Bulletin ILLINOIS 'Bitura,! History Survey BULLETII^ Root Infection of Woody Hosts with Verticillium albo-atrum NATURAL HISTORY SURVEY OCT 1 8 1974 E OF ILLINOIS ^RTMENT OF REGISTRATION AND EDUCATION rURAL HISTORY SURVEY DIVISION •ANA, ILLINOIS VOLUME 31, ARTICLE 6 ILLrNOIS a*tura.l History Sixrvey BULLETIN Root Infection of Woody Hosts with Verticillium albo-atrum d L. Born OF ILLINOIS RTMENT OF REGISTRATION AND EDUCATION URAL HISTORY SURVEY DIVISION \NA, ILLINOIS VOLUME 31, ARTICLE 6 AUGUST, 1974 STATE OF ILLINOIS DKPAUTMENT OF REGISTRATION AND F.DUCATION BOARD OF NATURAL RESOURCES AND CONSERVATION Dean Bakringer, Ph.D., Chairman; Thomas I'akk, Ph.D., Bioluyy; h. L. Sloss, Ph.D., Oeolayy ; Herbert S. GuTowsKY, Ph.D., Chemistry ; Rorebt H. A.xdeuson, B.S.C.E., Engineering ; Charles E. Olsibteu, Ph.D., Forestry; W. L. EVERITT, E.E., Ph.D., Uepnseitting the President of the University of Illinois; KLBElir II. Hadlbv, Ph.D., liepresentintj the President of Southern Illinois University. NATURAL HISTORY SURVEY DIVISION, Urbana, Illinois SCIENTIFIC AND Tl^CHNICAL STAKK Geokge Spruuel, Jr., Ph.D., Chief Alice K. An St'. Section of Economic Entomology William H. Luckmann, Ph.D., Entomoloyist and Head Willis N. Bruce, Ph.D., Entomologist Wavne L. Howe, Ph.D., Entomologist Stevenson Moore, 111, Ph.D., Entomologist^ Extension HuwAKi) B. Petty, Ph.D., Entomologist, Extension James E. Appleby, Ph.D., Associate Entomologist Edward J. Ahmbrust, Ph.D., Associate Entomologist Marcos Koi^a.n', Ph.D., Associate Entomologist Joseph V. Maddox, Ph.D., Associate Entomologist Ronald H. Meyer, Ph.D., Associate Entomologist Robert D. Pausch, Ph.D., Associate Entomologist Ralph E. Sechriest, Ph.D., Associate Entomologist John K. Bouseman, M.S., Assistant Entomologist GEORtiE L. Godfrey, Ph.D., Assistant Entomologist William G. Ruesink, Ph.D., Assistant Enton James R. Sanborn, Ph.D., Assistant Ento Douglas K. Sell, Ph.D., Assistant Entomologist John L. Weuberg, Ph.D., Assistant Entomologist Clarence E. White, B.S., Assistant Entomologist Keun S. Park, M.S., Assistant Chemist Sub E. Watkins, SupervisoTy Assistant Donald E. Kuiilman, Ph.D., Assistant Professor, Exten- sion RoscoE RandelLj Ph.D., Assistant Professor, Extension Tim Cooley, M.A., Assistant Specialist, Extension Kurt E. Redborg, M.S., Assistant Specialist John F. Walt, M.S., Assistant Specialist, Extension JE.iN G. Wilson, B.A., Supervisory Assistant Daniel P. Bartell, Ph.D., Research A:' weight 227 Leaf number 227 Stem height 227 Nitrogen content - 231 Water content of leaves 231 Leaf area 232 Discussion and Conclusions 232 This report is printed by authority of the State of Illinois. IRS Ch. 127, Par. 58.12. It is a contribution from the Section of Botany and Plant Pathology of the Illinois Natural History Survey. (58200—4,000—8-74) Effect of Temperature & Heat Treating on Development of V. albo- ATRUM IN Roots 234 i Materials and Methods 2341 Results 235 Effect of temperature 235 Effect of heat treating of soil 235 Discussion and Conclusions 235 Evaluation of Systemic Fungicides Against V. albo-atrum 2371' Materials and Methods 237 Laboratory studies 237 Greenhouse studies 238 Soil drenches 238 Foliar treatments 238 Root treatments 239 Results 239 Symptoms 239 Laboratory studies 239 Greenhouse studies 240 Soil drenches 240 Foliar treatments 243 Root treatments 243 Discussion and Conclusions 244 Summary 245 Literature Cited 247'; Index 249 Root Infection of Woody Hosts with Verticillium albo-atrum Gerald L. Bom \^RTICILLIUM WILT is a plant disease caused by the fungus Verticil- Hum albo-atrum Reinke and Berthold. This pathogen is peculiar in tliat it does not confine its attacks to one host, or a few closely related hosts, as is so frequently the case with most other pathogenic fungi; it attacks a large number of widely unrelated plants, many of which are of economic im- portance. The disease does not often occur in forest stands, but it is be- coming increasingly prevalent in plant- ings of ornamental trees and shrubs, particularly in temperate regions of the world. Symptoms of Verticillium wilt on woody hosts are ^'ariable and often difficult to recognize. Usually the first \isual s\'mptom is sudden wilting of foUage on one or several twigs on a branch. A yellowing of foliage some- times precedes wilting. Most plants exhibit leaf symptoms in early July, but some trees may first show symp- toms in early spring or late fall. Leaves on affected ash species may drop while still green and before noticeable yel- lowing or wilting has occurred. Other symptoms suggesting Verticil- lium wilt are decline in current twig growth, stunting, and dieback of indi- \idual twigs and branches. Occasion- ally trees such as maple and tulip tree de\-elop elongated dead areas of bark on the diseased branches or trunk. Water-soaked areas sometimes develop under the killed bark. Trees that develop a limited amount of branch wilt during the summer may show additional wilt and dieback the following year, and others may reco\er and not wilt in succeeding years. Trees that ha\e extensive wilt throughout the crown usually die before the end of the summer. The present study initiated in 1970 and completed in 1972 deals with ( 1 ) the influence of root wounds and age of wounds on infection, (2) penetra- tion and de\elopment of the fimgus in susceptible and resistant woody hosts, (3) analysis of the growth response of young tree seedlings after root infec- tion, (4) the influence of temperature and heat treating of soil on develop- ment of V. albo-atrum in excised roots, and (5) laboratory and greenhouse e\'aluation of fungicides against V. albo-atrum. This report is adapted from a thesis submitted to the Lhiiversity of Illinois in partial fulfillment of requirements for the degree of Doctor of Pliilosophy in Plant Pathology. ACKNOWLEDGMENTS Much of the work on this project was conducted with the cooperation of the Ilhnois Natural History' Survey staff and through the use of Sur\'ey facilities. The study was carried out under the super\dsion of Dr. J. C. Carter, Head of the Section of Botany and Plant Pathology at the Survey, to whom I express my greatest appreciation. Others on the Natural History Survey staff who deserve special recognition for helping to make this a successful project are Dr. E. B. Himelick, Dr. Dan Neel\', Dr. J. L. Forsberg, and Dr. J. L. Crane. Dr. Crane was es- pecially helpful in his assistance in microscopic photograph)'. I grateful!)' acknowledge the assis- tance given by Dr. Richard E. Ford, Professor of Plant Patholog)- and Head of Department, University of Illinois, and Mrs. Betty Nelson and Mrs. Bar- bara Little of the Surve)' staff in prep- aration of the original thesis report, 209 210 Illinois Natural History Survey Bulletin Vol. 31, Art. 6 and b\- O. F. Glissendorf, Survey Tech- nical Editor, who edited the manuscript for this Bulletin article. LITERATURE REVIEW The first reference to a wilt disease was made by Reinke & Berthold ( 1879). They isolated a fungus from potato plants with the KrauseUcrankheit dis- ease which they named Verticillium albo-atrum. Their investigations were not appreciated until 30 years later when Bhttrollkrankheit and KmiiseJ- krankheit were causing severe losses in the potato fields of Europe. Van Hook (1904) described a typi- cal case of wilt in ginseng {Panax quinquifoUum L. ) which he attributed to Acrostalagmtis aJbus Preuss. How- ever, this name is synonymous with the earlier name Verticillium which was estabhshed by Nees von Esenbeck (1816). Corda (1838) did not describe the genus Acrostalagmtis until 1838. Klebahn (1913) isolated a Verticil- lium from dahUa plants which he con- sidered distinct from V. albo-atrum, and he named this fungus V. daliliae Kleb. Since 1913 the relationship be- tween V. albo-atrum and V. dahliae has been the subject of much controversy. Many investigators have disagreed in their interpretations of the drawings and descriptions found in previous re- ports. Wollenweber (1929), Rudolph (1931), Presley (1941), Wilhelm & Taylor (1965), and Van den Ende (1958) maintained that the fungi that produce sclerotia and resting mycelium are members of a large variable species. Others, e.g., Klebahn (1913), Van der Meer (1925), Isaac (1949), and Smith & Walker (1930), have preferred to treat them as separate species. In 1957 Verticillium wilt was re- ported as affecting plants in at least 18 orders, 38 famihes, 98 genera, and 137 species in the temperate climates of the world (Caroselli 1957). Numerous papers in the past have dealt with factors that influence the incidence of Verticillium wilt. Nutri- tion, soil type, soil moisture, soil and 1 air temperature, and light have all been i shown to have an effect on the inci- dence of this disease (Amdt 1957; Edgington & Walker 1957; Gallegly , 1949; Oilman 1916; Ludbrook 1933; Wilhelm 1950). Although many workers have men- tioned wounds as a source of entry by , pathogenic fungi into the root system, little work has been done on the prob- lem. Selman & Buckley ( 1959 ) sug- gested that root injury facilitated fun- gus invasion and that deliberate injury with a scalpel was less harmful than nonnal transplanting. Their study made with transplanted seedlings, or with plants damaged by cutting, gave clear evidence that exposing injured roots to a conidial suspension of the fungus resulted in rapid systemic invasion of the host. Sehnan & Pegg (1957) failed 1 to show any appreciable increase in i infection as a result of deliberate root damage but, in these cases, the "un- damaged" control plants had been transplanted. Under normal conditions of root growth in soil, where the inocu- lum potential of the fungus may be expected to be relatively low, it seems probable that entry into the xylem ves- sels occurs largely through wounds. Armstrong & Armstrong (1958) found that a high incidence of infec- tion occurred when roots of herbaceous hosts were cut prior to inoculation with Fusarium spp. Also, the average num- ber of days for wilt to occur signifi- cantly decreased when roots were cut immediately prior to inoculation. Ful- ton ( 1952 ) showed that