not contain glucose. The wetwood organism likewise brings about an alkaline condition when it is grown in nutrient broth without dextrose. It would seem, therefore, that fermentation as it occurs in the tree goes on when no sugar or only traces of sugar are present. The validity of this assumption is further em- phasized by our failure to obtain tests for reducing sugars in any of the sap samples, or in any of the leach samples (p. 437) until after they had been autoclaved. Control Studies Control studies, which were started in 1940, included injection of chemicals into trunk wood, fertilization, and installation of trunk drains. The chemicals injected were Helioue, mercuric chloride, copper sulfate, silver nitrate, and 8-hydroxyquin- olin sulfate. The fertilizers used were 10-8-6 commercial fertilizer and urea. Helione Injections and Feeding in 1940.—Twenty elms, 3 to 10 inches d. b. h., were selected for this experiment. Nine of these trees had wilted in 1939 and all 20 in July, 1940. The 20 trees were treated. as follows: (1) Eight trees were injected with Helione—2 trees with a 1 : 200 dilution, 2 with a 1 : 500 dilution, 2 with a 1 : 750 dilution and 2 with a 1:1,000 dilution—and each re- ceived 250 ml. of solution. (2) Eight trees were injected with Helione in the same manner as in (1) but were also fed 10-8-6 fertilizer. (3) Four trees were fed 10-8-6 fertilizer but were not injected with Helione. Helione injections were made during August and September, 1940. Fertilizer was applied during September and Octo- ber by the punch bar meth(xl in amounts of 25, 35 and 40 pounds to 3—i--, 6- and 8-inch trees, respectively. Subsequent to treatment, seven of the eight trees injected with Helione, but given no fertilizer, wilted in 1 or more years: three in 1941, two in 1942 and three in 1944. One of the three trees that wilted in 1944 had wilted in 1941 also. None of the eight trees injected with Helione and given fertilizer wilted in 1941, but five of them wilted in 1942 and one of these five wilted also in 1943 and 1944. Wilt occurred in all four of the trees which were given no injections but were given fertilizer, two in 1941, one in 1942 and one each year, 1941—1944. Only one of the eight injected trees and three of the eight trees that were injected and fed did not wilt during the 4 years following treatment. Under the conditions of this experiment, injecting with Helione, feeding with 10-8-6 fertilizer or combin- ing both treatments did not appear to be effective in preventing wilt. Urea Feeding in 1941-42.—Urea was fed by the punchbar method to 10 elms, 3 to 5 inches d. b. h. Five of these trees were fed 15 pounds of urea each and five were fed 25 pounds of urea each. Each tree received half of its dosage of urea in October, 1941, and the other half in April, 1942. Previous to treatment, eight of the 10 trees had wilted in 1941, 440 Illinois Natural History Survey Bulletin Vol. 23, Art. 4 one in 1939 and one in both 1939 and 1941. Subsequent to treatment, only three of the trees wilted, 1942-1944, one in 1942 and 1944, one in 1943 and 1944 and one in 1944 only. The tree which wilted in 1942 and 1944 had received 25 pounds of urea; the other two had received 15 pounds of urea each. Drains Installed in 1942.—Drains were installed in 10 affected elms, 4 to 6 inches d. b. h, to determine whether provision of an artificial outlet for the ab- normally produced sap and gas in the trunk would prevent wilt. In eight trees the drains were installed on March 24, in one tree on May 13 and in one on June 17. Holes were bored into the trunks at an upward slant to within about 1 inch of the cambium on the opposite side. Sec- tions of pipe were then driven into the holes far enough to be firmly held, but an effort was made not to drive them into the wetwood-affected tissue. Only three of the trees fitted with drains wilted, 1942-1944. One wilted in 1942 and again in 1944, the other two in 1943. The tree which wilted in both 1942 and 1944 fluxed through cracks immedi- ately above and below the drain, and the bark adjacent to these cracks was killed. In the other two trees, it is possible that cracks in the wood, concealed beneath the bark, permitted seepage of toxic sap into current wood, whence it was distributed to branches and leaves, causing wilt. Injections in 1941 and 1942.