LIBRARY .THESlS I Michigan State University This is to certify that the thesis entitled THE ISOLATION, CHARACTERIZATION AND CONTROL OF RHIZOCTONIA CEREALIS CAUSING YELLOW PATCH ON POA PRATENSIS L. IN MICHIGAN presented by Cynthia L. Brown has been accepted towards fulfillment of the requirements for M.S. Plant Pathology degree in (I 7/2/flu. jor ”(professor Dr. J. M. Vargas, Jr. 0.7639 MS U is an Affirmative Action/Equal Opportunity Institution )VIESI_J RETURNING MATERIALS: Place in book drop to LJBRARJES remove this checkout from “ your record. FINES Will be charged if book is returned after the date stamped below. THE ISOLATION, CHARACTERIZATION AND CONTROL OF RHIZOCTONIA CEREALIS CAUSING YELLOW PATCH ON POA PRATENSIS L. IN MICHIGAN By Cynthia L. Brown A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1984 ABSTRACT THE ISOLATION, CHARACTERIZATION AND CONTROL OF RHIZOCTONIA CEREALIS CAUSING YELLON PATCH ON POA PRATENSIS L. IN MICHIGAN By Cynthia L. Brown A new disease was recognized on Poa pratensis in Michigan in 1981 and 1982. Symptoms developed during the spring and fall, appearing in patches or "frog-eye” patterns. A Rhizoctonia-like fungus was Consistently isolated from diseased foliar and crown tissues. The pathogenicity of these fungi was demonstrated in growth chamber and field studies. The fungi were characterized based on nuclear state, growth rate and cultural characteristics. The isolates were binucleate and produced buff pigmented mycelium and yellow to brown sclerotia. Hyphal anastamosis testing done with Ceratobasidium anastamosis group (CAG) testers revealed the isolates to anastamose only with the tester from CAGl, the group comprised of R. cerealis. The fungi were identified as ‘3. cerealis, reported to cause yellow patch of turfgrasses. Fungicides were screened for eventual use in a yellow patch management program. Iprodione, triadimefon, chlorothalonil, propiconazol and benomyl warrant further testing in field control studies. ACKNOWLEDGEMENTS I would like to express my gratitude to my major professor Dr. J. M. Vargas, Jr., and the members of my committee, Drs. Karen K. Baker and Paul E. Rieke, for their guidance throughout my studies. I also wish to thank Dr. Tom K. Danneberger and Ron Detweiler for their assistance. I am particularly grateful to Dr. David L. Roberts for his technical expertise and patient editing. Most important, I wish to thank my parents Cy and Gerry who are a never ending source of love and support. 11 TABLE OF CONTENTS Page LIST OF TABLES...... .............................................. ... v LIST OF FIGURES....... ...... . .......................... .............. vi IFTTRODUCTIOI‘ AND LITERATURE REVIEW‘.........OOOOCOOOOOO......OOOOOOOC 1 SECTION I. ETIOLOGY OF THE COOL SEASON BLIGHTING OF POA PRATENSIS L. IN MICHIGAN Materials and Methods....................... ..... .. ........ ....... 6 Diagnostic Description of Symptoms........................ ..... 6 Association and Isolation of Causal Organism. ........ .......... 7 Pathogenicity.................................................. 7 Growth chamber inoculations......... ...... ....... ........ ... 7 Field inoculations.......................................... 8 Environmental Conditions Associated with Natural symptom DevelopmentOO......0.00.0.0...0.0.0.0....OOOOOOOOOOOOOO 10 Results and Discussion............................................ 10 Diagnostic Description of Symptoms............................. 10 Association and Isolation of Causal Organism................... 15 Pathogenicity.................................................. 17 Growth chamber inoculations....................... ...... .... 17 Field inoculations.......................................... 17 Environmental Conditions Associated with Natural Symptom Development............................................ 22 SECTION II. CHARACTERIZATION/IDENTIFICATION OF THE RHIZOCTONIA SP. ISOLATED FROM COOL SEASON BLIGHTED POA PRATENSIS L. Materials and Methods........... ............. ............ ........ . 25 Cultural Characteristics...... ..... ............................ 25 Temperature-Growth Relations ..... ...... ....... ................. 25 Nuclear Staining................... ..... ....................... 26 Hyphal Anastamosis............................................. 26 RESUTtS and DiSCUSSionOOOOOOOOOO0.0......OOOOOOOOOOOOOOOOO00...... 28 Cultural Characteristics.......................... ............. 28 Temperature-Growth Relations.. ....... .......................... 28 Nuclear Staining ...................... .. ........ ............... 31 Hyphal Anastamosis...... ....................................... 31 The Identification of 3. cerealis .............................. 35 iii Page SECTION III. FUNGICIDE SCREENING FOR THE CHEMICAL MANAGEMENT OF R. CEREALIS ON A TURFGRASS HOST Materials and Methods ............................................. 