more infection occurred when the canes or roots of raspberry were injured prior to inocu- lation. Little has been reported on the anatomy of woody hosts affected with Verticillium wilt. Extensive histologi- cal work has been done on herbaceous hosts attacked by this pathogen. Ru- Aug., 1974 BoRN: Root Infection with V. alho-atrum 211 dolph ( 1931 ) reported that the fungus was found only in the xylem in the early wilt stage, and later invaded the pith, cambium, and cortex in the ad- vanced wilt stages. McWhorter (1962), working with Pelargonium infected with V. alho-atrum, found only traces of mycelium in tissues that had con- siderable discoloration. He rarely found large amounts of mycelium in diseased tissue. Therefore, the amount of mycelia in the vessels is not always indicative of the severity of wilt. Talboys ( 1958 ) observed that acute symptoms of hop wilt were associ- ated with extensive development of mycelium in the xylem vessels but sparse production of tyloses; con- versely, mild symptoms were associated with the development of limited my- celium but abundant tyloses in the ves- sels. Talboys ( 1964) suggested a simple explanation of the inverse correlation he had found between density of mycelium and frequency of tyloses in infected xylem vessels of the hop plant by postulating that a low concentra- tion of fungal metabolites in the xylem stimulates the formation of tyloses but that a high concentration inhibits for- mation. Until recently, spread of the fungus throughout the plant has received little attention. Sewell & Wilson (1964) con- cluded that V. alho-atrum conidia are transported in xylem sap of hops and occasionally they become lodged in vessels where they germinate and pro- duce more conidia. In cotton and tomatoes, conidia may spread through- out the plant in 12 hours to 6 days following inoculation (Garber 1957; Green 19.54). Many earlier workers noted that the hyphae are very slender and reduced in diameter at the point where they pass through the cell walls, but once through they swell to a much greater size ( Garber 1957; Garber & Houston 1966; Klcbahn 191.3; Reinke & Berthold 1879). In the vascular system, the fungus moves from one vessel element to another through pits (Garber 19.57; Garber & Houston 1966; Green 1954). There is an apparent inability' of the mycelium to penetrate new cellular growth lateral to the invaded cells as ri'pidly as the new cells are formed (Green 1954). Klebahn (1913) and Rankin (1914) reported microsclerotia in the vessels of infected plants. Talboys ( 1958 ) ob- served that penetration of vascular tissue of hop by V. alho-atrum de- pended on the amount of suberin in the endodermal cell walls. Garber & Houston (1966) observed gum-like de- posits in tolerant cotton plants which impeded the fungus from penetrating the vascular element. They reported that the splitting apart of cells was a mechanical process and not enyzmatic although they observed enzymatic ac- tion on the middle lamella of cell walls when tlie inoculum potential was high. Symptom appearance is variable, re- quiring days to many weeks after in- fection for expression. Yellowing of foliage and sudden wilting are usually the first \isual symptoms. General stunting accompanied by shortening of the internodes may accompany wilt. Young tomato plants infected with V. alho-atrtrm may show neither leaf yel- lowing nor wilting in the initial stages, but onl>' a stunting of the whole plant (Selman & Pegg 1957). Salman & Pegg (1957) found that 8 weeks after inoculation the dry weights of tomato leaves, stems, and roots were decreased by 72, 70, and 65 percent respectively. Of the growth charac- teristics studied, leaf area was most reduced by infection and this was due to a failure of the leaves to expand rather than to a reduction in leaf pro- duction. After infection, symptom develop- ment, and necrosis, the fungus may o\erwinter within die plant as micro- sclerotia. Benken & Khakimov (1964) obser\ed abundant microsclerotia of V. 212 Illinois Natural History Survey Bulletin Vol. 31, Art. 6 albo-atrum in veins and petioles of o\'er\vintering cotton leaves. The fungus spread unchecked in tlie field within the necrotic tissues of infected cotton seedlings and sporulated freely over the surface of the stems for a short distance above ground level, eventu- ally forming numerous microsclerotia in stems and roots. Nadakavukaren (1965) observed that V. albo-atrum microsclerotia survived best at low temperatures and high moisture levels. Heale & Isaac ( 1963 ) reported that rest- ing mycelium remained viable for 9 months in pieces of necrotic lucerne buried 12 inches (30 cm) in soil. Brinkerhoff ( 1969 ) observed that micro- sclerotia were elongated in leaves incu- bated at 28 to 30 C and round in leaves incubated at 18 C. V. albo-atrum sur- vived for relatively long periods in cot- ton tissue, and infested debris con- stituted a ready source of inoculum when incorporated into either sterile or nonsterile soil. Evans et al. ( 1966 ) suggested that further colonization by V. albo-atrum was arrested when cotton plants were plowed under prior to microsclerotial formation in the tissues. Many recent papers have shown the value of systemic fungicides for the control of vascular wilts. Most of the work has been done with Benlate (benomyl) and thiabendazole (TBZ). Schreiber et al. (1971) found that benomyl was taken up equally well when either applied as a drench or in- corporated directly into the potting media. The planting medium affected the concentration as well as the rate of accumulation of benomyl. Highest levels of accumulation of the fungitoxi- cant were in seedlings grown in media that had the lowest content of organic matter and the highest pH. Heat sterilization of soil prior to benomyl treatment resulted in greater accumula- tion of benomyl in elm seedlings than when the plants were grown in non- sterile soil. Erwin et al. (1971) found that the addition of thiabendazole to soil re- duced the incidence and severity of cotton wilt in plants subsequently inoc- ulated with V. albo-atrum. Rawlins & Booth (1968) reported that the addi- tion of surfactant Tween 20 increased the effectiveness of benomyl and thi- abendazole against V. albo-atru7n, prob- ably by increased absorption of the fungicide by the roots. Erwin et al. (1968) found that thiabendazole not only translocates from the roots to the stems of cotton plants but also can be detected in the bark. They concluded that thiabendazole diffused laterally from the xylem to the bark. Soil treatment, or seedling root dips with difolatan (Bankuti 1964) gave good control of Fusarium oxysporum f. sp. lycopersici and V. albo-atrum on tomatoes in greenhouse and field tests. Complete protection against V. albo- atrum was provided for seedlings planted up to 140 days in soil treated with difolatan. Applying systemic fungicides to the foliage and allovdng the chemical to be translocated downward may be the method used in the future. However, this method presents many problems. Many fungicides, such as benomyl, are extremely insoluble in water. Hock (personal communication) has been able to solubilize benomyl using inor- ganic acids, heat, and constant stirring. Buchenauer & Erwin ( 1971 ) found that benomyl and thiabendazole induced curative effects when sprayed on inocu- lated cotton plants that showed initial symptoms of Verticillium wilt. Both fungicides were detected by bioassay and chemical analysis in xylem tissue and in nontreated stems and leaves above the place of application. CODE OF VERTICILLIUM ALBO-ATRUM ISOLATES MATERIALS AND METHODS All isolates used throughout this study were obtained from actively wilt- Aug., 1974 BoEN: Root Infection wtth V. albo-atnim 213 ing hosts in Illinois. They were main- tained on freshly prepared potato dex- trose agar (PDA) tube slants and transferred periodically. An isolate used to inoculate a particular species was obtained earlier from another of the same species. Resistant species were inoculated with a mixture of all isolates. Below are the code numbers used in this study to identify each isolate. Also, the host, date of isolation, and location of host plant in Illinois are given for each isolate. Code Host Date Place 1 Sugar maple 1969 Urbana 2 Russian olive 1956 Wheaton 3 Redbud 1960 Urbana 4 Green ash 1961 Decatur RELATIONSHIP OF ROOT WOUNDS & AGE OF WOUNDS ON INFECTION MATERIALS AND METHODS Two hundred twenty each of 2-year- old bare-rooted sugar maples {Acer sacchanim Marsh.) and redbud {Cercis canadensis L. ) seedlings were selected as test plants. The plants were break- ing dormancy when received from a commercial nursery. The average height was 45 to 60 cm. The roots of each plant were washed with tap water and rinsed with distilled water prior to planting. The plants were potted in a medium-grade perlite and fertilized bi- weekly with a balanced liquid fertiUzer. Isolate 1 was used to inoculate sugar maple and Isolate 2 was used to inocu- late redbud. Type of Wound Two weeks after potting, 100 plants were remoxed from the perlite and treated. Treatments immediately pre- ceding inoculation included: (1) no wound, (2) abrasion, (.3) puncture, and (4) vascular incision. Wounds were made on tlie primary root ap- proximately 5 cm below tlie ground line. With the abrasion-t)'pe wound, the root surface w as injured by rubbing moist 400 grade carborundum against the root surface. Puncture wounds were made by forcing a balsam wood block, in which five pins were embedded, against the root. This produced pin prick wounds 3 mm deep into the root. The \ascular incisions were made by cutting a V-shaped wedge approxi- mately 0.5 cm deep into the root. When no wound was made, a mycelial disc was placed against the root sur- face. All treated plants were inoculated with a mycelial disc and the wound area covered witli vinyl grafting tape to prevent moisture loss. The control plants were treated identically except that a sterile agar disc was placed on the wounded area and covered with grafting tape. Twenty plants of each species were used for each treatment. After 30 days, all plants were re- moved from the pots and isolations were attempted from the plant roots and stems. Age of Wound In an additional experiment 120 plants of each species were tested to determine the importance of wound age on infection. Two weeks after potting, the plants were gently removed from the potting medium and V-shaped wounds were made on each plant ap- proximately 5.0 cm below the soil line on all plants. Fifteen plants were inoculated with a mycelial disc im- mediately after wounding and the wounds were covered with vinyl graft- ing tape. AU other wounds were wrapped widi vinyl grafting tape and the wounded plants replaced in perlite. At intervals of 1, 2, 4, 8, 16, and 32 days, 15 plants were removed from the potting mixture, inoculated at the wound site, rewrapped \\'ith grafting tape, and planted back in perlite. Thirty days after each inoculation date, the plants were removed from the pots and isolations were made from the roots and stems of each plant. 