N—I ' OVERDUE FINES: 25¢ per dew per item RETURNING LIBRARY MATERIALS: -————_.____________ ““‘Vl' Place in book return to remove w“ "5" charge from circulation records :54.” [Id-“\\ L Rafi 1 $1,953 335/.) 31*!“ ~. ~. .9 V1.1 ABSTRACT AN EXPLORATORY STUDY ON THE POSSIBILITIES OF THE CONTROL OF OAK WILT WITH CYCLOHEXIMIDE AND ITS DERIVATIVES By James L. Stewart The research reported in this paper was an exploratory effort to determine if cycloheximide or its derivatives could be applied to a tree in a manner that would give systemic control of the oak wilt disease. Water and No. 1 fuel oil were used as carriers for the anti- biotic when applied to the bark of the trees and water was used as the carrier for foliar applications. Penetration of the antibiotics was determined by bio-assays. Foliar applications of Acti-dione (cycloheximide) and five of its six derivatives, namely? oxime, acetate, acetoacetate, semi- carbazone, and thiosemicarbazone ranging from 5 to 50 ppm were made before and after inoculation with the oak wilt pathogen. Cyclo- heximide was the only one which gave some degree of preventive con- trol. Cycloheximide and the same five derivatives at 1 ppm were treated with equal quantities of juice extracted from oak stem wood to determine what effect the juice had on the antibiotics. Bio- assays indicated that the extracts increased the toxicity of thio- semicarbazone and semicarbazone but did not affect the others. This experiment was run three times with somewhat different results. Using fuel oil as the carrier, cycloheximide and the methyl- hydrazone derivative were applied to the bark of small trees by painting or spraying. The bio-assays were negative and it was con- cluded that oil is not a good carrier. Cheese cloths, saturated with Acti-dione in water, were wrapped around the trunks of small trees and if a bark wound was present under the cloth, penetration and uptake of the antibiotic resulted. Phytotoxic symptoms also occurred on these trees. If there were no bark wounds present, penetration apparently did not occur. 'AN EXPLORATORY STUDY ON THE POSSIBILITIES OF THE CONTROL OF OAK WILT WITH CYCLOHEXIMIDE AND ITS DERIVATIVES By James L. Stewart A THESIS Submitted to Midhigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1963 ACKNOWLEDGMENTS The author would like to thank Professor Forrest C. Strong and Professor Miriam C. Strong for their guidance and advice through- out the duration of this work, and for their suggestions during the writing of this thesis. He is also grateful to Dr. Donald J. deZeeuw, Dr. John E. Cantlon, and Dr. Victor J. Rudolph for their careful reading and their criticisms of this thesis. Thanks is also given to the Upjohn Company for financial assistance, materials and advice on methods, and to Dr. Jonathan W. Wright for some of the trees used during this work. Photographic assistance was given by Professor Forrest C. Strong and Mr. William H. Hillerman. These research studies were supported in part by the North Central COOperative Agriculture Experiment Station Project NC-22 oak wilt. ii TABLE OF CONTENTS PAGE INTRODUCTION 1 REVIEW OF THE LITERATURE 6 METHODS AND MATERIALS ' 10 I. Isolation of the Fungus II. Inoculation of trees III. Treatments A. Bark application of methylhydrazone in oil B. Bark application of Acti-dione in oil C. Cone and chisel method D. Bark application of Acti-dione in water E. Foliar application of chemicals in water F. Foliar application of Actispray in water IV. Bio-assay Methods V. Effect of Oak Stem Tissue Liquids on the Fungicidal Properties of Cycloheximide and its Derivatives EXPERIMENTS AND DISCUSSION OF RESULTS 16 I. Inoculations for Observation of Symptoms and Miscellaneous Purposes II. Foliar Applications of Cycloheximide and its Derivatives III. The Effect of Oak Stem Tissue Liquids on the Fungicidal Properties of Cycloheximide and its Derivatives IV. Foliar Application of Actispray in Water V. Bio-assay of Foliar Application of Actispray iii PAGE VI. Bark Application of Methylhydrazone in fuel oil VII. Penetration of the Bark by Chemicals in oil VIII. Penetration of the Bark by Acti-dione in water CONCLUSION 44 LITERATURE CITED 46 LIST OF TABLES PAGE Foliar application of cycloheximide and its derivatives. 19 Results showing effects of oak stem tissue liquids on the fungicidal properties of cyclo- heximide and its derivatives. 22 Effect of foliar application of Actispray, six days before inoculation, on the incidence of oak wilt. 26 Effect of foliar application of Actispray, three days before inoculation, on the incidence of oak wilt. ‘ 27 Effects of Actispray on the foliage. 28 Penetration of the bark by Acti-dione, at 12,800 ppm in oil, when applied to the trunk. 32 Possible penetration of red oak bark by Acti- dione in water. 36 Results of bio-assay of branch tips, cut one day and four days after treatment was begun, from red oak trees treated with Acti-dione in water. 37 Effect of bark wound of red oak on penetration by Acti-dione in water. 40 iv 10. 11. LIST OF FIGURES PAGE Cracked bark of a red oak under which a mat has developed. ‘ 4 Same tree with the bark removed exposing the mat. 4 Inoculated red oak saplings in a portion of the botany experimental field at Michigan State University. 5 A red oak tree treated with the cone and chisel method. 17 Nitidulidae beetles clustered around an inoculation point on a red oak stem. ' 20 Illustrating method of determining effect of oak stem tissue liquids on the antibiotic Acti-dione and its derivatives. 23 Bio-assay of cross sections cut from trunks that were.treated with Acti-dione in oil. .34 Red oak stem showing cheese cloth method of treatment. B 38 Bio-assay method A. 41 Bio-assay method B. 41 Tree number 9 showing phytotoxic effects. 42 INTRODUCTION (41) Although a strange dying of oak had been reported as (1) early as 1927, it was not until 1942 that a specific disease (21) causing the wilting of oak trees was described. In 1944, Henry described the fungus which causes the disease and named it Chalara guercina. Since that time the name has been changed to Endoconidio- (8) phora fagacearum by Bretz and finally to Ceratocystis fagacearum (30) by Hunt. This fungus attacks all of our native species of oak plus all (9,10,11) (7,17) exotic species thus far tested. Species of Castanea (4,42) will also host the fungus and it has infected species 0f.!