38 ‘In Vitro Fungicide Bioassay .................................... 38 ‘lfl Vivo Fungicide Bioassay. ................ . ................... 39 Results and Discussion ............................................ 4O ‘Ig Vitro Fungicide Bioassay.... ................................ 40 In Vivo Fungicide Bioassay................. .................... 42 FUngicide Screening for the Chemical Management of R. cerealis ........ .............. ........................... 47 SUMMARY AND CONCLUSIONS. ..................................... .. ...... 52 LIST OF REFERENCES..... ..... . ...... ... ..... . ................. ........ 57 APPENDICES APPENDIX I ................................................. . ...... 60 APPENDIX II ...... .. ...... .... ....... . ............................. 61 iv Table LIST OF TABLES Page Rhizoctonia 5p. associated with the cool season blighting of Poa pratensis L. in Michigan.................... 16 Results after 30 days of field inoculations using five Rhizoctonia-like fungi isolated from cool season blighted Poa pratenSiS LOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 21 Mycelial pigmentation of several Rhizoctonia isolates on PDA after 40 days at 23°C in the absence Of lightOOOO..................OOOOOOOOOOOOOOO0......O 29 Hyphal pairing among the Rhizoctonia sp. isolated from cool season blighted Poa pratensis L. and the Ceratobasidium anastamosis group (CAG) testers to observe the occurrence of hyphal anastamosis.............. 33 lgVitro fungiCide bioassay.’0.0.0..........OOOOOOOOOOOOOOOOO 41 Figure 3 and 4 10 LIST OF FIGURES Page The placement of inoculum was standardized in the field inoculations by using a piece of poster board. Within each block the wheat seed was placed at each of the four corners of the inside squarECOOCCOOOOOOOOOO......OOOOOOO......IIOOOOOOOOOOOOO...O 9 The patch and "frog-eye" symptoms associated with the cool season blighting of Kentucky bluegrassinf’11Ch1‘gan.......OOOOOOOOOOOOIOOO0.0.00.0000...O 11 Features associated with the diseased Kentucky bluegrass. (3) Dark fungal nwcelium and sclerotia are found on the crown, sheath and rhizome tissues of the diseased plants. (4) Plants with reddened leaf blades are present at the margins of the diseased patches........................................... 12 The field symptom of the less severe cool season fOTTarb]ight'.I.I.0.000000000000000000I..OOOOOOOOOOOO0.0.014 The cool season pathogens exhibited hyphal morpho- logical and cultural characteristics commonly associated with Rhizoctonia solani. These include typical hyphal branching and septation g (a) and shades of brown mycelial pigmentation (b).......... 18 Foliar blighting caused by Michigan Rhizoctonia TSOIate 21.” the fieldTnOCUTationSoococo-00000000000000...19 The average daily air temperature and rainfall which preceded the development of naturally occurring foliar blight symptoms around May 31, 1982....................................................... 23 Mycelial fragments of the isolates were placed parallel on the water agar coated slides. The area of hyphal contact was stained and observations were made for the occurrence of hyphal anastamosis ....... .. 27 The mycelial growth rates of several binucleate Rhizoctonia isolates incubated at different temperatures in the absence of light. MI-I is an average of the growth rates of Michigan isolates 1, 2, 3, 5 and 6.................................. 30 vi Figure 11 12 13 14 Page Nuclear staining of several Rhizoctonia isolates employing HCl-Giemsa technique. The cool season turfgrass pathogens were all found to be binucleate (a) and the R. solani isolate appeared multinucleate (b)........................................... 32 The cell wall and cytoplasmic fusion which indicate the occurrence of hyphal anastamosis............... 34 The effect of fungicides on the lg_vitr0 growth of two 3. cerealis isolates. Mycelial growth was measured after five days on PDA amended with 100 ug of the fungicide active ingredient/ml POA................ 43 The effect of fungicide applications on the development of foliar blight symptoms. The rate of each of the funigicides is as follows: Daconil 2787 (Chlorothalonil) 4 02/1000 sq.ft. Bayleton (Triadimefon) .5 02/1000 sq.ft. Tersan 1991 (Benomyl) 1 02/1000 sq.ft. CGA-64250 (Propiconazol) .5 02/1000 sq.ft. Chipco 26019 (Iprodione) 2 02/1000 sq.ft. Vorlan (Vinclozdin) 2 02/1000 sq.ft. Within each block, bars topped with the same letter are not significantly different according to Duncan's Multiple Range Test, P=0.05............. ........ 