214 Illinois Natural History Survey Bulletin Vol. 31, Art. 6 RESULTS Type of Wound No infection occurred on unwounded roots. Root wounds were a prerequisite for fungus entry into the plant (Table 1 ) . Any disruption of the periderm on the older roots which allowed the fun- gus to by-pass these tissues was suitable to fungal entry. The percentages of infection for abrasive, puncture, and vascular wounds were 75, 80, and 85 respectively on redbud, and 50, 55, and 80 on sugar maple. The most eflB- cient wound on both hosts was a vascu- lar wound which placed the pathogen in direct contact with the vessel mem- bers. Age of Wound Root wounds remained as infection courts up to 32 days on redbud and 16 days on sugar maple seedlings (Table 2 ) . As the age of the wound increased the number of plants infected through wounds decreased. Only 13 percent of the redbud plants became infected when inoculated at wound sites that were 32 days old and no infection oc- curred through wound sites 32 days old on sugar maple. Thirty two-day-old wounds had sev- eral lavers of dead cells which were oc- cluded with heavily pigmented ma- terials. This condition was a barrier against penetration by the fungus. Many vessel members adjacent to wounds were occluded with tyloses and wound reaction materials. Callus was beginning to form at the margins of the wound after 32 days. Wounds were not made on other areas of the root. Therefore, location of the wounds may have some signifi- cance because wounds made on older secondary tissue may require a longer time for initiation of repair tissue. Younger tissue, i.e., at the root tip or lateral roots, may heal faster and reduce the time a wound remains as an infec- tion court. DISCUSSION AND CONCLUSIONS The periderm consists of the phello- derm, phellem, and phellogen which completely surrounds the vascular cyl- inder of woody plant roots. The cells of the phelloderm are parenchyma and remain alive and active. The cells of the phellem become suberized, which renders them virtually waterproof, and at maturity they die, forming a rather impervious, protective layer around the outside of the root. The fungus gains entrance through Table 1 .—The effect of root wounds on number of redbud and sugar maple plants infected with Verticillium albo-atrum. Numier of Plants Infected Aug., 1974 BoRN: Root Infection with V. aJbo-atnnn 215 Table 2.—The effect of age of root wounds prior to inoculation with Verticillium albo- atrum. Age of Wound 216 Illinois Natural History Sur\'ey Bulletin Vol. 31, Art. 6 solution and genninated in perlite un- der glass. Twenty seedlings of each species in the 2-leaf stage were inocu- lated by dipping the roots into an ap- proximate 1 X 10"/ml conidial density of V. alho-atrum, by placing 3-mm blocks of PDA containing the fungus on selected areas, and by placing a Verticillium-inlested oat seed adjacent to a selected area. After inoculation, the seedlings were placed horizontally in 150-mm petri dishes containing sterile peat moss or planted in sterile soil in pots in the greenhouse. Selected seedlings were sectioned for microscopic examination at intervals after inoculation. The seedlings were removed from the petri dishes or soil and the portions to be sectioned were killed and fixed in FAA, dehydrated in tertiary butyl alcohol, embedded in paraffin, and sectioned at 12 to 15/x using the technique described by Jo- hansen ( 1940 ) . The sections were stained with thionin in phenol and counterstained with orange G. in ab- solute alcohol (Stoughton 1930), then examined under the microscope. RESULTS Fungus Growth on Root Surface The four genera of hosts used were essentially alike morphologically and no differences were detected in the way the fungus penetrated them (Table 3). The fungus colonized the exterior surface of the epideniiis (Fig. 1). The fungal growth was appressed over the entire epidermal surface with conidio- phores arising at right angles from the surface. Tissue around the area of penetration became necrotic. Brown Table 3.—Root colonization of susceptible redbud and green ash, and of resistant honey locust and sycamore seedlings, with Verticilliutn albo-atrum. Intensity of Colonization" in Susceptible and Resistant Plants in Specified Regions of Penetration Days Exposure to Inoculum Host Outer Root Tip Epidermis Cortex Inner Cortex Xylem Phloem'' 1 Redbud Green ash Honey locust Sycamore 2 Redbud Green ash Honey locust Sycamore 4 Redbud Green ash Honey locust Sycamore 6 Redbud Green ash Honey locust Sycamore 8 Redbud Green ash Honey locust Sycamore i " Symbols shown represent Infection intensity as follows : = no colonization ; 1 — slight colonization ; 2 r= moderate colonization ; and 3 = severe colonization. " Passage of the fungus through the phloem into the vessel members occurred but no phloem colonization occurred. Aug., 1974 BoRN: Root Inkectiok with V. (ilho-dfruiu 217 Fig. 1 ,—Verticillium microsclerotia completely colonizing the exterior surface of a green ash root (X 250). iifcrotic flecks could be seen extending above but not below the point of infec- tion. Root Tip Penetration The fungus penetrated the root cap within 48 hours. The hyphae pene- trated both intcrcelluliirly and intra- cdlularly, but intracellular penetration was most common. There was no ten- dency for the cells to separate, which might have occurred if a weakening of till' middle lamella took place, unless an I xiremely high inoculum potential oc- cuired on the root surface. In the region of root elongation and maturation, the fungus penetrated i through the epidermis. Penetration was ! either direct through the cell wall or j between the epidermal cells. The hyphae or germ tubes produced appres- sorium-like swellings over the epidermis ! within 48 hours. A penetration peg de- veloped from the appressorium and was I smaller in diameter than the parent hypha. Penetration in Root Hair Region In the epidermal area between the root liairs, the fungus penetrated at random, both inter- and intracellularly. Germ tubes developed over the root hairs but none was seen penetrating the root hairs. The base of the root hair frequently was penetrated but no fur- ther growth occurred. Penetration in Area of Lateral Root Formation Another avenue for fungus penetra- tion into roots is the area of lateral root formation. Rupture of the primary root tissue did not occur until the lateral root primordia were well de- \eloped. The fungus penetrated the torn areas where the lateral root emerged. Mycelia could be seen in the cortical layers of the lateral root but none was obser\ed in the xylem. At this point in the process of invasion no difierences were detected between the susceptible and resistant hosts. 218 Illinois Natural History Survey Bulletin Vol. 31, Art. 6 Cortical Invasion — Susceptible Hosts Most mycelial growth in the cortex was intracellular. Mass penetration re- sulted from a high inoculum potential at the invading point, and the mycehal de- velopment was centripetal (Fig. 2). Many hyphae at the point of penetra- tion formed appressorium-like swellings against the cortical cell wall and pene- trated to the next cell layer (Fig. 3a). Other hyphae that penetrated the corti- cal cells were constricted in diameter at the point of penetration (Fig. 3b). When the invasion of the inner corti- cal layers was limited to a few hyphae, no marked centripetal alignment of hy- phal strands occurred. Hyphal strands sometimes deviated from the centripetal development and developed tangen- tially and intercellularly for several cell layers and then penetrated directly through the wall. Cortical Invasion — Resistant Hosts Most mycelial growth in the cortex was intracellular. The mycelium was hyaline but became heavily pigmented after 3 days. After 8 days, most hyphae were dark brown, regularly septate, and swollen between the septa so as to appear torulose. These hyphal strands gave rise to microsclerotia by re- peated budding (Fig. 4a). Micro- sclerotia varied in shape, from elongate to irregularly spherical, and varied in size, from 15 to ISfx in diameter. These microsclerotia continued to enlarge, which caused cortical cells to be sepa- rated or expanded many times their nor- mal size ( Fig. 4b ) . Penetration of Vascular Region of Susceptible Hosts If the fungus penetrated the cortical cells of the susceptible hosts, it in- Fig. 2.—Longitudinal section of redbud cortex showing mass penetration of cortical cells resulting from a high inoculum potential at the Invasion point (X400). Aug., 1974 BoFN: Root Infection with V. allw-atnnn 219 Fig. 3 - -C'-rtic,-,! cells in longitudinal section. A) Appressorium-like swellings against the cortical cell wall IX 25001 . B) Hyphal constriction in diameter at the point of penetration through a cortical cell wall (X 20001 . 220 Illinois Natural History Survey Bulletin Vol. 31, Art. ^-^-^^^—-nT"^ Ar»| i '̂ Fig. 4.—Cortical cells of honey locust in longitudinal section. A) Dark brown, septate, budding hypha (X2200). 