—In August of 1941, 10 elms affected with wetwood were selected for this experi- ment. All 10 trees were wilting but none of them had wilted in 1939 or 1940. Five of them were injected with mercuric chloride and five with copper sulfate. The five trees injected with copper sulfate were 4 to 6 inches d. b. h. and each tree received 100 ml. of a 1: 1,000 dilution. The five trees injected with mercuric chloride were 3 to 10 inches d. b. h. One 3-inch tree received 50 ml., one 4-inch and two 5-inch trees received 100 ml. each, and one 10- inch tree received 200 ml. of a 1: 1,000 dilution. Each injection was made with a hand syringe, fig. 23, through a hole bored in the tree to within 1 inch of the cambium on the opposite side. One to four such holes were bored in each trunk, the num- ber of holes depending upon the size of the tree: e. g., one hole in a 3-inch tree, four holes in a 10-inch tree. The total dosage for any tree with more than one hole was divided equally among the several holes. From the results of bactericidal tests made in 1942, the amounts of mercuric chloride and copper sulfate injected into these 10 elms in August, 1941, were esti- mated to have been inadequate. The 1942 tests were made during February, March and April, and the following concentra- tions of materials were found to kill the wetwood organism : mercuric chloride, 1 : 150,000; copper sulfate, 1 : 1,000; silver nitrate, 1 : 50,000 ; and 8-hydroxyquinolin sulfate, 1 : 9,000. On J:he basis of this in- formation, the elms injected in 1941 were reinjected with the same chemicals — mercuric chloride and copper sulfate—in June, 1942. Also, additional elms were selected for injection with silver nitrate and 8-hydroxyquinolin sulfate. The amount of material to be injected into each tree was determined in the following manner. First, a wc'od core or boring was obtained with an increment borer, and the diameter of the wood af- fected with wetwood was measured on the core. Then the probable height of the affected wood in the tree was estimated. With these figures, the volume of affected wood was roughly calculated. The amount of chemical injected into each tree was sufficient to give a concentration that would kill the wetwood organism in a volume of water equal to the estimated volume of affected wood. Four elms — three which had wilted in 1940 and 1941 and one which had wilted in 1939 and 1941—were injected with a 1 : 500 dilu- tion of silver nitrate ; two of these trees were each given 200 ml., the third was given 300 ml. and the fourth 500 ml. Three elms which had wilted in 19-10 and 1941 were injected with a 1 : 200 dilution of 8-hydroxyquinolin sulfate; one of these trees was given 300 ml., the second 800 ml. and the third 1,000 ml. After the in- jections were made, the holes were closed with iron set-screws sterilized with 70 per cent alcohol. The five trees injected with mercuric chloride in 1942 did not wilt in that year, in 1943 or 1944. The five trees injected with copper sulfate and the four injected , with silver nitrate did not wilt in 1942 or' 1 August, 1945 Carter: Wetwood of Elms 441 1943, but two trees injected with copper sulfate and two injected with silver nitrate wilted in 1944. Two of the three trees injected with 8-hydroxvquinolin sulfate did not wilt in 1942, 1943 or 1944, but the third tree wilted in 1942 and again in 1943. These tests might be interpreted as indicating that mercuric chloride, copper sulfate, silver nitrate, and 8-hydroxyquin- olin sulfate may be of value in controlling wilt. However, it must be borne in mind that of 284 trees in Hinsdale that wilted, 1939—1943, only 73 wilted in more than 1 year. Also, the wetwood organism was isolated repeatedly, 1942—1944, from the wetwood-af^ected trunk wood of the trees which had received injections in 1941 and 1942, indicating that the organism was not killed throughout the affected wood by any of the materials injected. Later experiments indicated that most of the material injected went into the younger sapwood, especially the current-season wood, and not into the heartwood or older sapwood. Injections of mercuric chloride and copper sulfate made in August, 1941, caused the cambium to die back 0.5 to 1.5 inches around many of the injection holes. Most of these injured areas callused over in 1942. Cambial injury following the in- jections made in 1942 was negligible, since care was taken to prevent the solu- tions from coming in contact with the cambium around the injection holes. Miscellaneous Experiments Injections of Toxic and Stimula- tory Materials.