45 vii INTRODUCTION AND LITERATURE REVIEW Kentucky bluegrass (Poa pratensis L.) is the most important and widely utilized of the cool season turfgrass species (2). It is native to Eurasia and has become widely distributed throughout the cool temperate and transitional climates of the world (2). It is a perennial grass propagated primarily by seed; however, with vigorous rhizome development and an extensive root system, it is widely used in commercial sod production (2). When properly maintained, Kentucky bluegrass can form a high quality turf for lawns, athletic fields, golf courses and other general purpose turf areas. Considerable variability in leaf texture and color, growth habit, shoot density, rhizome development and particularly disease resistance exists among cultivars of Kentucky bluegrass (2). Some diseases that may cause severe problems on Kentucky bluegrass include rust, stripe smut, Fusarium blight, the snow molds, Pythium blight, dollar spot and brown patch. These diseases are of fungal etiology and can generally be managed by early diagnosis, the implementation of wise cultural practices and carefully timed fungicide applications. A new problem was recognized through the mid to late seventies when turfgrass researchers reported disease symptoms on Kentucky bluegrass, bentgrass (Agrostis spp.), and zoysiagrass (ggysla_spp.) (11,30,31). The symptoms appeared during periods of cool, wet weather and were described as patches of foliar blighted turf or chlorotic "frog-eyes”. The "frog-eyes" closely resembled those associated with the disease Fusarium blight which led to the suggestion that possibly a "cool weather Fusarium" was responsible (Vargas, personal communication). Fungi that resembled Rhizoctonia solani Kuhn in hyphal and cultural appearance were isolated from the chlorotic "frog-eyes” or foliar blight lesions (11,30,31). R, splani, the cause of brown patch of turfgrasses, is usually associated with periods of humid weather and air temperatures in the mid-eighties (10,36). The organism inciting the newly recognized disease problem was termed the "cool weather Rhizocton a" (31). In 1977 Sanders et al. (31) reported preliminary results of studies on the "cool weather Riizoctonia" turfgrass pathogens. The majority of the isolates were binucleate and believed to be species of Ceratobasidium. The Optimum temperature range for lg vitrg growth of the binucleate isolates was 21-23°C. Greenhouse pathogenicity tests conducted at 10, 17-20, 21-26 and 32-38°C demonstrated that the binucleate isolates were highly virulent on bentgrass over a wide range of temperatures. Nearly 100% foliar blighting occurred within the range of 10-26°C. In contrast, an isolate Of.3.'§ngfli causing brown patch on bentgrass was found to have an Optimal in xitrg_growth temperature of 28°C and caused no apparent symptoms on bentgrass below 17°C. These growth and pathogenicity differences between the binucleate and multinucleate Rhizoctonia spp. are in agreement with the temperatures at which the respective disease symptoms occurred in the field. The characteristics considered most critical for the accurate identification 0T.3-.§Qlflfli are multinucleate hyphal cells with prominent septal pore apparatus and the production of the Thanatephorus cucumeris sexual (perfect) state (13). In 1967 Parmeter et al. (26) reported the finding of R. solafli-like fungi isolated in the United States from a number of hosts including strawberry, potato and Bermudagrass. Comparative studies found that isolates with hyphal and cultural characteristics of R. solani could be separated into two distinct groups. One group had multinucleate hyphal cells and produced a.I. cucumeris sexual state, and the other group had_binucleate hyphal cells and produced a Ceratobasidium sexual state. The perfect state of the binucleate isoltes could be assigned to Ceratobasidium Rogers on the basis of fruiting habit, basidial morphology and repetitive spore germination as discussed by Talbot (33). On the average, the binucleate isolates had smaller hyphal diameters and slower growth rates than the multinucleate isolates. The identification of Rhizoctonia species is complicated by the fact that many of the Rhizoctonia fungi possess asexual structures that are morphologically similar (34). Incorrect identification of some fungi as 3. §91221_has undoubtedly occurred in the past because of the heavy reliance on features that are highly variable and not unique to R. 591321 (31). Such characteristics as branching patterns, dolipore septation and shades of brown mycelial pigmentation occur in numerous Rhizoctonia species (34). ‘R. cerealis was described by van der Hoeven (4) as being distinguishable from R. solani by its slower radial growth rate, smaller hyphal diameters and binucleate hyphal cells. 0f the four Rhizoctonia Spp. recognized as pathogens on cultivated turfgrasses, R, solagi and R. ggag Voorhees are multinucleate, 3. 