3) Microsclerotia causing cortical cells to be separated or expanded many times their normal size (X 250) . Aug, 1974 BoEN: Root Infection ^VITH V. albo-atnim 221 variably penetrated the endodermis and vessel members. The fungus grew to the endodemial layer within 4 days. The quantity of vessel meml^ers in- vaded appeared related to the number of points of entry and to the mass of mycelia that developed from the points of entry. The hyphae that penetrated the en- dodermis usually penetrated the vessel members through pits. The hypha nar- rowed to a thin, peg-like projection as it grew through the pit. Hyphae did not necessarily stop at the first vessel member contacted, but in many in- stances they grew out through a pit on the wall of the vessel into an ad- jacent vessel on the side opposite the entry point ( Fig. 5 ) . The mycelium was generally unbranched, hyaline, 3.5/x in width. No typical conidiophores were observed. VerticiUium conidia were observed in the xylem 8 days after inoculation. In most cases, the conidia appeared to be free-floating in the xylem stream and in no way connected with the mycelium present (Fig. 6 and 7). The conidia often were found lodged at the end walls of the vessel members (Fig. 8). No defense mechanism such as tyloses or gimi deposits was observed in the xylem members. The lack of a defense mechanism on susceptible hosts is in complete disagreement with other workers' data on hops and cotton (Table 4). Penetration of Vascular Region of Resistant Hosts Altliough the fungus penetrated the cortical cells, few hyphae penetrated the endodermis and vessel members. The quantity of vessel members in- Fig. 5.—Longitudinal section through the vascular cylinder of redbud showing a hypha vithin a vessel member (X 850) . 222 Illinois Natural History Survey Bulletin Vol. 31, Art. 6 Fig. 6.—Free-floating conidia of V. albo-atrum in a vessel member of green ash (X 500). Fig. 7.—Longitudinal section through a vessel member of a redbud root showing a V. albo-atrum condidium germinating (X 1700) . Aug., 1974 Born: Root Infection with V. albo-atrum 223 Fig. 8.—Longitudinal section showing lodged conidia at the end walls of the vessel member of a redbud root (X 2300) . Table 4.—A comparison of penetration and development of Verticillium albo-atrum in roots of herbaceous and woody hosts. 224 Illinois Natural History Survey Bulletin Vol. 31, Art. 6 were observed in the vessel mem- bers. The mycelium was hyaline, un- branched, and 2.7^ in diameter. The mycelia did not ramify throughout the vessel members as they did in the sus- ceptible hosts. No conidia could be seen in tlie xylem members although mycelium was present. Frequently, microsclerotia developed in the vessel members, completely plug- ging the vessel members (Fig. 9a). They arose from single hyphae by re- peated budding of heavily pigmented, thick-walled cells. The microsclerotial cells often grew through pit pairs and moved into adjacent vessel members where repeated budding took place (Fig. 9b and 9c). Germinated micro- sclerotial cells were also observed that grew through pit pairs into adjacent vessel members. The ray parenchyma was heavily colonized with microsclerotia. Germ tubes from microsclerotia grew from one parenchyma cell to another through pit pairs or plasmodesmata (Fig. 9d). This may be an avenue for lateral growth of the fungus outward from the central vascular cylinder. DISCUSSION AND CONCLUSIONS Conidia of V. albo-atrum germinated on the surface of both the susceptible and resistant roots and grew in random directions. Some germ tubes grew away from the host; others penetrated the epidermis. Although germ tube penetration occurred, most epidermal penetration was by either hyphae or genninated microsclerotia. Intercellular and intracellular penetration occurred within 48 hours after inoculation. Nel- son ( 1950 ) found that V. albo-atrum penetrated peppermint roots 6 hours after inoculation. Reid ( 1958 ) reported intercellular penetration but observed no intracellular penetration of melon roots by F. bulbigenum Cook and Massee. According to Anderson & Walker ( 1935 ) , F. conglutinans Wollenw. pen- etrated the cell walls of cabbage plants by mechanical pressure. Talboys ( 1958) found that the splitting apart of hop cells by V. albo-atrum was a mechani- cal rather than an enzymatic process. My evidence through visual observa- tion did not suggest that an enzyme was involved in either epidermal pene- tration or cortical invasion unless the cells were invaded by a mass of hyphae. This is in agreement with Garber & Houston ( 1966 ) on VerticiUium inva- sion of cotton. Direct penetration was either by constriction of a hypha as it passed through the wall or by a peg- like projection of an appressorium-like swelling. Garber & Houston (1966) noted similar structures in cotton cells invaded by VerticiUium. I did not observe penetration of root hairs although it has been reported by Smith & Walker (1930) for Fusarium invasion of cabbage roots and by Gar- ber & Houston ( 1966 ) for VerticiUium invasion of cotton roots. The areas of lateral root emergence were not important as infection courts. The fungus penetrated the lateral root and ramified throughout the cortical tissue, but no mycelia were found in- vading the vascular tissues. Many un- injured roots were invaded to the same cortical layers. Smith & Walker ( 1930) reported similar observations; however, Reid ( 1958 ) suggested that penetration ! of emerging lateral roots might provide • a mechanism for a vascular pathogen to < avoid the penetration barrier of the ^ endodermis. The progress of infection in the sus- f ceptible green ash and redbud and the resistant honey locust and sycamore was identical after the point of cortical colonization. The species were alike in morphology and were penetrated by the fungus in a comparable fashion. Differences in fungus growth were noted immediately as the fungus pro- gressed beyond the initial cortical col- onization. In the susceptible species, mycelia Auti., 1974 Born: Root Infection with V. alho-atrum 225 Fig. 9.—Longitudinal section through the vascular cylinder of a sycamore root. A) Ger- minating microsclerotium of V. albo-atrum which has completely plugged a vessel member (X550). B, C) Budding cells growing through pit pairs into adjacent vessel members (X 10001. D) Ray parenchyma heavily colonized with microsclerotia and microsclerotium germinating (X 1000) . i 226 Illinois Natubal History Survey Bulletin Vol. 31, Art. 6 ramified throughout the tissues and reached the endodermis and xylem ele- ments within 4 days. Conidia were found in the vessels of roots 8 days after inoculation. Lack of mycelial con- nections between fungus parts present in the xylem and conidia at secondary sites higher in the root system can be explained by conidial movement. It is reasonable to assume that free-float- ing conidia moved to secondary infec- tion sites and provided for rapid fungus dispersal throughout the plant. In some susceptible cotton plants Schnathorst et al. (1967) found that 30,000 conidia/ ml of tracheal fluid were present 96 hours after inoculation. In the resistant species, microsclerotia were produced in abundance in the cortex. These structures enlarged by repeated budding and ruptured the walls of the cortical cells. Few hyphae penetrated the endodermis and reached the xylem members. Few hyphae were found in the xylem members and conidia were not observed. Schnathorst et al. (1967) found that tolerant va- rieties of cotton depressed conidial numbers more than 20 fold. Talboys (1964) postulated that the xylem defense-response is much the same in different species and cultivars of plants. Since it is a generahzed re- sponse to physical damage and infec- tion, the difference in host resistance to vascular infection is constituted by a difference in response of the extra- vascular tissue at the early stage of infection. This I found to be only partially true. The endodermis pro- hibited mycelial penetration to some extent in the resistant hosts. However, a xylem-defense response took place after penetration of the vessel members. Few hyphae were found in the vessel members after penetration and no conidial production occurred. Beckman et al. (1962) inoculated bananas with Fiisariiim by means of a standard dose of microconidia introduced into the xylem elements and found a highly sig- nificant difference between the xylem- defense response of the resistant Laca- tan and susceptible Gros Michel ba- nanas. Therefore, Talboys' postulate should be expanded to include the in- fection sequence in the vascular sys- tem in trees. EFFECT OF ROOT INFECTION ON GROWTH RESPONSE OF REDBUD & GREEN ASH SEEDLINGS MATERIALS AND METHODS Redbud and green ash seeds were collected and germinated as previously described. After 3 weeks, 80 seedlings of each species were removed from the germination beds and the roots dipped in inoculum for 5 minutes. After root- dipping, 5 plants were planted in each of 32 No. 10 potting cans. Isolates 3 and 4 were used to inocu- late redbud and green ash respectively. Each isolate was grovra on PDA for 14 days at 24 C. The fungus and agar were macerated vdth water in a Waring blendor to produce a thick suspension of inoculum. An equal number of con- trol plants were root-dipped in a PDA blended suspension which did not con- tain the fungus and potted as described above. The plants were inoculated on March 29. The first samples of healthy and infected plants were taken on April 12 and at 2-week intervals thereafter until July 19. Ten plants per treatment were sampled on eight occasions mak- ing a total of 160 redbud and 160 green ash plants. The following data were obtained from each treatment: stem height, leaf area, total number of leaves produced, fresh and dry weights, water content of leaves, and nitrogen content of stems, leaves, and roots. Dry weights were obtained by drying the plant parts in an electric oven at 80 C for 72 hours. Leaf areas were determined by weighing a specific Aug., 1974 BoRN: Root Infeciion with V. albo-atnim 227 known leaf area as compared to the weight of the whole leaf. Micro-Kjeldahl determinations for toal nitrogen were made on bulk sam- ples of leaves, stems, and roots from healthy and infected plants. All data for stem