—An experiment was started on July 29, 1942, to determine whether materials toxic to the wetwood organism, as determined by bactericidal tests, would inhibit or kill bacteria in wet- wood-affected trunk wood and subsequent- ly reduce the pressure in the trunk. Also, materials known to stimulate fermentation were injected into other trees to see if pres- sure could be increased. The toxic materials injected were mercuric chloride, 1 : 500, copper sulfate, 1 : 200, silver nitrate, I 1 : 500, 8-hydroxyquinolin sulfate, 1 : 200 and Elgetol, 1 : 40. The stimulatory mate- rials injected were 5 per cent dextrose and nutrient broth plus 1 per cent dextrose. Sterile distilled water was injected as a check material. Each material was inject- ed into a single tree. A one-half inch hole was bored through the heartwood of each tree to be treated, to about 1 inch from the cambium on the opposite side. A gaugecock was installed in each hole. All injections were made with a hand syringe, fig. 23. After the solutions were injected, the gaugecocks were closed to keep these solutions in the trees. Pressures registered immediately be- fore and immediately after the injections were made and during the remainder of the growing season of 1942 and the season of 1943 are shown in table 5. Following the injection of each mate- rial, including distilled water, pressure temporarily increased. However, the pressure in most trees had returned to approximately the initial point within 9 hours after the injections were made. Trunk pressures increased during August and September of 1942 in all trees except the one which was injected with 5 per cent dextrose. Pressure readings obtained from each tree followed the normal pres- sure curve from June to mid October of 1943. Failure of the toxic materials to reduce pressure suggested that these materials either had not become distribut- ed throughout the bacteria-infected wood, that through some chemical change they were not toxic to the bacteria after being injected into the wood or that the quantity of materials was not sufficient to produce the desired result. Distribution of Malachite Green in Elms.—Malachite green was used to study the distribution of materials injected into elms. These tests were made August 31, 1943, on 8-year-old American elm trees growing in our experimental nurs- ery. Methods of supplying the dye were ( 1 ) through a hole bored under dye in the trunk, dye being held against the trunk in a cuplike container that kept the hole submerged while it was being bored and for 10 minutes afterwards; (2) by the hand syringe method, fig. 23 ; (3) by cut- ting the tree and immersing the cut trunk base in the dye; (4) by cutting the leader and immersing the cut end in the dye; and (5) by cutting a branch and immers- ing the cut end in the dye. Two trees were subjected to each method of treatment. Penetration of malachite green through a hole in the trunk submerged below the 442 Illinois Natural History Survey Bulletin Vol. 23, Art. surface of the dye was not extensive but was similar in both trees. In one tree the dye extended in its upward spread from 0.3 inch in 1938 wood to 16 inches in 1943 wood. It extended in its downward spread from 2 inches in 1941 wood to 5 inches in 1943 wood. The dye did not penetrate 1937 and older wood above the hole or 1940 and older wood below the hole. It spread laterally beyond the hole 0.1 inch in 1943 to 1941 wood. With the injection method, penetration of the dye w^as more extensive than in the test described in the preceding paragraph. The dye in one tree extended in its upward spread from 24 inches in 1939 wood to 36 inches in 1942 wood. It extended in its downward spread from 6 inches in 1938 wood to 12 inches in 1942 wood. It spread only in the midsummer and late summer portions of the 1938 wood, and laterally beyond the injection hole from 0.1 inch Table 5.