951533 Ryker and Gooch has four nuclei per cell and R. cerealis is binucleate (32). Burpee suggested that host specificity and anastamosis reaction may be of taxonomic value in identifying R. cerealis (6). In 1980, Burpee et al. (8) established seven Ceratobasidium anastamosis groups (CAG) based on hyphal anastamosis. Pairings were made among isolates of C, cornigerum (Bourd) Rogers, C. EEEEEé and related binucleate fungi having Rhizoctonia imperfect states. Seven CAG were established and the only CAG to exhibit any homogeneity with regards to the host of the isolates was CAG1. The isolates in CAGl were isolated from hosts of the Gramineae and included the "cool weather Rhizoctonia" turfgrass pathogens. Burpee (6) identified the "cool weather Rhizoctonia" turfgrass pathogens as R, cerealis van der Hoeven. .3. cerealis was originally described in 1977 as the causal agent of sharp eyespot lesions on wheat in the Netherlands (4). Identification was based on hyphal anastamosis between the isolates of CAGl and the R. cerealis type culture. The R. cerealis type culture failed to anastamose with CAGZ through CAG7 (6). Anastamosis has been useful in the identification of other fungi since hyphal anastamosis is considered evidence that two mycelia represent the same species (12). The R, cerealis isolates collected to date have been host specific to the Gramineae and make up the CAGl which is distinct from 3, 591331 and other Rhizoctonia spp. (6). Lipps and Herr (21) suggest that growth raté may facilitate in the separation of R. cerealis from other binucleate isolates. For the disease formerly known as cool weather brown patch caused by the ”cool weather Rhizoctonia" Burpee (6) proposed assigning the name "yellow patch" to the disease of cultivated turfgrasses caused by R, cerealis. Symptoms have been observed primarily on creeping bentgrass and Kentucky bluegrass (9), however, there have also been reports of symptoms occurring on tall fescue, zoysiagrass, Bermudagrass and St. Augustinegrass (6,17). Greenhouse host range studies indicated that perennial ryegrass (Lolium perenne) and tall fescue (Festuca arundinacea) were susceptible and may be potential hosts (7). Koch's postulates have been satisfied on a turfgrass host primarily for the foliar blight symptom and have been performed in the greenhouse and the growth chamber. No fungicides are currently labelled for management of R. cerealis on turfgrasses (17). Sanders et al. (31) reported significant radial growth inhibition of the "cool weather Rhizoctonia" lg vitro with chloroneb, chlorothalonil, benomyl 0r iprodione. Martin (24) examined the sensitivity of binucleate R. solani-like fungi isolated from tall fescue to benomyl, carboxin and PCNB. lfl‘llELQ radial growth inhibition was greatest on the benomyl amended POA. Van der Hoeven et al. (35) reported radial growth inhibition of several isolates of R. cerealis from wheat on benomyl amended PDA. There are no reports of any i_ vivo greenhouse or field fungicide evaluation studies. During 1981 and 1982 Kentucky bluegrass exhibited foliar blight and/or crown rot in many locations in Michigan. 3. solaniflike fungi were consistently isolated from the affected tissues. Macro symptoms in the field appeared as "frog-eyes" or patches of dead grass or round patches of foliar blighted turf. The symptoms appeared in the spring and fall which are seasons of the year normally associated with cooler environmental conditions. The objectives of the following investigations were to 1) describe the symptomatology, 2) determine if the etiology of this new disease in Michigan is R, cerealis, and 3) screen potential antifungal control chemicals. SECTION I ETIOLOGY OF THE COOL SEASON BLIGHTING OF POA PRATENSIS L. IN MICHIGAN Considerable confusion and speculation followed the recognition in the late seventies of unusual symptoms occurring during cool weather on Michigan home lawns. Symptoms of the disease were associated with Kentucky bluegrass sod during "cooler" environmental conditions; however, the etiology of the problem remained unknown. The objectives of these investigations were to 1) determine the causal agent of the symptoms and 2) provide a useful diagnostic description of the symptoms incited by the organism. Materials and Methods Diagnostic Descript on of Symptoms. Kentucky bluegrass plants affected with this disease were observed lg situ for macr0500pic symptoms at several locations. Photographs were taken and samples of diseased turfgrass were brought to the laboratory for microsc0pic observation. Numerous samples were also received by Michigan State University during the periods of spring and fall, 1981 and 1982. Leaf blades, crowns and roots were removed from samples and closely examined for any lesions and microorganisms with the aid of a binocular stereo microscope and a compound microscope. Association and Isolation of the Causal Organism. Diseased leaf and crown tissues were washed in tap water, surface disinfected in 10.0% sodium hypochlorite solution for 1-2 minutes, rinsed in sterile deionized water and placed in petri plates containing either potato dextrose agar (PDA, Gibco Laboratories, Madison, Wisconsin) or water agar (Bacto-Agar, Difco Laboratories, Detroit, Michigan). The plates were incubated in the laboratory under a 12 hour light/dark cycle at 