height, leaf area, and dry weight were analyzed statistic- aUy using a one-way analysis of vari- ance and student "T' tests. RESULTS Symptoms Fourteen days after inoculation, young inoculated plants were retarded in growth but no wilt symptoms were apparent. Two weeks later the plants were stunted and the leaves had failed to expand. Sectioned roots and stems showed extensive invasion of the vessel mem- bers by the fungus. The hyphae were confined to the primary xylem vessel members 16 weeks after inoculation. Dry Weight Infection markedly reduced dry-mat- ter production on both redbud and green ash seedlings. The mean values for the dry weight of whole plants for controls and infected plants are shown in Table 5 and Fig. 10. All weight data for leaves, stems, and roots were analyzed statistically and the mean values for the dry weights on all sampling periods after inoculation are given in Tables 6 and 7 and Fig. 11 and 12. When comparing healthy and infected plants, a significant difference in dry weight was evident for leaves and stems of redbud and leaves of green ash 14 days after inoculation. A significant diff^erence in dry weight of roots of both hosts occurred 28 days after inoculation. On July 19, 112 days after inoculation, the percentage dif- ferences for healthy and infected plants were 45, 53, and 47 for leaves, stems, and roots of redbud, and 36, 17, and 24 for leaves, stems, and roots of green ash, respectively. Leaf Number The mean values for the number of leaves for healthy and infected redbud and green ash plants are given in Table 8 and Fig. 13. The infected plants showed limited leaf production 28 days after inoculation, and thereafter the rate of leaf production difi^ered little in the two groups. Stem Height The mean values for stem height of healthy and infected redbud and green ash plants are given in Table 9 and Fig. 14. A significant difference in stem height of redbud and green ash was not evident until 42 days and 28 days after inoculation, respectively. The initial reduction in growth due to infection Table 5.—The dry weight of redbud and green ash seedlings infected with Verticillium albo-atrum. 228 Illinois Natural History Subvey Bulletin Vol. 31, Art. 6 Fig. 10. — The dry weights of green ash and redbud seedlings after inoculation with V. albo-atrum. Table 6.—Dry weights of leaves, stems, and roots of redbud seedlings infected with Verticillium albo-atrum. Aug., 1974 BoRN: Root Infection with V. albo-atrum 229 Fig. 4.CH 1 " "o. » 3.0- 9 2.0 ROOTS ChKk— tion with V. albo-atrum. -The dry weights of leaves, stems, and roots of redbud seedlings after inocula- 230 Illinois Natural History Survey Bulletin Vol. 31, Art. 6 14 28 42 56 70 84 98 H2 DAYS 15-1 Aug., 1974 Born: Root Infection with V. albo-atrum 231 Table 7.—Dry weights of leaves, stems, and roots of green ash seedlings infected with Verticillium albo-atrum. 232 Illinois Natural History Survey Bulletin Vol. 31, Art. 6 27-1 24- z < s; 21 £ 18 U 28 42 56 70 84 98 DAYS 14 28 42 S6 70 84 98 DAYS Fig. 13.— Influence of root infection of red- of leaves produced per plant, bud and green ash seedlings on total number Table 9.— Influence of root infection on stem height of redbud and green ash seedlings. Aug., 1974 BoRN: Root Infection wtth V. alho-atrum 233 & E — 10- 35- 234 Illinois Natural History Survey Bulletin Vol. 31, Art. 6 Table 1 1 .—The water content of redbud and green ash leaves in response to root infec- tion with Verticillium albo-atrum. Aug., 1974 Born: Root Infection with V. alho-atrum 235 Isolates 3 and 4 were used to inocu- late redbud and green ash respectively. Each isolate was grown on PDA for 14 days at 24 C. The fungus mycelia and agar were macerated with water in a Waring blendor to produce a thick suspension of inoculum. The control plants were root-dipped in a PDA solu- tion without the fungus. After the roots were dipped, the plants were potted in perlite and al- lowed to grow for 14 days. The plants were then removed from the perlite and the roots were excised at the ground line. To study the effects of temperature on microsclerotial development, the ex- cised roots were incubated at continu- ous temperatures ranging from 5 to 35 C at 5-degree intervals. The roots were wrapped in moist paper towels and then placed in capped bottles to maintain a moist atmosphere. Cultures of the fungus on PDA were grown at the same range of tempera- tures. Observations were made on the production of microsclerotia. The influence of the soil microflora on microsclerotial formation was de- termined by incubating whole roots in sterile and nonsterile soil in capped bottles. Two soil-moisture levels were used. One level approximated field capacity; the other approximated one- half field capacity. The temperature was maintained at 25 C for the 28-day test. Both tests had four root systems per treatment rephcated three times. Ob- servations were made at 7-day intervals for 35 da>'s. For microscopic observa- tion, roots were cut into small pieces, sectioned on a freezing microtome, and stained in cotton blue. RESULTS Effect of Temperature Abundant microsclerotia were ob- served in roots after 14 days incubation at 15, 20, 25, and 30 C. Microsclerotia did not develop at 35 C and were not observed in roots incubated at 5 and 10 C until after 35 days. The micro- sclerotia tend to develop as compact balls of dark-walled cells (Fig. 15). At the lower temperatures, indi\'idual microsclerotia tended to be elongated, and some were reduced to single strands of rounded, dark-waUed cells. Although the fungus failed to form microsclerotia on PDA at 35 C, a limited amount of mycelial growth occurred. After 14 days' growth, abun- dant microsclerotia were produced ( Fig. 16) at 15, 20, 25, and 30 C. Fewer microsclerotia developed at 30 and 10 C. Little growth occurred on PDA after 14 days at 5 C, but measurable hyphal growth occurred after 35 days. Thus, microsclerotial development on PDA closely paralleled development in moistened roots at similar tempera- tures. Effect of Heat Treating of Soil Microsclerotia de\'eloped in dead roots incubated in both steamed and nonsteamed soil. Moisture levels near the field capacity of the soil were more favorable for microsclerotial develop- ment. Relatively few microsclerotia de- veloped in nonsterile soil at the low moisture level. Although microsclerotia developed uniformly and more abun- dantly in steamed soil, appreciable numbers of microsclerotia were found in nonsteamed soil. DISCUSSION AND CONCLUSIONS The microsclerotia of V. alho-atrum develop rapidly at 15 to 30 C in excised green ash and redbud roots after being incubated at high moisture levels. Microsclerotia were produced at 5 C, but a longer incubation period was re- quired. Temperature requirements for microsclerotial development on PDA and in dead host tissue were similar. The development of microsclerotia at low temperatures is important in inoculum increases in overwintering de- 236 Illinois Natural History Survey Bulletin Vol. 31, Art. 6 Fig. 15.—V. albo-at-rum microsclerotia consisting of compact balls of dark-walled cells on dead root tissue (X 250) . Fig. 16.—V. albo-afrum microsclerotial development on PDA (X 250) Aug., 1974 Born: Root Infection with V. alho-airum 237 bris. Evans et al. (1966) found large numbers of microsclerotia in o\'er- wintering cotton stalks where fall and winter \\'eather temperatures were rela- tival)' low and there was sufficient moisture. The range of temperatures at which microsclerotia form permits the fungus to compete favorably with or- ganisms that decompose root debris. Bom (1971) found that heat-treating the soil increases symptom develop- ment because of a decrease in compe- tition \\'ith other fungi. The present study indicates that witli high temperature and low soil moisture prior to microsclerotial development, the inoculum le\'el was significantly re- duced. EVALUATION OF SYSTEMIC FUNGICIDES AGAINST V. ALBO-ATRUM MATERIALS AND METHODS V. albo-atnnn Isolates 1 and 2 were used throughout this study. Inocula for laboratory studies were prepared by growing the fungus for 14 da>'s at 24 C in petri dishes containing PDA. Greenhouse experiments were initi- ated in March and ran through June. The day and nighttime temperatures were approximately 25 and 16 C, re- spectively. The soil consisted of a mix- ture of equal parts by Aolume of loam, peat, and river sand, steamed at 100 C for 4 hours. Soil pH varied from 6.5 to 7.2. Inocula for infesting soil were pro- duced by growing the fimgus for 14 days at 24 C in petri dishes containing PDA. The fungus mats, containing both microsclerotia and conidia, were frag- mented in tap water in a Waring blendor for 2 minutes. The fungus was added to the soil at the rate of one culture mat in 100 ml of water 20,000 g of soil. The soil was stirred after add- ing the inoculum to distribute the fun- gus uniformly throughout the soil. To determine the inoculum potential in the soil, the soil mixture was air-dried and screened to break up large particles. One-g samples were diluted with sterile water to 10' g ml, and 1 ml aliquots plated out on PDA -\- streptomycin. This measured an inoculum potential of 250,000 propagules/g of dry soil. When plants were inoculated di- rectly, a V-shaped wound was made \\'ith a scalpel on the primiry root ap- proximately 5 cm below the soil line. A 5-inm mycelial disc was inserted under each flap, pressed in place, and wrapped with \'inyl grafting tape to pre\'ent drying of the inoculum and wound area. The fungicides subjected to labora- tory and greenhouse evaluation were: Bcnlate 50 percent WP [Methyl-1- ( butylcarbamoyl ) 2-benzimidazolecar- bamate]=benomyl; Thiabendazole 60 percent WP [2-4-( thiazolyl) benzimi- dazole]=TBZ; Bra\o-6F .54 percent (tetrachloroisophthalonitrile); and Vita\'ax 75 percent WP (5, 6-dihydro- 2-methyl-l, 4-oxathiin-3-carboxanilide ) . Laboratory Studies The four fungicides were tested in vitro to detenninc the antifungal ac- tivity of each against V. alho-atrum. Concentrations of 1,000, 500, 100, and 10 //g ml ( active ingredient ) aqueous sus- pension of each fungicide were pre- pared and 10-mm \Miatm-n filter discs were soaked for 5 minutes in each con- centration. Sterile PDA culture plates were seeded with a conidial suspension and discs from each fungicide were placed on the seeded culture plates, two per plate. This was replicated four times using a factorial arrange- ment of treatments (4 trials x 4 fungi- cides x 4 levels) in a completely ran- dom design with four plates per treat- ment combination. Zones of inhibition were measured after 4 or 5 days, at which time growth on control plates had entirely co\ered the agar surface. Laboratory bioassays were conducted on plant materials used in fungicide 2.38 Illinois Natural History Survey Bulletin Vol. 31, Art. 6 tests in the greenhouse. The plants were severed at the base of the stem and divided into three regions: (1) tenninal, characterized by fully ex- panded terminal leaves; (2) center; and (.3) bottom, located about 5 cm above the severed base of the stem. Leaf discs (9 mm diameter) and wood and bark sections ( 100 mm long ) from each region were frozen at —10 C for 24 hours prior to being placed into petri dishes which contained 15 ml PDA seeded with a conidial suspension of V. albo-atrum. After the plates were incubated at 24 C for 7 days, the di- ameters of the zones of inhibition were measured to determine the relative con- centration of fungitoxicant present in the sample. Greenhouse Studies Soil Drenches.