—Pounds pressure per square inch registered by elms injected with various materials. August, 1945 Carter: Wetwood of Elms 443 in 1938 wood to 0.5 inch in 1942 wood. It did not penetrate 1943 or 1937 and older wood. However, 1943 wood was blocked off by the screwed-in syringe. Penetration of malachite green was most extensive in the two trees that had their trunk bases submerged in dye for 3 hours. Both trees were about 18 feet tall. The dye spread to the top of the trunk and into some branches in the 1943 spring and early summer wood but only 9 feet up the trunk in the 1943 late sum- mer wood. It spread upward 8 feet in 1942 wood, 7 feet in 1941 and 1940 wood, 6 feet in 1939 wood and 4 feet in 1938 late summer wood. The dye did not penetrate 1937, 1936 or 1935 wood, or 1938 spring and early summer wood. Evidently, the 1935—1937 wood had become inactive. The 1943 or current-season wood, es- pecially the spring and early summer wood, was the most active in carrying the dye to various parts of the tree. From the cut leader submerged in mala- chite green for 3 hours, the dye spread down the trunk 60 inches in 1943 spring wood, 42 inches in 1942 and 1941 wood and 36 inches in 1940 and 1939 wood; 1939 wood was the oldest in this portion of the trunk. The dye spread into branch- es through the 1943 spring and early summer wood and reached the branch tips and penetrated the leaf petioles. These branches were within 4 feet of the cut on the leader and measured 6 inches to 5 feet long. Penetration of malachite green in a 5- vear-old branch, cut 30 inches from the trunk and immersed for 3 hours, was mainly in 1943 wood. The dye spread from 22 to 24 inches back from the cut in 1942-1939 wood. In 1943 wood, mainly n the spring and early summer wood, it ipread from the base of the immersed branch down the trunk 1 foot and up the :runk 6 feet. The dye, as it spread up the crunk, was carried out into lateral branch- es through the 1943 spring and early •ummer wood. With all methods of injection except :he hand syringe, the distances to which Tialachite green penetrated were greatest n current-season wood, especially that ;0rmed in spring and early summer. The jiye failed to penetrate heartwood, be- :ause, as MacDougal, Overton & Smith ^1929) found, the heartwood of certain Table 6.—Analysis of Hinsdale, Illinois, parkway soil in which elms affected with wet- wood were wilting. 444 Illinois Natural History Survey Bulletin Vol. 23, Art. 4 elm is pH 6.0 to 7.5, according to Spur- way (1941). Elm Seeds.—Seeds were collected from 10 wetwood-affected elms in Hins- dale and planted in the spring of 1941 to ascertain if the disease was transmitted through the seed. Nine of these trees had wilted previous to 1941, three in 1939, one in 1940 and five in both 1939 and 1940. Verticillium had been isolated from the three trees which wilted in 1939 and from the one tree which had not wilted. The seedlings that were obtained grew nor- mally. Growth Associations.—Growth of the wetwood organism in association with four fungi — Verticillium albo-atrum R. & B., Dothiorella ulmi V. & M., Coniothy- rium sp. and Alternaria sp.—was studied because these fungi were found occasional- ly in trees affected with wetwood. Each fungus to be tested was planted at the center of a Petri dish of potato dextrose agar, and each test was run in duplicate. Four days later, the wetwood organism was planted in streaks along two sides and about 20 mm. beyond each growing fun- gus colony. The plates were held 30 days for observation. The wetwood organism was inhibitory to growth of Verticillium and Dothiorella upon contact. However, this inhibition was overcome in 12 days, and thereafter both Verticillium and Dothiorella grew slowly over the bacterial colonies and spread over the agar beyond. The wet- wood organism produced only a slight, temporary inhibition of growth of Conio- thyrium and Alternaria. This inhibition occurred when the fungus and bacterial colonies were in contact with each other. Both Coniothyrium and Alternaria grew through and over the bacterial colonies, and the bacteria spread along the hyphae of both fungi. In each test, the bacterial colonies spread more rapidly from the fungus colony than toward it. Conclusions From wetwood-affected elms in