§lE§ through artificial inoculations. In the red oak species group, death of the tree can be com- paratively rapid as complete wilting has been observed within four weeks after inoculation. In this group, the foliage symptoms usually appear in the upper part of the tree or at the end of lateral branches and progress rapidly downward and through the entire crown. The leaves first become dull, pale green with some curling, and later turn a bronze color starting at the tips and proceeding towards the petiole. Defoliation may be slight.but usually occurs as the wilting progresses. Sucker growth may be stimulated, but these soon wilt. Trees which are infected late in the season may leaf out the next spring, but these leaves soon wilt and die. Dur- ing the following late fall or early spring, mycelial mats may be produced on dead trees between the bark and the sapwood and growth of the mat soon cracks the bark. Conidia and ascospores are pro- duced on these mats. (Figure l and 2). A fermenting apple odor is 2 produced which attracts insects, squirrels, birds, and other animals which may offer a means of spread for the fungus. The fungus progresses more slowly in the white oak group and it may take several years for a white oak to die. Symptoms are usually localized on single branches while other branches remain healthy. No mats have thus far been found on white oak. Often a brown discoloration of the vascular tiSSue accompanies infection. The oak wilt disease is indigenous to the United States and was originally thought to be endemic to the upper Mississippi River Valley. However, its later recognition over much of the eastern deciduous forest region led to governmental and private support for research and control of the disease. Pennsylvania, Maryland, Virginia, North Carolina, Tennessee, Kentucky, West Virginia, Ohio, Michigan, Indiana, Illinois, Wisconsin, Minnesota, Iowa, Missouri, Kansas, Oklahoma, Nebraska, and Texas now report oak wilt locations. Due to this demonstration of wide occurrence, it was recognized in 1950 as a concern of national importance and funds were made available by the U.S. Forest Pest Control Act which enabled the Division of Forest Pathology of the U.S.D.A. to begin limited survey. Research on the problem.was, however, left to the support of private oak-using industries. Ten trade associations representing these industries sponsored a national oak wilt committee which chartered a three-year intensive research program. This aammittee obtained advice and assistance from a group of outstanding plant pathologists led by .AHJ. Riker of the University of Wisconsin. In 1954, federal funds Veere made available through the Regional Research Cooperative pro- ;ject (oak wilt) NC-22 which was revised in 1956; it was with the aid 3 of'a portion of these funds that the research reported in this paper has been carried on. In Michigan, this disease is presently confined to the southern part of the lower peninsula. Its presence was discovered in 1951(39) but to date appears not to have spread extensively. The work at Michigan Agricultural Experiment Station has been mostly centered around attempts to find a chemical control for the oak wilt disease. The field experiments are being carried out at the botany experimental field at Michigan State University. (Figure 3). In Michigan, the spread of the disease has not become as ex- tensive as in some other states, largely because of the excellent control program by the co-Operative efforts of the State of Michigan Departments of Agriculture and Conservation, and Michigan State Uni- versity. This program consists of locating each infected oak and killing it and all trees of the same species group within a fifty- foot radius of the infected tree by frilling and treating with 2 2,4-D ester. This control program has made it impossible to extend any experiments to actual field conditions. Figure 1. Cracked bark of a red oak under which a mat has developed. Figure 2. Same tree with the bark re- moved exposing the mat. The gray raised portion is the sterile pressure pad and the tan area surrounding this on the wood is the fertile mycelium that produce conidia and asco- spores. Figure 3. Inoculated red oak saplings in a portion of the botany experi- mental field at Michigan State University. The brown leaved trees have been inoculated with the oak wilt fungus. REVIEW OF THE LITERATURE Other than some general reports on the finding of oak wilt in various states, and a few papers on research findings, little was published on this disease prior to 1951. Perhaps the most important paper is that of Henry and Riker.(22) In 1947 they found that wound inoculations with the oak wilt pathogen produced symptoms when introduced into the vascular tissue of the roots, stems, branches, twigs, and leaves, and that the fungus was dis— tributed throughout the crowns of wilting trees including branches that did not show symptoms. Then in the early 1950's, with the availability of funds and organization of the national‘oak'wilt committee, research was greatly increased. In 1951, Beckman and Kuntz(:5) using poisons, dyes, and radio active iodine, found that the killing of one tree with a poison caused the death of surround- ing trees. This pattern of death, especially for red oaks, had been noted at oak wilt locations, and gave support to the theory of tree-to-tree spread of the disease through root graft unions. In the same year, Bretz(7 ) discovered that the fungus was patho- genic to Chinese chestnut and went on to identify many other (9,10,11) hosts. Research on the control of the disease was also being carried (27) on. During 1951, Hoffman published on control by chemicals, and in 1952 (28) reported that subsurface injection of chemicals was more effective than trunk or foliar sprays, and those used as preventives were more successful than those used as curatives. In 1952, much information was published on the fungus itself. (24) Hepting, T0018 and Boyce found the fungus to be interfertile 7 and heterothallic. All but one of their isolates were self-sterile (25) hermophrodites. Later in the same year, Hepting designated the two compatible types of Chalara quercina as A and B. The perithecial (82> stage of the life cycle was found, named, and described by Bretz. (13) Barnett, Staley, and True found that mycelial mats occurring beneath the loose cracked bark of trees killed by the disease are ready for spermatization and are capable of producing perithecia when conidia of the opposite compatible type are introduced. These mats consist of two parts; a center pad and an outer part. (Figure 2). In 1952, Morris and Fergus<36> discovered that conidia and peri- thecia are produced only on the outer part of the mat and not on the center pad. The following year, Curl, Stessel, and Zucherman<15) stated that the center pad provides the pressure to crack the bark. Fergus (18) studied the production of these mats and determined that neither size of tree nor site seemed to have any effect