21:1°C and examined periodically for the growth of potential pathogens. Pure culture, isolates of fungi were obtained by transferring hyphal tips to PDA and water agar slants and stored at 4°C. Pathogenicity. To determine the pathogenicity of the isolated fungi, inoculations were performed in a growth chamber. Field inocula- tion studies were conducted in an attempt to duplicate disease symptoms under natural conditions. All inoculum was prepared by autoclaving 100 ml of whole wheat seeds and 75 ml of distilled water in a 500 ml Erlenmeyer flask for one hour on two consecutive days. Colonized agar disks from pure cultures of each isolate were transferred to the flasks and incubated at 21¢1°C for 14 days. Inoculum was removed from the flasks and stored in plastic bags at 4°C until use. Growth chamber inoculations: Inoculations were made by placing three wheat seeds infested with an isolate at the base of the leaf blades of six week old 'Touchdown' Kentucky bluegrass plants grown in the greenhouse in three inch plastic pots. The plants were fertilized twice with a moderate rate of complete soluble fertilizer and trimmed to a height of 2 1/2-3" every 10 days. Autoclaved, non-fungus-infested wheat seeds were used to inoculate the control plants. Each treatment was replicated four times. After inoculation the plants were misted with sterile distilled water and placed in plastic bags in temperature controlled growth chambers. The temperature and relative humidity inside the bags were maintained at 20:2°C and 100%, respectively. The photoperiod in the growth chamber was 12 hours. The pots were misted once daily with sterile deionized water to maintain leaf wetness. Four days after the inoculations were made the pots were removed from the plastic bags, disease severity evaluations were made and attempts were made to reisolate the fungi from the necrotic tissues. The inoculation procedure was repeated one time. Field inoculations: Field inoculations were made on a block area of two year old seeded 'Fylking' Kentucky bluegrass located at the Michigan State University Hancock Turfgrass Research Center in East Lansing, Michigan. Ten pieces of infested wheat seed were placed at the base of the plants within the thatch layer at each inoculation site. Placement of the inoculum was standardized within each 4 X 4' block by using a piece of poster board from which a square had been cut out of the center (Figure 1). The inoculum was placed at each of the four corners of the center square. One square was used for the four replications of each isolate. Inoculations were done on September 14, 1982, using Michigan isolates 1 through 5 and autoclaved, non-fungus-infested wheat seeds to inoculate the control block. The inoculation area was fertilized twice with urea during the summer of 1982. One pound of nitrogen per thousand square feet was applied in June and another pound at the end of July. Following the inoculations the area was irrigated only as necessary to prevent wilting. No cover was placed over the inoculated area. Thirty days following inoculation disease symptom ratings were made. Disease symptom ratings were made by examining two variables: the width of the Figure 1. Cr: \.. “'. I. ‘ . . ivfik \wa’g. . _ ‘1‘ ‘3 it . ' ’52h.» .1 Y ' , ”.4 v \ .Wq‘lifit'u. 4f The placement of inoculum was standardized in the field inoculations by using a piece of poster board. Within each block the wheat seed was placed at each of the four corners of the inside square. 10 affected patch and the severity of the damage within the patch as described on page 21. Attempts were made to isolate the fungi from necrotic tissues. Environmental Conditions Associated with Natural Symptom Develop- BEER: Measurements of the daily rainfall and air temperature were recorded throughout the spring of 1982 at the MSU Hancock Turfgrass Research Center. The amount of rainfall was measured by a rain gauge and the average daily air temperature was calculated as an average of twenty-four hourly readings recorded by a hygrothermograph. Results and Discussion Diagnostic Description of Symptoms. Two distinct symptoms atypical for that time of the year were observed on Kentucky bluegrass in Michigan. The first symptom was noted to occur on sodded turfs. The plants were killed in either a patch or "frog-eye" pattern (Figure 2) and under microscopic examination exhibited a reddish-brown crown and root necrosis. Dark colored sclerotia were abundant on the necrotic tissues (Figure 3). Dark colored fungal mycelium was also found to be present on sheath and crown tissues, growing along the junctures between epidermal cells. Plants showing no apparent symptoms exhibited neither the rot or the presence of the fungal structures. Plants with reddened leaf blades were often present in the outer margins of the diseased patches (Figure 4). N0 wilting stage was associated with symptom development. Little recovery occurred during the season of injury. These symptons are similar to those of yellow patch as described by Joyner (17) and cold temperature brown patch as described by Hirrell and Shurtleff (15). The patch and "frog-eye" symptoms associated with the cool season blighting of Kentucky bluegrass in Michigan. Figure 2. 