Illinois a bacterium was isolated, and with it the wetwood disease was reproduced experi- mentally. The bacterium is quite similar to Erwinia salicis Day, the watermark disease bacterium, and to Pseudomonas lignicola Westerdijk & Buisman (1929), but because of certain differences it is regarded as a separate species and has been named Erwinia nimipressuralis, new species. Erwinia nimipressuralis inhabits mainly the heartwood and older sapwood of the trunk. Fermentation by this organism in the infected wood liberates gas which, if it does not escape, produces high pressure. Sap accumulates in the infected wood and produces the water-soaked condition which gives rise to the name wetwood. Much of the abnormal gas and sap can be drained out of the affected wood by the installation of drains. Radial cracks may occur, prob- ably during the winter months, in wet- wood-affected trees. These cracks may or may not reach the cambial region. If they extend to the cambial region, they serve as avenues of escape for the sap and the gas generated in the infected wood. Sap and gas flow out through the trunk cracks, killing the surrounding cambium and forming pockets between the bark and wood. The bark external to these cracks may split ; then the sap and gas seep out, and the sap flows down the trunk. In many trees, cracks form in branch crotches, and sap and gas escape through these cracks. Likewise, wounds caused by the removal of branches may allow the sap and gas to escape. The flowing out of the sap and gas through these vents is com- monly called fluxing, and the escaping sap and gas are known as flux. Wilt develops wherever sufficient quan- tities of the wetwood sap are taken up in the current-season wood and carried to the branches. The severity and extent of wilt appear to depend largely upon the amount of toxic sap that is carried into the branches. Some trees show yellowing of leaves and premature leaf drop in July and August without the development of wilt. Trees that wilt may show symptoms any time during July and August; oc- casionally they show symptoms in late June and early September also. Although wilting branches may lose all of their foliage, if no twig or branch dieback oc- curs they may produce a new crop of foliage within 3 weeks. If, however, severe wilt occurs, not only do the leaves wilt and fall but the twigs and branches die,, back to larger laterals. I There is very little spread of wetwoou, into the roots of elms, and usually it does I August, 1945 Carter: Wetwood of Elms 445 not spread below the region of grafting in Moline or other grafted types of elms. In the experimental work reported here, buds, leaves, branch and trunk phloem, trunk cambium and current trunk sap- wood inoculated with the wetwood organ- ism did not become infected. Trunk heart- wood and older sapwood inoculated with fermenting bacteria became infected, and topical wetwood was produced. However, the trees in which the heartwood became infected did not wilt during the 3 years they were under observation. In the trunks of elms affected with wet- wood, high pressures develop, whether the trees wilt or not. These pressures common- ly reach 5 to 30 pounds per square inch, and are highest in trees that do not flux. In trees which flux freely, there usually is very little or no pressure. A pressure of 60 pounds per square inch was recorded in one elm in 1942. This tree fluxed freely in 1943 and, while fluxing, developed a pressure of only 1 to 2 pounds. Pressure is absent in February and March but be- gins to develop in April and early May. It increases until August or September; then decreases until late December or January. Pressures recorded in artificially infected greenhouse elms followed the gen- eral annual pressure cycle observed in naturally infected elms and were too low to be measured with a gauge when the trees were dormant. In artificially infected greenhouse elms, a diurnal pressure cycle was observed. The maximum pressure occurred about 1 1 : 00 A.M. in June, July and August and about 1:00 P.M. in October, and the minimum pressure occurred usually between 7:00 P.M. and 11 :00 p.m. The amount of pres- sure that developed in the greenhouse trees was influenced by air temperature. When : the temperature fell much below the opti- mum temperature range of the wetwood organism—23 to 30 degrees C. or 75.2 to 86.0 degrees F.