—Three hundred twenty seedlings each of sugar maple and Russian olive were used as plant material. The seedlings were 2 years old, bare-rooted, and 45-60 cm in height. The seedlings had no previous treatment and were just breaking dor- mancy. Plants wound inoculated or placed in infested soil were potted 2 weeks prior to fungicide treatment. Control plants were treated identically, but without the fungus. Eight different treatment combinations for each of the four fungicides were tested; with 36- treatment combinations arranged as a 4x3x3 factorial [four fungicides X three levels (two rates and a con- trol)] X three infestations (with and without fungus) in a completely ran- domized design giving a total of 320 observations per species. The eight treatments were: (1) infested soil, non- treated plants; (2) wound inoculated, nontreated plants; (3) infested soil, plants treated with 1,.500 /jg/nil; (4) infested soil, plants treated with 500 /xg/ml; (5) wound inoculated, plants treated with 1,500 /xg/ml; (6) wound inoculated, plants treated with .500 /xg/ml; (7) noninfested soil, plants treated with 1,500 /ug/ml; (8) non- infested soil, plants treated with 500 /tg/ml. Each plant was placed in a No. 10 potting can. In each pot, 200 ml of the fungicide at the designated concentration were applied as soil drenches three times at weekly inter- vals. Water was applied and the soil kept moist by watering when required. All fungicide treatments had 10 plants per treatment except that the benomyl treatments had 25 plants per treatment. Disease control was calculated by using the following formula. Percent disease control= Disease Disease incidence — incidence in control in treated X 100 Disease incidence in control The noninfested treated pots were used for detection of fungicide phyto- toxicity on seedlings. Foliar Treatments.—A benomyl derivative was applied to the foliage of sugar maple and Russian olive seed- lings to evaluate its effectiveness as a foliar fungicide. Solutions of the beno- myl derivative were prepared as fol- lows: benomyl (5.0 g of active chemi- cal) was dissolved in 100 ml of 85- percent concentrated lactic acid over heat and brought up to a liter with distilled water ( 5,000 /xg/ml ) ; benomyl (5.0 g of a.c. ) was dissolved in a liter of distilled water over heat in which 2 ml of concentrated sulfuric acid had been added (5,000 /^g/ml); benomyl ( 5.0 g of a.c. ) was suspended in a liter of distilled water (5,000 ;ag/ml). The pH of the benomyl-lactic acid-water solution was 1.2-1.5, and of the beno- myl-sulfuric acid-water solution was 2.5-3.0. Each formulation of the benomyl de- rivative was applied to an equal num- ber of plants 2 weeks prior to soil in- festation. Another group of plants was treated with each formulation 2 weeks after soil infestation. Foliage was dipped twice to run-off in late after- noon to retain moisture on the foliage as long as possible. The fungicide was Aug., 1974 BoRN: Root Infection with V. aJbo-atrum 239 prevented from contaminating the soil by the placing of a cardboard cover on the top of each can before dipping. To determine if the benomyl deriva- tive could be translocated from the place of application to new growth in sugar maple seedlings, fohar dips were applied to localized areas. A benomly- lactic acid-water solution was prepared as previously described. Treatments with 5,000 fxg/ml were applied in three different ways — to the top three leaves, applied to leaves on the lower two branches, and apphed to all leaves on one side of the plant. The agar diffusion bioassay method was used to detect fungitoxic chemicals in 9-mm leaf discs above and below the area of treatment or in 10-mm sec- tions of xylem tissue. Root Treatments.—Benomyl, thi- abendazole, Bravo-6F, and Vitavax were apphed as root dips to evaluate each fungicide as a prophylactic against root penetration by the pathogen. Four liters of each fungicide were fonnu- lated at 1,500 (ng ml in distilled water. Ten plants of each species were al- lowed to stand in each fungicide for 5 minutes. Only the roots were covered with the fungicide. After 5 minutes each plant was removed from the dip, shaken to remove excess liquid, and planted in infested soil. Each plant was potted in a No. 10 potting can. Data on phytotoxicity and symptom development were recorded. RESULTS Symptoms Initial wilt symptoms occurred with- in 7-10 days on both sugar maple and Russian olive seedlings after being in- oculated by the wound method. When the plants were placed in infested soil, symptoms occurred within 12 to 14 days. The progression of symptom de- velopment was the same regardless of the inoculation method. The leaves rapidly lost their turgidity within 2-3 days. Browning of the leaves and pre- mature leaf drop occurred soon after the leaves had wilted. Unlike larger trees where only a branch or several branches may wilt, these seedlings wilted quickly and completely. Laboratory Studies With the paper disc bioassay in vitro, benomyl and TBZ were highly inhibi- tory at a concentration of 10 /^g/ml ( Table 13 ) . Vitavax was somewhat less fungitoxic, and Bravo-6F was much less active. As the concentration of each fungicide decreased the zone of inhibition decreased proportionately ( Fig. 17 ) . The minimum concentration of benomyl and TBZ that inhibited growth was 0.01 and 0.1 /xg/ml, respec- tively. The minimum concentration of Vitavax was 0. 1 lUg/ ml and for Bravo-6F it was 1 /xg/ml. In PDA plates containing benomyl or TBZ, conidia germinated but failed to grow more than a few microns in Table 13.—Paper disc bioassay of fungicides against Verticillium albo-atrum in vitro. Concentration' 240 Illinois Natural HisTom- Survey Bulletin Vol. 31, Art. 6 length. Wiien single conidia were tran.s- ferred from the.se plates to PDA slants after 10 days, more than 90 percent ga\e rise to established colonies. Greenhouse Studies Soil Drenches.—When benomyl, TBZ, Vitavax, and Bravo-6F were ap- Fig. 17.—Filter paper disc bioassay of four fungicides for the control of V. albo-atrum illustrating zones of inhibition outward from filter discs. A) Benomyl. B) Thiabendazole. C) Bravo-6F. D) Vitavax (l=10Mg/mI; 2=100 Mg/ml; 3 = 500 /i^g/ml; 4=1,000 Mg/ml). Aug., 1974 BoFN: Root Infection with V. albo-atniin 241 plied as soil drenches to sugar maple and Russian olive seedlings, each gave some degree of disease control except Bravo-6F at 500 ^.g ml (Table 14). Benomyl, TBZ, Vitavax, and Bravo-6F, in descending order, were effective when applied 2 weeks after soil infesta- tion. Benomyl at 1,.500 /xg, ml gave the best control of Verticillium wilt of both sugar maple and Russian oHve seed- lings. The fungicide concentration, whether at 1,500 /xg/ml or 500 /xg/ml, at the time of application made little difference in the percentage of disease control. Benomyl at 1,500 /xg/ml and 500 /ig/ml gave 47.5 and 42.5 percent disease control, respectively, on sugar maple seedlings. Differences were no- ticed when comparisons were made be- tween fungicides and fungicide con- centrations. On Russian olive seedlings, TBZ at 500 /xg/ml gave the same amount of control as Vitavax at 1,500 /ig/'ml. Benomyl at 500 fig, ml gave less control than TBZ at 1,500 /xg/ml. Therefore the rate of soil application of any one fungicide is important in the control of the disease. Bioassay of terminal, center, and lower leaves of plants treated with a soil drench with each fungicide showed the highest accumulation of the fungi- Table 14.—Effect of soil drenches for the control of Verticillium wilt of sugar maple and Russian olive seedlings. 242 Illinois Natural History Survey Bulletin Vol. 31, Art. 6 toxiciint in the lower leaves and stems (Tables 15 and 16). Benomyl was de- tected in higher concentrations than all other fungicides in both leaves and stems whether it had been apphed at 1,500 or 500 |tg/ml. Bravo-6F could Table 1 5.—Effect of soil drenches on uptake and translocation of fungitoxic materials by sugar maple seedlings. Aug., 1974 BoBN: Root Infection with V. albo-atnim 243 not be detected in any plant tissue above ground. A higher concentration of the fungitoxicant accumulated in the sugar maple seedlings than in the Russian olive seedlings. The bioassay of foliage and wood from the sugar maple produced zones of inhibition ap- proximately twice as large as those fiom Russian olive seedlings. Foliar Treatments.