12 Q -o‘ \. , sheath and rhizome tissues of the (3) Dark fungal mycelium and sclerotia are (4) Plants with reddened leaf blades Mr... L s . .. {... _ . D I Features associated with the diseased Kentucky are present at the margins of the diseased patches. found on the crown diseased plants. bluegrass. Figures 3 and 4. 13 The second symptom is a blighting which affects foliar tissues. It has been reported infrequently in Michigan, occurring primarily on seeded turfstands. The blighting occurs in round patches 15-100 cm in diameter and causes the turf to become tan to bleached in color (Figure 5). The discoloration of the turf results from the extensive foliar blighting. Foliar lesions ranged in size from 0.5 to 2.5 cm in length, and usually encompassed the entire width of the blade. The affected area on the leaf was straw to ash brown in color and often surrounded by a darker colored border. The crown and root tissues of these plants appeared healthy and within one month the foliar lesions had been mowed off, leaving little indication of the earlier present necrotic symptom. These symptoms are similar to those of yellow patch as described by Burpee (6) and those caused by the "cool weather Rhizoctonia" as described by Sanders et al. (31) and Dale (11). Early observations suggested an apparent difference in the severity and occurrence of symptoms on sodded versus seeded lawns. Five of the six early reports of this disease problem were on sodded lawns less than five years old. Upon consideration of these two methods of lawn establishment two major factors are noted which any be contributing to the variation. First, the turfgrass species and cultivars used in sodded and seeded lawns in Michigan are usually different. The infected sod has been reported to be Kentucky bluegrass blends of three or four of the new "improved" cultivars such as 'Touchdown', 'Cheri', 'Adelphi', 'Victa', and 'Baron'. In contrast, seed purchased by the homeowner for the planting of a lawn usually contains the seeds of common or older Kentucky bluegrass cultivars, fine leaf fescue or ryegrass, but little or none of the "improved" Kentucky bluegrass cultivars. The fact that the cultivars and 14 Figure 5. The field symptom of the less severe cool season foliar blight. 15 plant species which are used on sod and seed are different suggests the role genetic factors may play in disease development. A factor which may contribute to the increased susceptibility of some sod plantings to this disease is the adverse conditions under which sod is sometimes established. Proper site preparation is critical for the successful establishment of any turf area (3,28) and it is not unusual to visit a site where the topsoil was removed and sod laid directly onto the clay subsoil. This type of soil compacts easily and is not favorable to root development (3,28). Sodded turf is often associated with rapid thatch accumulation. The disadvantages of a thick thatch are numerous and include decreased heat, cold and drought tolerance and increased insect and disease problems (2). The fact that disease symptoms have not been reported to occur before the sod has been cut and removed from the farm and that a sod farmer may sell his sod to several different customers and only one customer's lawn develops disease symptoms suggests that establishment conditions may play a role in predisposing potentially susceptible plants to this disease. Association and Isolation of the Causal Organism. Several isolates of a Rhizoctonia sp. were obtained from the infected tissues as listed in Table 1. It was noted that the isolation of the Rhizoctonia Sp. was more difficult from crown than from foliar tissues. Water agar supported more rapid growth of the Rhizoctonia sp. and facilitated in the separation of the Rhizoctonia Sp. from the other fungi present on the plant tissues. Tentative identification of the fungi as Rhizoctonia Sp. was based exclusively on the hyphal morphological and cultural characteristics as described for R. solani by Parmeter (27). These characteristics include 1) branching near the distal septum of cells in young vegetative hyphae, 16 Table 1. Rhizoctonia Sp. associated with the cool season blighting of Poa pratentsis L. in Michigan Isolate Designation Geographical Location Plant Tissue 1 Lansing, Michigan Foliage 2 Detroit, Michigan Crown 3 Lansing, Michigan Crown 4 Flint, Michigan Foliage 5 Pontiac, Michigan Foliage 6 Lansing, Michigan Foliage 17 2) constriction of the branch and the formation of a septum in the branch near the point of origin and 3) some shade of brown pigmentation in culture (Figures 6a