—pressure in the trees 1 decreased. The pressure recorded in a j greenhouse elm not affected with wetwood never exceeded 21 mm. of water, approxi- mately 0.03 pound per square inch, as I measured by a water manometer, but the maximum pressure recorded in one green- i house tree affected with wetwood was I 14.8 pounds. The diurnal rise and fall of I the pressure curve for a normal tree, of the trunk temperature curve and of the air temperature curve corresponded close- ly- Samples of gas from wetwood-affected elms contained approximately 46.4 per cent methane, 33.8 per cent nitrogen, 14.3 per cent carbon dioxide, 4.5 per cent oxy- gen and 1.0 per cent hydrogen. Carbon monoxide and illuminants were absent. Samples of sap from wetwood-affected elms contained phosphates and an abun- dance of potassium. Although tests were made for them, the following elements and compounds were not found: calcium, chlorides, copper, iron, magnesium, man- ganese, sulfates, zinc, nitrates, nitrites, ammonia, starch, reducing sugars, indole and erythrodextrin. The sap and diseased wood of wetwood- affected elms are alkaline, while both sap- wood and heartwood of normal trees are acid in reaction. Erwinia nimipressuralis has an optimum temperature range of 24 to 30 degrees C, 75.2 to 86.0 degrees F., a maximum tem- perature of 37 degrees C, 98.6 degrees F., and a minimum temperature of 5 de- grees C, 41 degrees F., or less. Summary 1. Our investigation of wetwood of elms was started in 1939. In Hinsdale, 1939-1943, 284 elms wilted because of wetwood infections in their trunks. None of the 284 elms which wilted died, and only 73 wilted in more than 1 year. 2. Wetwood infection has been found in the American elm {Ulmus americana L.) and its cultivated varieties Moline and Littleford, and in the slippery elm (Ulmus fulva Michx.), the English elm {Ulmus procera Salisb.) and the Siberian elm {Ulmus putnila L. ). Samples from 346 elms located in 21 counties of Illinois were cultured, and the wetwood organism was isolated from 239 of them. Of the 346 trees, 292 showed wilt at the time the samples were taken. 3. Histological studies show the wet- wood bacterium to inhabit principally the vessels and ray-cells of the trunk. It does not grow in sufficient abundance to cause general clogging of the conducting tissues and it does not cause disintegration of the tissues it inhabits. 4. Wetwood and flux were produced by trunk wood inoculations with this bacte- 446 Illinois Natural History Survey Bulletin Vol. 23, Art. 4 rium. Wilt was not produced by these in- oculations, nor by inoculations of buds, leaves, shoots or branches. Infection was not obtained by patch grafting with dis- colored inner bark taken from diseased trees. 5. Sap from wetwood caused young trees to wilt when it was introduced into their current-season wood. It appears that grajash brown streaks in the current-sea- son wood are caused by the discolored sap and not by the wetwood organism. The bacterium was not often isolated from wilting branches. 6. Trunk pressures in wetwood-affect- ed elms commonly reached 5 to 30 pounds per square inch, and were highest in trees that did not flux. A pressure of 60 pounds per square inch was recorded in one tree in 1942. Pressures in wetwood-affected elms began to develop in April and early May, increased until August or Septem- ber and then decreased until late Decem- ber or January. Pressures were not detect- ed in February and March. In artificially infecteid greenhouse trees, pressures fol- lowed a diurnal cycle with a maximum between 11:00 a.m. and 1:00 p.m. and a minimum between 7 : 00 and 1 1 : 00 p.m. 7. Gas from affected elms contained methane, carbon dioxide, oxygen, hydro- gen and nitrogen. 8. Sap from affected elms contained phosphates and potassium. 9. The pH determinations showed the sap and discolored wood of wetwood trees to be alkaline, the wood of healthy trees to be acid. 10. The bacterium which causes wet- wood is a fermenting facultative anaerobe of the genus Erwinia. It is named Erwinia nimipressuralis, new species. 