on their ’production, but moisture content of the wood did. No mat production was found when the moisture content was below 60.7 percent. He observed mats in March, May, June, August, September, and November. With the understanding of mat production, the question of how spores were made available to the outside was answered. Wind was not thought to be a vector because the spores are produced in a sticky substance and are protected from the wind by a wall of bark. Thus the search for possible vectors was increased. The spread of this disease was divided into local spread and long distance spread. Local spread was said to take place through (23) root grafts as far back as 1944 by Henry, Moses, Richards, and Riker. (40) True, Judy, and Ross, in 1955 suggested a possible test for root 8 graft unions by the rate of absorption of solutions through freshly- cut stumps. Overland or long distance spread was known to occur because trees had become infected with no known oak wilt tree within possible range of root grafts. An example of this is the report by Guyton‘zO) of finding a blazed red oak infected, with the nearest known diseased tree 1/4 mile away. In the absence of wind disseminated spores, this type of spread suggests some kind of insect as being the (13) vector. In 1952, Craighead and Morris reported on the possible (31) importance of several insects. During 1953, Jeffery found that trees wounded in the spring became infected, and that Nitidulidae and Diptera were the two most abundant insects attracted to the (16) fresh wounds. The work of Dorsey, Jewell, Leach, and True during the same year with four species of Nitudulidae supported the belief (6 ) that nitidulids are important vectors in nature. In 1954, Boyce found that nitidulid beetles released 500 feet away reinfested oak (32) wilt mats. Jewell stated that conidia are viable after passing through the intestinal tract of certain Nitidulidae. Himelich and (26) Curl proved that squirrels are able to transmit the disease. In (38) 1956, Stambaugh and Fergus, working on the longevity of spores, found that ascospores picked‘up by the nitidulid beetles remained (33) viable over winter but conidia did not. Jewell reported on experi- ments done in 1953-54 which showed that contaminated nitidulid beetles could bring about infection of wounded trees and that these insects were quickly attracted to fresh wounds during the spring months. In 1957, Kuntz and Drake<35) reported that if wounds were immediately painted with liquid asphalt roofing compound or with house paint, the (12) trees did not become infected. In 1958, Buchman showed that the oak bark beetle Pseudopityopthorus minutissimus was capable of trans- mitting the disease. With the knowledge of how the oak wilt disease may spread, and of the conditions necessary for this spread, researchers in several states worked out control programs. "Although these differed some- what, they were mostly based on the principles of breaking the root graft chain and killing the infected tree so that the wood dries out before the production of spore-bearing mats can occur. This type of control program is being carried out currently in Michigan. Much work is also being done on chemical controls. Hoffman(29) obtained promising results with Monsanto 4370, Vancide 51, Monsanto 4376, and oxyquiniline benzoate. Two years later, in 1955, Fergus, Cole, and Stambaugh<19) found that Acti-dione completely inhibited germination of endoconidia and ascospores at a concentration of 1.0 microgram per ml., which is 1 ppm., and greatly reduced mycelial growth in culture. In 1957 Philps, Kuntz, and Riker<37) found that Olegomycin, Acti-dione, and the acetate derivative of cycloheximide could delay symptoms for as long as three months. 10 METHODS AND MATERIALS I. Isolation of the Fungus from Infected Trees Freshly wilting branches of trees showing oak wilt symptoms were cut and taken into the laboratory. Ideal branches were from 1/4 to 1/2 inch in diameter and cut to a length of about eight inches. The lab bench was sterilized with 95% alcohol. The branch was dipped in alcohol and flamed. This surface-sterilized the branch; care was taken not to allow the wood to get too hot. A knife was dipped and flamed and used to remove the bark. After flaming the knife again, slices were cut into the wood at a diagonal to the longitudinal axis. Then with flamed forceps, each slice, called a chip, was placed on malt agar in a petri dish. These dishes were placed in an incubator at 21 to 23° C. where they were kept until discarded. In about 5 to 7 days after the chips were placed on the agar, the fungus had grown out far enough to be identified. -Pure cultures were trans- ferred to test tube slants of malt agar and were kept at the same temperature for use as stock cultures. II. Inoculation of Trees with the Fungus A. Solid agar method For inoculations, a small wound deep enough to enter the sapwood was made. A piece of malt agar about 3 mm. square, cut from a petri dish containing sporulating mycelium of the oak wilt fungus, was placed on the wound. A piece of masking tape was then put over the wound to prevent loss of moisture and to keep insects out. Imple- ments used for inflicting the wounds were a knife, a chisel, or an increment hammer. Two inoculations were made on each tree, one on the main stem or trunk, and the other on a branch. 11 B. Agar paste method. The malt agar and mycelium of five different isolates growing in petri dishes were blended with 100cc of distilled water in a Waring Blendor. This made a smooth‘paste which contained more moisture and gave a more uniform inoculum than the solid agar method.’ A knife was used to make a small gouged wound deep enough to penetrate the sapwood. A small amount of the inoculum paste was placed in the wound with an eye dropper, and masking tape was used to cover the wound. As in the first method, each tree was inoculated in two different places. This method was easier to use than the first method, but both resulted in high percentage of infection. III. Treatments A. Bark application of the methylhydrazone derivative of cycloheximide in fuel oil by spraying. Red oaks and white oaks from 2 to 4 inches in diameter were treated with the methylhydrazone derivative of cycloheximide (formulation 174) at 600 ppm in No. 1 fuel oil. A one-quart compressed air Sprayer (Sure Shot) was used to thoroughly wet the bark of the trunk up to about three and one half feet from the ground. There was no way to determine the amount of chemical that remained on the bark because of runoff of the chemical. B. Penetration of the bark by Acti-dione (cycloheximide) in fuel oil. lkcti-dione (formulation 173), in