and b). Pathogenicity. Growth chamber inoculations: Michigan isolates 1 through 6 caused disease symptoms in all of the performed inoculations. The fungi had grown out from the wheat seeds within 24 hours and within 48 hours watersoaking of the leaf tissues had occurred. Leaves became matted together with mycelium and the watersoaked areas turned tan to golden-orange in color.' Lesions were present on 40-50% of the leaf blades after four days in all four of the inoculation pots tested with each isolate. No lesions were present on any of the control plants. Ten blades with necrotic lesions were removed from the inoculation pots of each isolate and surface disinfected for 2 minutes in 10.0% sodium hypochlorite solution. The blades were rinsed in sterile deionized water and plated out on PDA. A Rhizoctonia Sp. was isolated from 100% of the necrotic blades. A Rhizoctonia Sp. was not isolated from ten blades from the control plants. Field inoculations: Several of the isolates tested caused symptom development in the field inoculations. The infected plants occurred in round patches ranging in diameter from 1-14 cm. Symptom severity within each patch varied from a few foliar lesions to nearly 100% foliar blight as shown in Figure 7. Foliar symptoms included bleached lesions with dark borders and reddened leaf blades. Some plants exhibited stem discoloration and a limited number of plants were killed; the crown and root systems heavily rotted. No dark mycelium or sclerotia were present on any of the necrotic tissues. The control inoculations showed no season pathogens exhibited hyphal morphological and cultural characteristics commonly associated with Rhizoctonia solani. These include typical hyphal branching and septation (a) and shades of brown mycelial pigmentation (3). Figure 6. The cool 19 A .l.‘ JV» r Blighting caused by Michigan Rhizoctonia isolate 2 in the field inoculations. Figure 7. 20 evidence of foliar lesions or leaf reddening. The data from the field inoculations are presented in Table 2. A number of blighted plants were removed from the patches. From these plants ten necrotic blades and four crowns were washed in tap water for four hours, surface disinfected in 10% sodium hypochlorite for 1-2 minutes, rinsed in sterile deionized water and plated out on PDA. A Rhizoctonia Sp. was isolated from 100% of the leaf tissue tested. No Rhizoctonia Sp. was isolated from the crowns. An additional attempt to isolate a Rhizoctonia Sp. from three partially rotted crowns failed. No Rhizoctonia Sp. was isolated from blades or crowns of plants from within the control block. The preceding growth chamber and field inoculations have demonstrated the pathogenicity of the isolated Rhizoctonia Sp. on a Egg pratensis host. The production of symptoms similar to those of natural infections supports an association of these fungi with the cool season blighting of turf in Michigan in 1981 and 1982. Based on the demonstrated pathogenicity of the Rhizoctonia Sp. isolated from cool seson blighted turfgrass it is hypothesized that these fungi are iSolates of I'cool weather Rhizoctonia" or R. cerealis, the causal agent of the turfgrass disease yellow patch. The success of the inoculation technique is of paramount importance due to the difficulty researchers have had conducting field studies. Although there was considerable variation between the replicates of each isolate this may have been due to some variation in the inoculum such as unequal amounts of mycelial growth on the seeds. If that was the problem, it could be easily rectified and this inoculation method used in fungicide and fertility studies. At the present time there are no Table 2. Results after 30 days of field inoculations using five Rhizoctonia-like fungi isolated from cool season blighted Egg pratensis L. Diameter of Symptom Severity Block No./Isolate No. Affected Area Within Measured Area (cm) (as described below) 1 / 5 7 1 9 2 12 2 2 / 4 6 1 6 1 8 3 12 1 3 / Control - _ 4 / 2 6 2 5 1 14 5 8 3 5/1 _ _ 11 1 5 1 6/3 _ _ Symptom Severity Scale: Symptoms just visible; foliar lesions or reddening. up to 25% of the plants within measured area blighted. 25-50% of the plants within measured area blighted. 50-75% of the plants within measured area blighted. 75-100% of the plants within measured area blighted. 