11. Control measures tested included feeding with 10-8-6 fertilizer and urea, in- stallation of drains in trunks, and the in- jection of mercuric chloride, copper sul- fate, silver nitrate, 8-hydroxyquinolin sul- fate and Helione. Neither the use of fer- tilizers nor the injection of any of the chemicals appeared to be effective. The installation of drains, however, gave at least temporary control of flux. LITERATURE CITED Beilmann, August P. 1935. The use of instruments in tree diagnosis. Natl. Shade Tree Conf. Proc. 11: 18-26. 1940. An attempt to record internal tree-trunk pressures. Mo. Bot. Gard. Ann. 27(3) : 365-70. Burkholder, Walter H. 1932. Carbohydrate fermentation by certain closely related species in the genus Phytomonas. Phytopath. 22(8) : 699-707. Conner, H. A., W. H. Peterson and A. J. Riker 1937. The nitrogen metabolism of the crown gall and hairy root bacteria. Jour. Ag. Res. 54(8) : 621-8. Connors, Charles H., and Victor A. Tiedjens 1941. Chemical gardening for the amateur. Wh. H. Wise and Company, New York. 225 pp. Cook, Mel. T. 1918. Common diseases of shade and ornamental trees. N. J. Ag. Exp. Sta. Circ. 98. 27 pp. Crandall, Bowen S. 1943. Bacterial infection and decay of the inner wood of winter-injured young London plane trees. Phytopath. 33(10) : 963-4. Crandall, Bowen S., Carl Hartley and R. W. Davidson 1937. Wetwood. (Abstract.) Phytopath. 27(2) : 126. Crocker, William 1931. The effect of illuminating gas on trees. Natl. Shade Tree Conf. Proc. 7: 24-34. Crocker, William, P. W. Zimmerman and A. E. Hitchcock 1932. Ethylene-induced epinasty of leaves and the relation of gravity to it. Boycd Thompson Inst. Contrib. 4(2) : 177-218. Dodge, A. W. 1937. Slime flux. Arborist's News 2(7) : 1-2. August, 1945 Carter: Wetwood of Elms 447 Elrod, R. P. 1942. The frouiw/a-coliform relationship. Jour. Bact. 44(4) : 433-40. Cuba, E. F. 1934. Slime flux. Natl. Shade Tree Conf. Proc. 10: 56-60. 1942. Slime flux of trees. Arborist's News 7(3) : 17-8. Hansen, Emil Chr. 1889. Ueber die in dem Schleimflusse lebender Biiume beobachteten Mikroorganismen. Centbl. f. Bakt. u. Parasitenk. 5(19) : 632-40, (20) : 633-7, (21) : 693-6. Hartley, Carl, and B. S. Crandall 1935. Vascular disease in poplar and willow. (Abstract.) Phytopath. 25(1): 18-9. Holtz, Wilhelm 1901. Beitrag zur Kenntnis der Baumfliisse und einiger ihrer Bewohner. Centbl. f. Bakt., Parasitenk. u. Infektionskrank. Abt. II, 7(4) : 113-28, (5, 6) : 179-89, (7):229- 38, (8) :274-81, (9, 10) : 338-50. Krone, Paul R. 1937. The reaction of greenhouse plants to gas in the atmosphere and soil. Mich. Ag. Exp. Sta. Spec. Bui. 285. 35 pp. Large, John R. 1944. Alcoholic flux or white slime flux of tung trees. U. S. Dept. Ag. PI. Dis. Rep. 28(1) :35-6. Ludwig, F. 1886. Ueber Alkoholgahrung und Schleimfluss lebender Baume, verursacht durch Endomyces Magnusii n. sp. und Leuconostoc Lagerheimii n. sp. Hedwigia 25(5) : 168-72. 1888. Der braune Schleimfluss. Centbl. f. Bakt. u. Parasitenk. 4(11): 323-4. 1890. Zwei parasitologische Mittheilungen. II. Eine profuse Gummose der Eichen. Centbl. f. Bakt. u. Parasitenk. 8( 14) : 424-5. MacDougal, Daniel T. 1932. The pneumatic system of trees. Carnegie Inst. Wash. Yearbook 31: 192-3. 1936. Studies in tree-growth by the dendrographic method. Carnegie Inst. Wash. Pub. 462. 256 pp. 1938. Tree growth. Chronica Botanica Company, Leiden, Holland. 240 pp. MacDougal, Daniel T., J. B. Overton and Gilbert M. Smith 1929. The hydrostatic-pneumatic system of certain trees: movement of liquids and gases. Carnegie Inst. Wash. Pub. 397. 99 pp. MacDougal, Daniel T., and Ear! B. Working 1933. The pneumatic system of plants, especially trees. Carnegie Inst. Wash. Pub. 441. 87 pp. Massee, George 1897. Slime flux. Kew Bui. Misc. Inf. 132:423. 1907. Additions to the wild fauna and flora of the Royal Botanic Gardens, Kew. Saccharomycetaceae. Kew Bui. Misc. Inf. 6:240. May, Curtis 1942. A note on slime-flux in American elm. Arborist's News 7(7) : 52-3. Merck and Company, Inc. 1940. The Merck Index. Fifth edition. Merck and Company, Rahway, New Jersey. 1,060 pp. Ogilvie, Lawrence 1924. Observations on the "slime-fluxes" of trees. Brit. Mycol. Soc. Trans. 9: 167-82. Priestly, J. H. 1930. Studies in the physiology of cambial activity. III. The seasonal activity of the cambium. New Phytol. 