concentrations from 400 ppm to 9,600 in.No. 1 fuel oil, and as it comes from.the stock supply (12,800 ppm), was painted on the bark of small red and bur oaks. ,After a period of time, cross sections of stems from under the area 12 of treated bark, 6 inches above the treated area, and 12 inches above the treated area were cut and the bark was carefully removed. These cross sections were then subjected to bio-assay method A (page 14). Treated trees were also left in the field to observe any phytotoxic effects. C. Cone and chisel method. A water-tight acetate cone was placed around the trunks of diseased trees. This cone was then filled with the oxime derivative of cyclo- heximide at 150 ppm in water. A 1/4-inch chisel was used to wound the tree below the level of the solution. D. Bark penetration of cycloheximide in water. Four-layered pieces of cheese cloth cut to about 8 inches by 5 inches were dipped into chemical solutions at specified concentrations in water and wrung or allowed to drip until they contained about 15 to 20 cc of the solution. One of these cloths was then wrapped around the trunk of each tree. Saran wrap was used to cover the cheese cloth to retard evaporation and a piece of masking tape was placed around the bottom of the Saran Wrap to hold it and the cheese cloth in place and to reduce runoff of the solutions. (Figure 8). At given intervals after treatment, the trees were cut and taken into the laboratory. The wrappings were removed and the treated bark ‘washed in running water for about a half minute. After removing the bark, cross sections of the stems were cut from directly under the treated area, at 6 and 12 inches above this area and from branch tips. These cross sections were then subjected to bio-assay methods A and B (page 14). The trees used varied from 1/2 to 1/4 inch in diameter. Treated 13 trees were also left in the field to observe any phytotoxic effects. E. Foliar application of chemicals in water. Cycloheximide and its following derivatives: oxime, thiosemicarbazone, semicarbazone, acetoacetate, and acetate at concentrations from 5 to 50 ppm in water were used. About 30 grams of soybean flour per gallon of solution or'Tween 20 were used as a spreader and sticker. A small hydraulic sprayer operating at 300 pounds pressure with a -mist nozzle was used, and the foliage was thoroughly wetted. F. Foliar application of Actispray (cycloheximide in water soluble tablet form) in water. 1. Actispray at 5 ppm, 10 ppm, 15 ppm, and 25 ppm in water with Ice per gallon of Tween 20 added was sprayed on the foliage of small red oaks whose stems ranged from 1/2 to 1 inch in diameter. Plastic bags were placed over part of the crowns to prevent the chemical from reaching part of the foliage. After a period of time, cross sections from the trunk, cross sections of a stem from the sprayed part of the foliage, and cross sections of a stem from the bagged part of the foliage were used in bio-assay method A (page 14 ). 2. Trees of the same size were sprayed with Actispray in the same concentrations as above. Some of these trees were inoculated with the oak wilt fungus prior to being sprayed and some of them were inoculated after being sprayed. The purpose of the experiment was to determine if the treatment had any effect on the syndrome of oak wilt. Unsprayed but inoculated trees and sprayed but uninoculated trees were used as controls. IV. Bio-assay methods. 14 A. Cross sections of treated and untreated trees or small 13 mm Schleicher and Schuell analytical filter papers wetted with the solutions being tested, were planted on freshly poured agar plates which were seeded with a yeast that is very sensitive to cycloheximide. Then, after being incubated at 30°C. overnight, any halos (clear areas around the sections or discs where the yeast growth was inhibited) were measured (figures 6 and 9). B. Small branches of treated oak trees were cut so they would fit into 6-inch test tubes. The bark of these twigs was removed and the twigs were placed in the test tubes with 1 cc of distilled water. Stoppers were put in the test tubes which were then auto- claved at 15 pounds pressure for 15 minutes. After cooling and under sterile conditions, mycelium and spores of the oak wilt fungus were placed on the end of the sterile twigs. If the antibiotic was in the twig at a sufficient concentration, the fungus would not grow (Figure 10). Twigs from.untreated trees were used as controls. V. Effect of oak stem tissue liquids on the fungicidal properties of cycloheximide and its derivatives. Oak stems varying from.1/4 to 1/2 inch in diameter were cut into 1/2 inch sections. These were placed in a cylinder and pressed at 5,000 to 10,000 pounds pressure in a small hydraulic press. The sap which was squeezed from the wood was collected and mixed with an equal quantity of each derivative of cycloheximide and allowed to stand at 30°C. for 16 hours. After this interval, 13 mm Schleicher and Schuell analytical filter paper discs were dipped in a solution and placed on yeast Seeded plates (Figure 6). These were then incubated overnight at 30°C. after which the results were 15 checked according to bio-assay method A (page 14). Checks con- sisting of the derivatives with distilled water were also made. l6 EXPERIMENTS AND DISCUSSION OF RESULTS I. Inoculations for observation of symptoms and miscellaneous purposes. On June 11, 1960, five white oaks were inoculated. Two which developed symptoms were cut on August 11, 1960 and the trunks laid on the ground so that they would dry more slowly than if standing in an attempt to induce mat production. By July of the next year, no mats had been produced and these trunks were discarded. The three remaining trees were still healthy on August 15, 1962. On August 2, 1960, twelve white oaks and twelve red oaks were inoculated. By June 29th of the following year, all but three of the inoculated red oaks were dead or nearly dead. Of these three trees, one showed no disease symptoms. The remaining two, which were infected but still had some green leaves, were treated with the oxime derivative of cycloheximide at a concentration of 150 ppm. in water using the cone and chisel method of application (Figure 4). Although the foliage of these two trees was more than half dead at time of treatment, both trees were still alive at the onset of fall dormancy. They both failed to leaf out the following spring, but did not produce mats. Of the nine red oaks that died in the early summer, only two produced mats. It is possible that the dead tree tissues were too dry for mat production(18) by the time cool weather arrived. Of the twelve white oaks inoculated on August 2, 1960, eight were healthy, two were dead (of unknown cause), and two had developed typical oak wilt symptoms at the time of the writing (September, 1962). 