—A foliar ap- plication of benomyl, dissolved in lactic acid or sulfuric acid, 2 weeks prior to soil infestation gave the best control ( Fig. 18 ) . Benomyl suspended in water gave less control than either applica- tion of benomyl dissolved in acid. When the application of benomyl was delayed for 2 weeks after soil infesta- tion, little control occurred. All foliar applications, regardless of formulations, gave better control if they were applied prior to soil infestation. Benomyl, or a benomyl derivative, was detected moving upward to areas of new growth after it had been ap- plied to localized areas at 5,000 /xg ml. After applications had been made to the top three leaves of sugar maple seedlings, a fungitoxic material could be detected in the treated leaves, but no fungitoxic material was found mov- ing downward in the wood. After a benomyl derivative was applied to the lower two branches and leaves, a fungi- toxic material was found in the foliage and vascular wood of the treated area, and also in the untreated foliage and wood above the point of application. When applications were made to leaves on one side of the plant, a fungitoxic ma- terial was found adjacent to the treated area and upward in the nontreated areas. Therefore, a benomyl derivative was translocated from the treated areas to adjacent nontreated areas above the point of application. No fungitoxic ma- terials were detected below the point of application. Root Tre:atments.—Root infection of sugar maple and Russian olive seed- lings can be reduced and symptom ex- pression delayed by dipping the roots with fungicides before placing them in infested soil (Table 17). Benomyl, TBZ, Bravo-6F, and Vitavax all gave some degree of control against Verticil- 10- 9. 8. 7 244 Illinois Natural History Survey Bulletin Vol. 31, Art. 6 Table 17.—Effect of root treatments on symptom expression of sugar maple and Russian olive seedlings planted in Verticillium albo-atrum-infested soil. Aug., 1974 BoRN: Root Infection with V. albo-atmm 245 the addition of acids, the fungitoxicant was water soluble, and could be taken up more readily and translocated throughout the plant. The fungitoxicant must be locahzed in the plant parts be- fore the host-pathogen interaction pro- duces gums and tj'loses, blocking the upward movement of the fungitoxicant. Once wilt symptoms occurred and the vascular system was occluded, trans- location of the fungitoxicant was re- duced regardless of the formulation. The time of fungicide application is critical for the fungitoxicant to be dis- tributed throughout the plant before the fungus can become established. The critical time of application was similar for foliar treatments and soil drenches. When fungicides were tested as pro- phylactic root dips, each gave some degree of control. Initial wilt symp- toms were delayed as much as 3 weeks with benomyl. The delaying of root infection may allow wounds to be oc- cluded with wound material before the fungus can become established at the wound site. Tliis method of control may be of value when used on bare- rooted nursery materials. More work is needed to detennine how fast these systemic fungicides will move in the plant and how long they will remain active. Additional work is needed to determine the critical time of application and if higher concentrations will be more effecti\e but not phyto- toxic to the host. SUMMARY VerticilUum albo-atrum Reinke and Berthold is a widespread and destruc- tive vascular pathogen. It is peculiar in that it does not confine its attack to one host, or a few closely related hosts, but attacks a large number of widely unrelated plants, many of which are of economic importance. Tiie wound most conducive to in- fection was a vascular wound which allowed the pathogen to come in direct contact with the vessel members. No infection took place unless a wound was present on the root. Root wounds remained as infection courts up to .32 days on redbud and 16 days on sugar maple seedlings. As the age of the wound increased, the number of plants infected through wounds decreased sharply. In the susceptible hosts, the patho- gen rapidly colonized the cortex, endo- dermis, and vessel members. Conidia were produced in abundance within 8 days. The pathogen in the resistant hosts readily colonized the cortex, but few hyphae were found in the vessel members. Conidia were not present in the vascular system. Microsclerotia were found in both the cortex and vascular cyUnder of the resistant hosts. Infection leads to a significant re- duction in dry-matter production, stem height, and leaf area of the plants. The nitrogen content was lower in infected redbud and green ash steins, but higher in leaves and roots. There was no definite pattern of water content be- tween infected and healthy redbud and green ash seedlings. Frequently the water content of the infected seedlings was above that of the healthy controls, but not consistently. Abundant microsclerotia were ob- served in roots after 14 days when incubated at 15, 20, 25, and .30 C. Microsclerotia were observed after 35 days at 5 and 10 C, but no micro- sclerotia were observed at 35 C. Micro- sclerotia developed in dead roots incu- bated at 25 C in both steamed and nonsteamed soil. A moisture level near the field capacity of the soil was more favorable for microsclerotial develop- ment than was a lower soil moisture. The in vitro assay of the toxicity of the fungicides by the agar diffusion method appears to be quantitative. There is a proportional increase in the size of the zone of inhibition with in- crease in quantity of the fungicide. 246 Illinois Natural History Sijbvey Bulletin Vol. 31, Art. 6 Benomyl, TBZ, and Vitavax, when applied as soil drenches, reduced symp- tom development after plants had be- come infected. The inability of any fungicide to give 100-percent control may be due to the host-pathogen inter- action producing tyloses and gum-like material which prevents the fungitoxi- cant from being translocated to the foliage. Fungicides which show no systemic action are of little value in controlling Verticillium wilt when they are applied as a soil drench. Benomyl which had been solubilized in either an organic or inorganic acid and applied to the foliage gave better control than a benomyl suspension in water. With the addition of acids, the fungitoxicant was water soluble, and could be taken up more readily and translocated throughout the plant. When the fungicides were tested as prophylactic root dips, all delayed symptom expression and gave some degree of control. Benomyl delayed initial wilt symptoms as much as 3 weeks. The delaying of root infection may allow wounds to be occluded with wound material before the fungus can become established at the wound site. LITERATURE CITED Anderson, M. E., and J. C. Walker. 1935. Histological studies of Wisconsin Hollan- der and Wisconsin ballhead cabbage in relation to resistance to yellows. Journal of Agricultural Research 50:823-836. Armstuoxg. G. M., and J. K. Armstrong. 1958. Effect of cutting roots on the inci- dence of Fusarium wilt of cotton, toma- toes, cowpeas, and other plants. Phyto- pathology 48:341. (Abstr.). Aendt, C. H. 1957. Temperature as a fac- tor in the infection of cotton seedlings by ten pathogens. Plant Disease Reporter Supplement 246:63-84. Bankliti, M. M., and W- D- Thomas, Jr. 1964. Control of Fusarium and Verticil- lium wilt with defolatan. Phytopathology 54:1431. (Abstr.). Beckman, C. H.. S. Hamos, and M. E. Mace. 1962. The interaction of host, pathogen, and soil temperature in relation to sus- ceptibility to Fusarium wilt of bananas. Phytopathology 52:134-140. Benken, a. a., and A. Khakimov. 1964. Vertitsilleznaya infektsiya v list'-yakh Khlopchatnika ( Verticillium infection in cotton leaves). In Review of Applied Mycology 44:292. Born, Gerald L. 1971. Heat treatment of soil enhances Verticillium wilt infection of barberry and redbud. Plant Disease Reporter 55:996-997. Brinkebhoff, L. a. 1969. The influence of temperature, aeration, and soil micro- flora on microsclerotial development of Terticillium albo-atrum. in abscised cot- ton leaves. Phytopathology 59:805-808. Buciienauer, H., and D. C. Erwin. 1971. Control of Verticillium wilt of cotton by spraying foliage with benomyl and thia- bendazole solubilized with hydrochloric acid. Phytopathology 61:433-434. Caroselli, Nestok E. 1957. Verticillium wilt of maples. Rhode Island Agricul- tural Experiment Station Bulletin 335:5. CoRDA, A. C. J. 1838. Icones Fungorum hucusgue cognitorum, 2 (Prague). Edgington, L. v., and J. C. Walker. 1957. Influence of soil and air temperature on Verticillium wilt of tomato. Phytopath- ology 47:594-598. Erwin, D. C, J. J. Sims, D. E. Borum, and J. R. Childers. 1971. Detection of the systemic fungicide, thiabendazole in cot- ton plants and soil by chemical analysis and bioassay. Phytopathology 61:964-967. , , and J. PAKTRinGE. 196S. Evi- dence for the systemic fungitoxic activ- ity of 2-(4-thiazolyl) benzimidazole in the control of Verticillium wilt of cotton. Phytopathology 58:860-865. Evans, G., W. C. Snyder, and S. Wilhelm. 1966. Inoculum increase of the Verticil- lium wilt fungus in cotton. Phytopath- ology 56:590-594. Fulton, Robert H. 1952. Studies on Ver- ticillium wilt of raspberry. Phytopath- ology 42:8 (Abstr.). Gallbigly, M. E. 1949. Host nutrition in relation to development of Verticillium wilt of tomato. Phytopathology 39:7. (Abstr.). Garbee, R. H. 1957. The penetration and development of Verticillium albo-atruni Reinke and Berthold in the cotton plant. Ph.D. Thesis. University of California. 60 p. , and Byron R. Houston. 1966. Pen- etration and development of Verticillium albo-atrum in the cotton plant. Phyto- pathology 56:1121-1126. Gilman, J. C. 1916. Cabbage yellows and the relation of temperature to its occur- rence. Annals of Missouri Botanical Garden 3:25-84. Green, Ralph J. 1954. An investigation of the wilting phenomenon in Verticillium wilt of tomato Lycopersicum esculentam Mill. Dissertation Abstracts 14(6) :915- 916. Heale, J. B., and Ivor Isaac. 1963. Wilt of Lucerne caused by species of Verticil- lium. Annals of Applied Biology 52: 439-451. Himelick, E. B. 1969. Tree and shrub hosts of Verticillium albo-atrum. Illinois Nat- ural History Survey Biological Notes 66. 