29(5) : 316-54. Reynolds, Ernest S. 1939. Tree temperatures and thermostasy. Mo. Bot. Gard. Ann. 26(3) : 165-255. ;Society of American Bacteriologists, Committee on Bacteriological Technic I 1939. Manual of methods for pure culture study of bacteria. Leaflets 2:3-15; 4:3-23; 5:2-20. Seventh edition. Society of American Bacteriologists, Geneva, New York. Spurway, C. H. 1941. Soil reaction (pH) preferences of plants. Mich. Ag. Exp. Sta. Spec. Bui. 306. 36 pp. Stautz, Walter 1931. Beitrage zur Schleimflussfrage. Phytopath. Ztschr. 3(2) : 163-229. 448 Illinois Natural History Survey Bulletin Vol.23jArt.4 Stone, George E. 1916. Shade trees. Mass. Ag. Exp. Sta. Bui. 170. 264 pp. Tubeuf, Karl Freiherr von, and William G. Smith 1897. Diseases of plants induced by cryptogamic parasites. Longmans, Green and Company, New York. 598 pp. Ward, H. Marshall 1901. Diseases in plants. The Macmillan Company, New York. 309 pp. Westerdijk, Johanna, and Christine Buisman 1929. De lepenziekte. Rapport over het Onderzoek Verricht op Vorzoek van de Neder- landsche Heidemaatschappij. Nederland. Heidemaatsch. te Arnhem. 78 pp. •:,.,'.(>,/ . Recent Publications A.—ILLINOIS NATURAL HISTORY SURVEY BULLETIN. Volume 21, Article 6.—Preliminary Investigation of Oak Diseases in Illinois. Bj J. Cedrie Carter. June, 1941. 36 pp., frontis. + 51 figs., bibliog. (Bound witl Article 7.) Volume 21, Article 7.—A Needle Blight of Austrian Pine. By Robert L. Hulbary| June, 1941. 6 pp., frontis. + 3 figs., bibliog. (Bound with Article 6.) Volume 21, Article 8.—Duck Food Plants of the Illinois River Valley. By Frank C| Bellrose, Jr. August, 1941. 44 pp., frontis. + 35 figs., bibliog., appendix. Volume 22, Article 1.—The Plant Bugs, or Miridae, of Illinois. By Harry H. Knight. September, 1941. 234 pp., frontis. + 181 figs., bibliog., index." $1.25. 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September, 1943. 33 pp., frontis. + 24 figs., bibliog. (Bound with Article 6.) Volume 23, Article 1.—The Caddis Flies, or Trichoptera, of Illinois. By Herbert H. Ross. August, 1944. 326 pp., frontis. + 961 figs., bibliog., index. $1.50. Volume 23, Article 2.—Duck Populations and Kill. By Frank C. Bellrose, Jr. Novem- ber, 1944. 46 pp., frontis. + 27 figs., bibliog. Volume 23, Article 3.—Overfishing in a Small Artificial Lake; Onized Lake near Alton, Illinois. By George W. Bennett. May, 1945. 34 pp., frontis. 4- 15 figs., bibliog. I B.—ILLINOIS NATURAL HISTORY SURVEY CIRCULAR. 32.—Pleasure With Plants, By L, R. Tehon. November, 1942. (Second printing, with revisions.) 32 pp., frontis. + 9 figs. 33.—Controlling Peach Insects in Illinois. By S. C. Chandler and W. P. Flint. August, 1939. 40 pp., frontis. + 32 figs. 34.—Rout the Weeds! Why, When and How. By L. R. Tehon. August, 1943. (Third printing.) 47 pp., color frontis. + 13 figs. 35.—Diseases of Small Grain Crops in Illinois. By G. H. Boewe. September, 1939. 130 pp., frontis. + 47 figs. 36.—Planting and C)are of Shade Trees. By J. E. Davis. March, 1941. 23 pp., frontis. + 16 figs. 37.—Outwitting Termites in Illinois. By W. E. McCauley and W. P. Flint. August, 1942. (Second printing.) 23 pp., frontis. + 19 figs. 38.—Windbreaks for Illinois Farmsteads. By J. E. Davis. February, 1942. (Second printing.) 24 pp., frontis. + 19 figs. 39,—^How to Collect and Preserve Insects. By H. H. Ross. May, 1944. (Second printing, with additions.) 55 pp., frontis. + 63 figs. 40.—Control of Roundheaded Apple Tree Borer. By S. C. Chandler and W. P. Flint. April, 1942. 8 pp., 6 figs. C—ILLINOIS NATURAL HISTORY SURVEY MANUAL. 1.—Fieldbook of Illinois Wild Flowers, By the staff. March, 1936. 406 pp., color frontis. + 349 figs., index. $1.50. 2.—^Fieldbook of Illinois Land Snails. By Frank Collins Baker. August, 1939. 166 pp., color frontis. + 170 figs., 8 pis. $1.00. 3.—Fieldbook of Native Illinois Shrubs. By Leo R. Tehon. December, 1942. 307 pp., 4 color pis. + 72 figs., glossary, index. $1,25. List of available publications, about 400 titles, mailefi on request. Address orders and correspondence to the Chief ILLINOIS NATURAL HISTORY SURVEY Natural Resources Building, Urbana, Illinois Payment in the form of U, S. Poat Office money order made out to State Treasurer of Illinois, Springfield, Illinois, must accompany requests for those publications on which a price is set.