17 Figure 4. A red oak tree treated with the oxime derivative of cycloheximide in water by the cone and chisel method. 18 The two trees which were treated with the oxime derivative, using the chisel-cone method may have had their life prolonged at least two months. Both of these trees showed advanced oak wilt symptoms at the time of treatment and yet death was retarded. Re- tardation of death may not have been caused by the treatment as it is possible that genetic resistance or some other factor was res- ponsible. The fact that no mats were produced on these two trees cannot be positively attributed to the treatment because there were too many other variables such as temperature and moisture. This treatment technique was not used in further experiments because large enough trees were not available. II. Foliar applications of cycloheximide and its derivatives (1961). On the dates indicated in Table 1, red oak trees from 1/4 to 1/2 inch in diameter were sprayed with the various chemicals dissolved in water. About 30 grams of soy bean flour per 10 gallons of water was used as a sticker - spreader. The experiment, treatments and results follow: Leaf scorch was very light except when Actispray was used. This material caused severe leaf scorch. Many nitidulid beetles were observed clustered around the tape covering inoculation points (Figure 5). Table 1. Foliar application of cycloheximide and its derivatives dissolved in water (1961). of 8 Spray dates No. Treatment tree Actispraya l3 5 ppm ' Actispray 10 10 ppm No treatment 3 Acetate 8 10 ppm Acetate 9 50 ppm No treatment 4 Acetoacetate 5 10 ppm Acetoacetate 8 50 ppm No treatment 2 Semicarbazone 10 10 ppm Semicarbazone 8 50 ppm No treatment 7 Oxime 5 ppm 10 Oxime 10 ppm 7 Oxime 11 25 ppm Oxime 10 50 ppm 6/10/61 6/27/61 6/10/61 6/27/61 6/10/61 6/27/61 6/10/61 6/27/61 6/10/61 6/27/61 6/10/61 6/27/61 6/10/61 6/27/61 6/10/61 6/27/61 6/10/61 6/27/61 7/11/61 6/10/61 6/27/61 7/11/61 6/27/61 7/11/61 6/27/61 7/11/61 Inoculation dates 6/12/61 6/12/61 6/12/61 6/12/61 6/12/61 6/12/61 6/12/61 6/12/61 6/12/61 6/12/61 6/12/61 6/12/61 6/12/61 6/12/61 7/16/61 7/16/61 No. of trees inoculated 9 No. of trees infected 9 a Actispray is cycloheximide in water soluble tablet form. 61 I'll! . (VI-Ill'ii 20 Figure 5. Nitidulidae beetles clustered around an inoculation point on a red oak stem. 21 Discussion: Without replication, these results are useful only for selecting chemicals, dosages, and application techniques for more adequate testing. Actispray was the only treatment that had any effect on infection of the trees by the oak wilt fungus. A more extensive experiment using Actispray will be discussed later. III. The effect of oak stem tissue liquids on the fungicidal properties of cycloheximide and its derivatives. Wood tissue liquids were extracted from samples of red and white oak wood and then treated according to method V. (Figure 6). The results, based on three series of experiments performed at different times, are presented in Table 2. Discussion: ~The purpose of this experiment was to indicate which derivatives should be included in field trials and which ones could be eliminated. The data given in Table 2 indicate the possi- bility that the oak.liquid extracts may increase the toxic properties of the semicarbazone and the thiosemicarbazone derivatives. These two derivatives along with Acti-dione should be included in a well replicated field experiment. However, due to the unavailability of a large number of trees, it was decided to test just Acti-dione, the only compound that gave any promising results in the previous field experiment. IV. Foliar application of Actispray in water, with one cc. per gallon of Tween 20 added, before and after inoculation. A. On June 4, 1962, forty 3-5 year old red oaks were inoculated Vfllth the oak wilt fungus. Eleven days later the Actispray was applied to the foliage just after sundown when transpiration was at a mini- mum o In" I, 'I o .o .o o .o .o o .o .o . o .o .o mumumo< n .m .N n .o .o m .w .o oaonmnumoHEom o .o .o, o .o .o o .o .o o .o .o maoumnumUHEmmoaee o .o .o o .o .o o .o .o o .o .o accumuesesseumz as .eH .ms OH .m .m m .m .w a“ .5 mm m¢oae-auo< .H mowuom amulm. ENNIM. maulm. mom xmo moans mom xmo can mo 00 m.o msam mo 00 m.o mSHm nous: CH owuofinwuum .Emm‘m um oquHnHucm mo 00 m.o Hmfiuoumz .mo>wum>wuov mow was ooflewxosoHoho wo mowuuomoum Hmowowwcnm onu do moHSUHH osmmwu Eoum xmo mo muoommm .N oHan Table 2. Effects of oak stem tissue liquids on the fungicidal properties of cycloheximide and its derivatives. Material 0.5 cc of antibiotic at 2 ppm. plus 0.5 cc of red oak sap plus 0.5 cc of white oak sap Series 1. mime 59; 7, 7b Methylhydrazone O, O, O Thiosemicarbazone O, O, 0 Semicarbazone 6, 8, 9 Acetate 0, 0, O Acetoacetate 0, O, O Oxime O, O, 0 Water 0, 0, 0 Series II; Acti—dione 11, ll, 11 Methylhydrazone O, O, O Thiosemicarbazone 6, 5, 4 Semicarbazone 7, 6, l Acetate O, O, 0 Acetoacetate O, 0, 0 Oxime O, O, 0 Water 0, 0, 0 Series III. Thiosemicarbazone 0, 0, 0 Semicarbazone 4, 4, 2 antibiotic in water 1 ppm 2 ppm _5 ppm 10 ppm W 9, 9, 10 13, 14, 16 o, 0, 0 0, o, o 5, 6, 0, o, o 0, 0, o 19, 19 2, 5, 7 15, 13 0, 0, o 0, 0, 0 O, 0, 0, 0, 0 0, o, 0 o, 0, 0, o, 0 0, o, 0 o, o, 0, 0, o 7, 7, 7 0, o, 0 2, 2, 2 0, 0, 0 0, o, 0 0, 0, o 0, 0, 0 o, 0, 0 o, 0, 0 o, o, o 2, 2, 2 7, 8, 8 a Values represent the halo diameter minus the disc diameter (13 mm.) in millimeters. b With the exception of Thiosemicarbazone and semicarbazone at 10 ppm, three discs were planted for each combination. ZZ 23 Figure 6. Illustrating method of determining effect of oak stem.tissue liquids on the antibiotic Acti-dione and its derivatives. Circular filter pads were saturated with 1/2 cc of stem tissue extract plus 1/2 cc of antibiotic in water at 2 ppm. Pads were placed on plain agar plates seeded with a yeast. Halos show where growth of yeast has been inhibited. Left is Acti-dione; right is thiosemi- carbazone. 