8 p. Isaac, Ivor. 1949. A comparative study of ipathogenic isolates of Verticillium. Brit- ish Mycological Society Transactions 32: 137-157. Johansen, D. a. 1940. Plant microtech- nique. McGraw-Hill Book Co., Inc., New York. 523 p. Klebahn, H. 1913. Beitrage zur Kenntnis der Fungi Imperfecta I. Eine Verticil- lium - Krankheit auf Dahlien. Mykolo- gisches Zentralblatt 3:49-66. LtTDBROOK, W. V. 1933. Pathogenicity and environal studies on Verticillium hadro- mycosis. Phytopathology 23:117-154. McWhorter, Frank P. 1962. Disease symp- toms in Pelargonium infected with Ver- ticillium. Plant Disease Reporter 46: 349-353. Nadakavukaren. II. J. 1960. The effect of soil moisture and temperature on survi- 248 Illinois Natural History Survey Bulletin Vol. 31, Art. 6 val of Verticillium microsclerotia. Dis- sertation Abstracts 21(3) :419. Nees von Esenbeck, C. G. 1816. Das Sys- tem der Pilze and Schwamme. Stahel- schen Buchhandlung, Wurzburg. 329 p. Nei-son, R. 1950. Verticillium wilt of pep- permint. Michigan Agricultural Experi- ment Station Bulletin 221. 259 p. Presley, J. T. 1941. Saltants from a mono- sporic culture of Verticillium albo-atrum. Phytopathology 31:1135-1139. Rankin, W. H. 1914. Thrombotic disease of maple. Phytopathology 4:395. Rawlins, T. E., and J. A. Booth. 1968. Tween 20 as an adjuvant for systemic soil fungicides for Verticillium in cotton. Plant Disease Reporter 52:944-945. Reid, J. 1958. Studies on the Pusaria which causes wilt in melons. Canadian Journal of Botany 36:394-410. Reinke, J., and G. Berthold. 1879. Die Zersetzung der Kartoffel durch Pilze. Untersuchungen des Botanischen Labora- toriums der Universitat Gottingen 1:1- 100. Rudolph, B. A. 1931. Verticillium hadro- mycosis. Hilgardia 5:197-353. ScHNATHORST, W. C, J. T. Presley, and H. R. Carns. 1967. Determination of the internal inoculum potential of Verticil- lium albo-atrum. in cotton plants. Phyto- pathology 57:101. ScHREiBER, L. R., W. K. Hock, and B. R. Roberts. 1971. Influence of planting media and soil sterilization on the uptake of benomyl by American elm seedlings. Phytopathology 61:1512-1515. Selman, I. W., and W. R. Buckley. 1959. Factors affecting the invasion of tomato roots by Verticillinm albo-atrum. British Mycological Society Transactions 42:227- 234. , and G. P. Pegg. 1957. An analysis of the growth response of young tomato plants to infection by Verticillium albo- atrum. Annals of Applied Biology 45: 674-681. Sewell, G. W. p., and J. F. Wilson. 1964. Occurrence and dispersal of Verticillium conidia in xylem sap of the hop (Humu- lus lupulus L.). Nature 204:901. Smith, Rose, and J. C. Walker. 1930. A cytological study of cabbage plants in strains susceptible or resistant to yel- lows. Journal of Agricultural Research 41:17-35. Stouohton, R. W. 1930. Thionin and orange G. for the differential staining of bacteria and fungi in plant tissue. An- nals of Applied Biology 17:162-164. Talboys, p. W. 1958. Association of tylosis and hyperplasia of the xylem with vas- cular invasion of the hop by Verticillium, albo-atrum. British Mycological Society Transactions 41 : 249-260. . 1958. Some mechanisms contrib- uting to Verticillium-resistance in the hop root. British Mycological Society Transactions 41:227-241. . 1964. A concept of the host-para- site relationship in Verticillium wilt disease. Nature. London. 202:361-364. Van den Ende, G. 1958. Untersuchungen iiber den Pflanzen-parasiten Verticillium, albo-atrum R. & B. Acta. Bontanica Neer- landica 7:665-740. Van der Meer, J. H. H. 1925. Verticillium wilt of herbaceous and woody plants. Meded. Landbouwhogeschool Wagenin- gen. 28:1-82. Van Hook, J. M. 1904. Disease of ginseng. Cornell Agricultural Experiment Station Bulletin 219:165-186. WiLHEXM, S. 1950. Verticillium wilt in acid soils. Phytopathology 40:776-777. , and J. B. Taylor. 1965. Control of Verticillium wilt of olive through nat- ural recovery and resistance. Phytopath- ology 55:310-316. Wollenweber, H. W. 1929. Die Wirtelpilz- Welkekrankheit (Verticillose) von Ulme, Ahorn and Linde usw. Arb. Biol. Reich- sanstalt Land-u. Porstwirtsch. Berlin- Dahlem. 17:273-299. INDEX A Acrostalagmus albus, 210 Appressorium, 217-218, 224 B Bananas Gros michel. 226 Lactan, 226 Benomyl, 237 Bravo 6P, 237 Budding, 224 C Cabbage, 224 Code of Verticillum isolates, 212-213 Conidia, 211, 216, 221, 224. 226, 240, 245 Cortical invasion resistant hosts, 218 susceptible hosts, 218 Cotton, 224, 226 D Disease control determination of, 238 Dry weight reduction of, 227 E Endoderrais, 211, 221. 223, 226 Fungal penetration lateral root, 217 intercellular, 217 intracellular, 217 root hairs, 217 root tip, 217 Fusarium species bulbigenum, 224 conglutinans, 224 oxysporium f. sp. lycopersici, 212 Green ash, 215, 226 Microsclerotial formation of excised roots effect of temperature on, 235 effect of heat on, 235 Mycelium, 217 N Nitrogen content in leaves. 231 determination of, 227 Panax quinguafoUum, 210 Pelargonium, 211 Peppermint, 224 Periderm, components of phellem, 214 phelloderm, 214 phellogen, 214 Pit, 221 Plasmodesmata, 224 Potato dextrose agar (PDA), 213 R Ray parenchyma, 224 Redbud, 213, 215, 226 Relationship of root wounds on infection type of, 213 age of, 213 Root cap, 217 Root elongatium, 217 Statistical analysis one way analysis of variance, 227 student "T" test, 227 Stem height mean value of, 227 Sycamore, 215 Systemic fungicide treatments foliar treatments, 243 root treatments, 243 soil drenches, 240 Honey locust, 215 Hyaline, 218. 224 Inoculum potential determination of, 237 Lateral root, 217 Leaf area determination of, 226 mean value of, 232 Leaf number mean value of, 227 M Maple, sugar, 213 Microsclerotia, 211-212, 218, 224, 226, 235,245 Thiabendazole, 212, 237 Trachieds, 215 Tyloses, 211, 214-215, 246 V Vascular invasion resistant hosts, 221 susceptible hosts, 218 Verticillium clahUae, 210 Vltavax, 237 W Water content of leaves determination of. 231 Wilt, symptoms of woody hosts, 209 tomatoes, 211 Wounds as sources of pathogen entry, 213 Some Publications of the ILLINOIS NATURAL HISTORY SURVEY I BULLETIN Volume 30, Article 7.—A Comparative Study of Two Components of the Poinsettia Root Rot Complex. By Robert S. Perry. August, 1971. 35 p., index. Volume 30, Article 8.—Dynamics of Condi- tion Parameters and Organ Measure- ments in Pheasants. By William L. An- derson. July, 1972. 44 p., index. Volume 31, Article 1.—The Effects of Sup- plemental Feeding and Fall Drawdowns on the Largemouth Bass and Bluegills at Ridge Lake, Illinois. By George W. Ben- nett, H. Wickliffe Adkins, and William F. Childers. January, 1973. 28 p., index. Volume 31, Article 2.—The Reproductive Cycle of the Raccoon in Illinois. By Glen C. Sanderson and A. V. Nalbandov. July, 1973. 57 p., index. Volume 31, Article 3.—Nutritional Re- sponses of Pheasants to Corn, with Spe- cial Reference to High-Lysine Corn. By Ronald F. Labisky and William L. An- derson. July, 1973. 26 p., index. BIOLOGICAL NOTES 75.—Illinois Birds: Turdidae. By Richard R. Graber, Jean W. Graber, and Ethelyn L. Kirk. November, 1971. 44 p. 76.—Illinois Streams: A Classification Based on Their Fishes and an Analysis of Factors Responsible for Disappearance of Native Species. By Philip W. Smith. November, 1971. 14 p. 77.—The Literature of Arthropods Asso- ciated witii Soybeans. I. A Bibliography of the Mexican Bean Beetle, Epilachna varivestis Mulsant (Coleoptera: Coc- cinellidae). By M. P. Nichols and M. Kogan. February, 1972. 20 p. 78.—The Literature of Arthropods Associ- ated with Soybeans. II. A Bibliography of the Southern Green Stink Bug, Nezara viridula (Linneaus) (Hemiptera: Pen- tatomidae). By N. B. DeWitt and G. L. Godfrey. March, 1972. 23 p. 79.—Combined Culture of Channel Catfish and Golden Shiners in Wading Pools. By D. Homer Buck, Richard J. Baur, Charles F. Thoits III, and C. Russell Rose. April, 1972. 12 p. 80.—Illinois Birds: Hirundinidae. By Rich- ard R. Graber, Jean W. Graber, and Ethelyn L. Kirk. August, 1972. 36 p. 81.—Annotated Checklist of the Butterflies of Illinois. By Roderick R. Irwin and John C. Downey. May, 1973. 60 p. 82.—Lactate Dehydrogenase Isozymes of Darters and the Inclusiveness of the Genus Percina. By Lawrence M. Page and Gregory S. Whitt. May, 1973. 7 p. 83.—Illinois Birds: Laniidae. By Richard R. Graber, Jean W. Graber, and Ethelyn L. Kirk. June, 1973. 18 p. 84.—Interactions of Intensive Cultures of Channel Catfish with Largemouth Bass in 1-Acre Ponds. By D. Homer Buck, Richard J. Baur, and C. Russell Rose. February, 1974. 8 p. 85.—The Literature of Arthropods Associ- ated with Soybeans. III. A Bibliography of the Bean Leaf Beetles, Gerotoma trifurcata (Forster) and C. ruficorni.i (Olivier) (Coleoptera: Chrysomelldae). By M. P. Nichols, M. Kogan, and G. P. Waldbauer. February, 1974. 16 p. 86.—Illinois Birds: Tyrannidae. By Rich- ard R. Graber, Jean W. Graber, and Ethelyn L. Kirk. February, 1974. 56 p. 87.—The Literature of Arthropods Associ- ated with Alfalfa. I. A Bibliography of the Spotted Alfalfa Aphid, Therioaphit, maculata (Buckton) (Homoptera: Aphi- dae). By D. W. Davis, M. P. Nichols, and E. J. Armbrust. February, 1974. 14 p. 88.—The Literature of Arthropods Associ- ated with Alfalfa. II. A Bibliography Of the Sitona Species, (Coleoptera: Cur- culionidae). By W. P. Morrison, B. C. Pass, M. P. Nichols, and E. J. Armbru8| February, 1974. 24 p. CIRCULAR 47.—Illinois Trees and Shrubs: Their In- sect Enemies. By L. L. English. July, 1970. (Fifth printing, with alterations.) 91 p. 51.—Illinois Trees : Selection, Planting, and Care. By J. Cedric Carter. August, 1966. 123 p. 52.—Fertilizing and Watering Trees. By Dan Neely and B. B. Himelick. Decern- ber, 1971. (Third printing.) 20 p. 53.—Dutch Elm Disease in Illinois. By J. Cedric Carter. October, 1967. 19 p. ,- List of available publications mailed on request No charge is made for publications of the Illinois Natukal History Survey. A single copy of most publications will be sent free to anyone requesting it until the supply be- comes low. Costly publications, more than one copy of a publication, and publications in short supply are subjects for special correspondence. Such correspondence should identify the writer and explain the use to be made of the publication or publications. Address orders and correspondence to the Chief, Illinois Natural History Survey