24 10 ppm 10 inoculated trees and 4 uninoculated trees 15 ppm. 10 inoculated trees and 5 uninoculated trees 25 ppm 10 inoculated trees and 6 uninoculated trees Results: The sprayed trees began to show leaf scorch in about a week. The two higher concentrations caused severe injury to the leaves, and the 10 ppm spray caused enough injury so that by the date oak wilt symptoms should have appeared on the sprayed trees, it could not be detected. The inoculated but unsprayed trees developed wilt symptoms in 3 to 4 weeks after inoculation. The uninoculated but sprayed trees showed leaf scorch but did not die. By August 1, 1962 every inoculated tree in this part of the experiment, whether sprayed or unsprayed, was dead and the cause was determined by tissue plantings to be the oak wilt pathogen. B. On June 15, 1962 eighty 3-5 year old red oak trees were sprayed with varying concentrations of Actispray dissolved in water plus Tween 20 at 264 ppm. 10 ppm 20 trees 15 ppm. 35 trees 25 ppm 25 trees On June 21, 1962, 17 of the first group, 30 of the second group, 20 of the third, and a group of 10 unsprayed trees were inoculated. The leaves of the sprayed trees developed leaf scorch as before and wilt symptoms on the unsprayed trees appeared in three to four weeks. Because of the leaf scorch it was impossible to visually determine if the sprayed trees were infected. On August 7, 1962, tissue plantings were made from each inoculated tree to 25 determine if the fungus was present. Results appear in Table 3. C. On June 26, 1962 ninety 3-5 year old red oak trees were sprayed as follows: Actispray at 5 ppm plus Tweena 23 trees Actispray at 10 ppm plus Tween 35 trees Actispray at 15 ppm plus Tween 20 trees Tween alone 20 trees On July 29, 1962, (three days later), 21 of the first group, 30 of the second, 14 of the third, 10 of the fourth, and 2 un- sprayed trees were inoculated. Leaf scorch developed on all of the trees sprayed with Acti- spray with effects of the two higher concentrations quite severe. Tissue plantings of the inoculated trees were made on August 10, 1962. Results appear in Table 4. D. Effects of the chemical to the foliage (reading made August 24, 1962). (Table 5). Discussion: The results of this experiment show that Actispray applied to the foliage of small red oaks may delay symptoms and may even prevent infection. All of the trees in part A died due to oak wilt even though they were sprayed. Note that these trees were sprayed eleven days after inoculation. Part B showed the best control and these trees were sprayed six days before inoculation. Part C gave some control and these were sprayed three days before inoculation. This indicates that any control obtained here is a preventive control and not a cure o a Tween 20 was used at 264 ppm. 26 Table 3. Effect of foliar application of Actispray, six days before inoculation, on the incidence of oak wilt (6/15/62). Concentration of No. of trees No. of trees chemical (ppm) inoculated infected Z infection 10 17 3 17.6 15 19 4 21.0 25 3O 13 43.4 no treatment 10 10 100 27 Table 4. Effect of foliar application of Actispray, three days before inoculation, on the incidence of oak wilt (6/26/62). No. of No. of Concentration of trees in- trees in- % chemical (ppm) oculated fected infection ,Actispray at 5, plus Tween8 21 12 57.2 ,Actispray at 10, plus Tween 14 8 57.1 .Actispray at 15, plus Tween 30 16 53.4 Tween 10 10 100 2N0 treatment 2 2 100 a. Tween 20 was used at a rate of 264 ppm. 28 Table 5. Effects of Actispray on the foliage. Date curling and new Treatment Applied leaf scorch defoliation leaves Actispray 10 ppm 6/15/62 medium none healthy Actispray 15 ppm 6/15/62 severe none healthy .Actispray 25 ppm 6/15/62 severe light healthy ,Actispray 5 ppm 6/26/62 light none healthy .Actispray 10 ppm 6/26/62 medium none healthy Actispray 15 ppm 6/26/62 severe none healthy 29 Possibly the time or number of days between treatment and inoculation is also important as indicated by the difference in per- centage of infection in parts B and C. Why infection increased with increasing concentrations of Actispray in Part B is not clear. This might have been clarified had greater numbers of trees been available for this experiment. V. Bio-assay of foliar application of Actispray. Small red oaks were treated as follows with Actispray plus one cc. of Tween 20 per gallon of solution (July 18, 1962). 4 trees 5 ppm 4 trees 10 ppm 4 trees 15 ppm 4 trees 25 ppm Treatment was according to section IV - B in Methods and Materials. Sections were cut 4 and 24 hours after treatment. Results: No halos developed, indicating that the antibiotic did not enter the leaf tissue. Discussion: Because Actispray demonstrated some possible con- trol (Tables 3 and 4), it is believed that the antibiotic was present in the tissue in this experiment, but the bio-assay was not sensitive enough to detect it. VI. Bark application of the methylhydrazone derivative of cycloheximide (formulation 174) at 600 ppm. in eastern No. 1 fuel oil.a In early June 1960, nine red oaks and ten white oaks were a Eastern No. 1 oil is a paraffin hydrocarbon type oil and western No. 1 oil has an asphalt base. Western source oils have an ability to hold greater amounts of Acti-dione in solution. *1 . . ' h . ‘ V I ~ . . . D ‘ 7 Va A O - ,1 I \ _ ._. O I 30 treated. These trees varied from 1 to 3 inches in diameter, and the trunks were sprayed to a height of 3 feet from the ground. One week later, six of the red oaks and six of the White oaks were inoculated with isolates of the oak wilt fungus. The purpose of the experiment was to determine if the treatment would prevent infection or at least prevent mat formation. Results:A All of the red oaks develOped oak wilt symptoms in three to four weeks after inoculation, and produced mats in the fall of the same year. Mat production was abundant and formed on branches as small as 1/4 inch in diameter. Two of the white oaks were completely wilted by the 26th of September and the trees died. Although the oak wilt fungus was isolated from both of these trees their death probably was not entirely due to oak wilt because of the sudden, atypical onset. One of the sprayed but uninoculated white oaks also died in this manner. Unidentified wood borers had entered the trunks of these three trees, but it was not determined if they had caused the death of the trees. The other four inoculated white oaks were still living but showed typical oak wilt symptoms for white oak at the time of this writing (September, 1962). Other than the three white oaks that died suddenly, none of these white oak trees showed any apparent damage due to the treatment. (?19 Discussion: Fergus, Cole, and Stambaugh ; and Philps, (37) Kuntz, and Riker found that Acti-dione has adverse effects on the oak wilt fungus in vivo and invitro and is phytotoxic. The results of this experiment demonstrate that Acti-dione had no apparent effect on the fungus or the tree. It was therefore concluded that 31 the chemical either did not penetrate the bark, or, upon penetration, became chemically altered so that it no longer adversely affected these two organisms, at least to the degree that could be readily detected. VII. Penetration of the bark by chemicals dissolved in oil. A. In August, 1961, red and white oaks 1/4 to 1/2 inch in dia- meter were treated with Acti-dione (cycloheximide or the oxime or acetate derivative of cycloheximide, all at 12,800 ppm. which is undiluted. The materials were swabbed on the trunks with cheese cloth from the ground level to a height of about 8 inches. Trees were cut from 2 to 4 hours after treatment and cross sections of the trunks assayed by bio-assay method A. Results appear in Table 6. The plates containing cross sections of the trees treated with oxime or acetate developed no inhibition zones (halos). B. On September 21, 1961, the thiosemicarbazone and semicar- bazone derivatives and Acti-dione, all at 6,400 and 600 ppm. in Standard Oil's No. 9 oil were applied to 3 red and 3 white oaks in the same manner as above. No halos appeared around cross sections of these trees. C. On August 11, 1962, 1/4 to 1/2 inch diameter red oak trees were treated with Acti-dione in western No. 1 fuel oil as follows: 6 trees 400 ppm 6 trees 800 ppm 6 trees 1600 ppm 6 trees 3200 ppm 6 trees 6400 ppm 6 trees 12,800 ppm (concentrate from stock supply) 32 Table 6. Penetration of the bark by Acti-dione, at 12,800 ppm in oil, applied to the trunk. Date cross section cross section halo Treated species level (inches) diameter (mmo diameter (mm) 8/7/61 red oak u.b.a 10 16, 14, 18d 8/7/61 red oak 6b 9 o, o, 0 8/7/61 white oak u.b. 9 16, 15, 6 8/7/61 white oak 6 7 7, 11, 9 8/11/61 red oak u.b. 7 8, 0, 0 8/11/61 red oak 6 7 0, 0, 0 8/11/61 white oak u.b. 6 ll, 10, 15 8/11/61 white oak 6 6 2, 4, 1 a u.b. stands for the area under the treated bark b 6 means 6 inches above the treated area c exclusive of cross section d three cross sections from each area were planted on each plate 33 At four hours and twenty-four hours after treatment, two trees of each set were cut and the bio-assay technique was used to analyze them. Only cross sections located directly under the treated bark area were planted on the yeast plates. Two trees in each set were left uncut in the field to observe any phytotoxic effects. Results: concentration halo diameter minus (ppm) cross section diameter (mm) 3200 0, 5 12,800 1, 1 No other cross sections developed halos and it appeared that the halo around the cross section from the tree treated with 3200 ppm. was due to contamination by the antibiotic chemical because the inhibition zone was restricted to a small part of the circumference of the cross section (Figure 7). By August 23, 1962, no definite phytotoxic symptoms were apparent on the trees in the field. Discussion: Part A of this experiment appears to support the idea that Acti-dione in oil will penetrate the bark of red and white oak trees, at least at this high concentration. It also suggests that the chemical moved up in white oak but apparently did not in red oak. The reason for this is not clear. It has been observed in greenhouse watering that white oak requires more frequent watering than red oak. This could indicate a greater rate of transpiration, thus a greater movement of liquids within the plant. Parts B and C indicate that the oils which were used are not suitable carriers for penetrating the bark with cycloheximide. 34 Bio-assay of cross sections cut from trunks that were treated with Acti-dione in western No. 1 fuel oil. After removing the bark, cross sections were cut and placed on the agar plates seeded with a yeast. 35 White oaks were not used in B and C because they were not available. VIII. Penetration of the bark by Acti-dione in water. On August 10, 1962, 1/4 to 1/2 inch diameter red oaks were treated with Acti-dione in water as follows by wrapping cheese cloths containing the chemical around the trunks (Figure 8). 6 trees 250 ppm. 6 trees 500 ppm. 6 trees 1000 ppm. 6 trees 3000 ppm. 6 trees 6000 ppm. 6 trees 12,000 ppm. At four and 24 hours after the cheese cloths, saturated with Acti-dione, were applied, two trees in each set were cut and bio- assay A was used to determine if penetration and upward movement by the chemical resulted. (Table 7). Cross sections from branch tips were also cut and assayed at 24 hours and four days after treatment was started (Table 8) (Figure 9). The remaining two trees in each set were left uncut to observe possible phytotoxic effects. On August 23, 1962, 10 of the 12 trees left for phytotoxicity study showed severe leaf scorch and appeared nearly dead. The other two, one treated with 6,000 ppm and the other with 3,000 ppm, were healthy and showed no leaf scorch. Upon removal of the cheese cloth it was observed that there were no pruning wounds or bark wounds of any kind under the cheese cloth on the two healthy trees. The ten dying trees had wounds under the cheese cloth. 36 Table 7. Possible penetration of red oak bark by Acti-dione in water. ) Cross section Concentra- . levelp(inches) tion (ppm) Halo diameter minus cross section diameter_(mm)fl 4-hour cut 24-hour cut 12a 250 o, o ( 5, 6)° o, o (14, 14) 6 250 1, 0 ( 9, 7) 0, 0 (17, 18) b u.b. 250 2, 0 (10, 10) 0, 0 (19, 20) 12 500 0, 0 ( 2, 1) 0, 0 ( O, 0) 6 500 O, 0 ( 5, 5) 4, 3 ( 0, 0) u.b. 500 l, 0 ( 6, 6) ‘ 5, 5 ( 0, l) 12 1000 O, 0 (21, 19) 4, 4 ( 0, 0) 6 1000 0, 0 (18, 18) 6, 7 ( 0, 0) u.b. 1000 0, 1 (l7, l7) 3, 4 ( 0, l) 12 3000 0, 0 ( 0, 0) 23, 23 (18, 18) 6 3000 0, 0 ( 0, 0) 22, 22 (l7, l7) u.b. 3000 9, 9 ( 0, ll) 18, 16 (20, 20) 12 6000 15, 15 ( 0, 0) 0, O ( 8, 8) 6 6000 12, 12 ( 0, 0) 0, 0 (13, 12) u.b. 6000 l3, l3 ( 2, 2) 15, 5 (10, 7) 12 12,000 26, 26 ( 1, 0) 26, 25 (21, 24) 6 12,000 30, 30 ( 0, 6) 21, 21 (22, 23) u.b. 12,000 32, 32 (11, 14) 26, 26 (23, 24) p a Numbers indicate the distance above the treated bark area in inches, b u.b. stands for the area under the treated bark. c The numbers in parenthesis represent one tree and the other numbers represent the other tree. .._ -w, 2 “fl .. -_ .— - “TV “7‘. -< .mwF—- .........—- , .\I \ 7| vli..1n--.l. l '1‘ F101: [£11.11 r. a . . , . . a . Ill.“ 11:11-1- v.1]: .,. '11 111 37 Table 8. Results of bio—assay of branch tips, cut one day and four days after treatment was begun, from red oak trees treated with Acti-dione in water. Concentration Halo diameter minus cross section diameter (mm). (ppm) one day8 ' four days Branch No. 1 Branch No. 2 250 9, I 7b 14 9 500 O, O O 3 1000 l, l 5 11 3000 13, 13 0 0 6000 15, 15 14 21 12,000 20, 20 19 24 a Different trees were used on the two different days. b Two cross sections were planted from each branch. 38 Figure 8. Red oak stem.showing cheese cloth method of treatment. Pieces of cheese cloth were saturated with the solution and wrapped around the trunk. Saran Wrap was then placed over the cheese cloth to reduce run off and evaporation. Masking tape was used to secure the Saran wrap. 39 Discussion: Tables 7 and 8 show that Acti-dione, once in the sapwood of a red oak, will move up and be distributed through the crown of the tree without losing its toxicity to the yeast. Whether the Acti-dione penetrated the oak is not proven here. In order to wrap the cheese cloths around the trunks, small branches had to be pruned off. The fact that the ten trees which showed phytotoxic effects contained wounds under the cheese cloth wraps and the two which showed no effect did not, supports the belief that Acti-dione entered the sapwood through these wounds and did not directly penetrate the bark. In order to answer this question, another experiment was carried out. A total of 14 trees were treated with Acti-dione on August 17, 1962. An attempt was made to place the cheese cloth wraps so that no pruning wounds would have to be made. This was impossible on three trees. The cheese cloths held about 100 cc. of solution when placed on the trunks. Bio-assay method A was used and the results appear in Table 9. Bio-assay method B was also used to analyze branches from the crowns of these trees. The fungus grew on all of the branch pieces except those cut from trees 9 and 13 (Figure 10). This substantiates the results obtained with the other bio-assay method. Discussion:l Trees 9 and 13 had wounds under the cheese cloths, and both of these trees demonstrated heavy phytotoxic symptoms (Figure 11). Branches from them also gave positive results in the bio-assays. Tree No. 8 had one small wound under the cheese cloth and demonstrated slight phytotoxic symptoms. None of the unwounded Table 9. Effect of bark wounds of red oak on penetration by Acti-dione in water. Tree No. 7 10 11 12 13 14 Concentration (ppm) 125 125 125 125 250 250 250 250 500 500 1000 1000 1000 1000 Condition of stem under cheese cloth no wounds one small wound wounds no wounds no wounds no wounds no wounds no wounds no wounds no wounds no wounds no wounds wounds no wounds Interval to Halo dia. (mm)a bio-assay lst branch 2nd branch 2 days 0 0 4 days 0 0 2 days 0 0 4 days 0 O 2 days 0 0 4 days 5 5 2 days 0 0 4 days 0 0 2 days 0 O 4 days 0 0 2 days 0 O 4 days 0 0 2 days 0 0 4 days 0 0 2 days 0 0 4 days 0 0 2 days 0 O 4 days 0 O 2 days 0 O 4 d ay 3 C \~ 2 days 0 0 4 days 0 O 2 days 0 0 4 days 0 0 2 days 2 20 4 days 9 10 2 days 0 0 4 days 0 O Phyto- toxicity none slight heavy none none none none none none none none none heavy none a Values represent the halo diameter minus the stem cross section diameter. OF 41 Figure 9. Illustrating bio-assay method A. Debarked cross sections of stems from treated trees were placed on yeast seeded agar plates. Halos demonstrate that the antibiotic was present in the wood tissue. Figure 10. Illustrating bio-assay method B. TWigs from treated trees were placed in test tubes with 1 cc of distilled water and autoclaved. Spores and mycelium.of the oak wilt pathogen were then placed on the twigs. The fungus did not grow on those twigs that contained the antibiotic as indicated by tubes 3 and 7 counting from.the left. 42 Figure 11. Illustrating the phytotoxic effects of Acti-dione. Cheese cloth, saturated with Acti-dione at 125 ppm.in water, was placed around the trunk of tree 9 which had been wounded. The phytotoxic effect is demon- strated by the color of the foliage. 43 trees gave any evidence that the antibiotic penetrated the bark. The author believes that these results give strong evidence that the chemical did not penetrate the bark directly but entered the sap stream through pruning wounds. 44 CONCLUSIONS From the results obtained in experiments discussed in this paper, the following conclusions can be drawn. I. Fuel oil as a carrier for Acti-dione used as a possible control for the oak wilt disease, does not aid the fungicide to penetrate the bark. In unpublished work by Forrest C. Strong with elm, American chestnut, and maple, repeated applications of oil to the bark were found to be injurious to the trees. Where the oil was put over bark wounds, death of the trees often resulted. On the basis of the findings in the present study, it was concluded that oil was not satisfactory as a carrier for Acti-dione in treatment of oak trees even though oil is used successfully as the carrier for Acti-dione for the control of white pine blister (34) rust. II. Acti-dione as a cure for oak wilt. Results showed that trees treated as soon as 11 days after inoculations were made or when symptoms developed, continued to die. It is known that once symptoms deve10p in red oak, the fungus can be isolated from any part of the crown.(22) This means that any chemical that would cure the disease would probably have to not only kill the pathogen, but reverse the death-producing processes already taking place in the tree. Acti—dione apparently does not accomplish the latter. Therefore, it is concluded that Acti-dione is not a practical cure for the oak wilt disease. III. Acti-dione as a preventive of oak wilt. Promise that Acti-dione may be a preventive was demonstrated by some of the experiments reported in this paper. It is believed 45 that more extensive experiments should be conducted, using various techniques for getting the Acti-dione into the sapwood through the bark and by foliar applications, and also varying the time between treatment and inoculatiOn. IV. Practicality of a preventive chemical for the control of oak wilt. A preventive control could be very useful in the field where the disease is endemic. It would still be necessary of course to kill all the known diseased trees in each location. However, if a chemical preventive could be applied to the surrounding oaks, it 5 would save the growing stock that is now being sacrificed under the present control program used in Michigan. It is also doubtful that the cost of a systemic chemiCal control would be any greater than that of frilling and applying 2,4-D ester to the adjacent healthy trees. Oak wilt in Michigan is almost entirely a woodland problem, and as far as the controlling of woodland diseases are concerned,- prevention would be the best answer. 10. ll. 12. 13. 14. 46 LITERATURE CITED Anonymous. 1942. Oak wilt a fungus disease. What's New in Farm Science. Wisconsin Agr. Expt. Sta. Bull. 455: 76-76. Anonymous. 1961. Oak wilt control, Michigan 1961. Mich. Dept. Agr., Mich. Dept. Cons., Mich. State Univ. Multi- lithed. 7 pp. Barnett, H.L., J.M. Staley, and R.P. True. 1952. Mycelial mats of Chalara quercina on killed oak wilt trees as a potential source of perithecia in nature. Phytopathology 42: 531-532. Bart, George J. 1957. Susceptibility of non-oak species to Endoconidiophora fagacearum. PhytOpathology 47: 3. Beckman, C.H. and J.E. Kuntz. 1951. 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