01-9me H II II II II 22 evaluations regarding the susceptibility of various Kentucky bluegrass cultivars to disease symptoms caused by these Rhizoctonia Sp. Field inoculations on different cultivars of seeded and sodded turf may also be useful in establishing the role of genetic factors in disease development. Environmental Conditions Associated with Natural Symptom Develop- mgpp. The rainfall and air temperature conditions which preceded the appearance of the Rhizoctonia Sp. incited foliar blight symptoms on a seeded block of 'Fylking' Kentucky bluegrass in Michigan were recorded (Figure 8). This area did not receive any irrigation. The natural symptoms (Figure 5) appeared over the weekend of May 31, 1982. The average daily air temperature was between 19 and 23°C for approximately five days before the time of symptom appearance. A temperature pattern very Similar was noted between the 12th and 16th, however, no symptoms developed. Although air temperatures were similar during these two time periOds the symptoms appear to have developed only after an increase in the amount of rainfall. This is preliminary data, however, it supports the hypothesis that these Rhizoctonia fungi are "cool weather Rhizoctonia" as described by Sanders et al. (31). 23 Fimire £3. The average daily air temperature and rainfall which preceded the development of naturally occurring foliar blight symptoms on or close to May 31, 1982. 24 SYMPTOMS 7 JUNE ,/////////////25 31 ,/’//////////////////53/55/2555?. 3’3x/é55/5/5/755/55’2?2‘ 9/553553/3/55 24 1'7 10 Y 139 T M“ h r H6 T2 1 R 1 ER .- In 1 NV .....n .9 Km. 1 cc 0 c- No DUN HI 2“ 2 ma 1 DH SP 4 d 4i.- 1 d 4 q 4 R 0 0 O 0 8 6 4 2 meruz_u 2::— Figure 8. SECTION II CHARACTERIZATION/IDENTIFICATION OF THE RHIZOCTONIA SP. ISOLATED FROM COOL SEASON BLIGHTED EQA‘ERATENSIS L. In the previous findings a Rhizoctonia-like fungus was consistently associated with cool season blighted Kentucky bluegrass in Michigan. The objectives of these investigations are to characterize and identify the isolated Rhizoctonia fungi. Culture on agar, temperature-growth relations, nuclear state and hyphal anastomosis are characteristics that ‘ are commonly used in identifying Rhizoctonia spp. Materials and Methods Cultural Characteristics. Agar disks 5 mm in diameter were cut from the margins of actively growing colonies and transferred to 15 mm X 100 mm plastic petri plates containing 20 ml of PDA. The plates were incubated in darkness at 21:1°C. After 40 days the cultural and sclerotial characteristics of each of the Michigan isolates, the Ceratobasidium anastamosis group testers (CAGl-CAG7) and an isolate of R, £91331 were compared. The CAG testers were obtained from Patricia Sanders, Pennsylvania State University, University Park, Pennsylvania and Brian Olsen, Cornell University, Geneva, New York. Temperature-Growth Relations. To compare the 13 31559 growth rates of each isolate at various temperatures agar disks 5 mm in diameter were cut with a cork borer from the margins of actively growing colonies and 25 26 transferred to 15 mm X 100 mm plastic petri plates containing 20 ml of PDA. Each isolate was incubated at temperatures of 10, 16, 23 and 28°C in the dark. Colony diameter was measured at 24 hour intervals for up to 6 days. Each treatment was replicated three times. Nuclear Staining. Each isolate was stained using an HCl-Giemsa nuclear staining technique developed by Herr (13). This technique allows the determination of the number of nuclei in vegetative cells. An isolate known to be 3. pplppi was stained as a control. Hyphal Anastamosis. To observe hyphal anastamosis a technique described by Herr and Roberts (14) was employed. The method involves the pairing of unknown isolates with known tester isolates followed by examination of the point of contact between the hyphal growth of the two isolates. Autoclaved microscope slides were dipped in molten autoclaved 2% water agar and pfip for the maintenance of moisture. 3 mm X 15 mm sections of the fungal isolates to be paired were removed from the margins of colonies actively growing on PDA and placed approximately 2.5 cm apart on the agar-coated Slide (Figure 9). The petri plates were covered, placed in plastic bags and incubated at room temperature. When the advancing hyphae from the opposing colonies had made contact and were slightly overlapped the Slides were removed from the Petri plate. A drop of 0.05% cotton blue was placed on the area of hyphal contact, covered with a cover slip and examination for hyphal anastamosis was made. The Michigan isolates were opposed in all possible combinations and with known testers from CAGI (Ceratobasidium anastomosis group) through CAG7. Figure 9. 27 Mycelial fragments of the isolates were placed parallel on water agar coated slides. The area of hyphal contact was stained and observations made for the occurrence of hyphal anastamosis. 28 Results and Discussion Cultural Characteristics. Variation existed in the mycelial and sclerotial characteristics of the Rhizoctonia isolates examined, but some trends were apparent (Table 3). With the exception of isolate 4, all of the Michigan Rhizoctonia fungi isolated from the cool season blighted turfgrass produced mycelium which was buff in color. The pigmentation of isolates increased with time to darker shades of brown (Figure 6). Yellow to brown pigmented sclerotia formed on the culture surface of Michigan isolates 1, 2, 3, 5 and 6. Michigan isolates 1, 3 and 6 showed obvious zonation. Michigan isolate 4 produced mycelium white in color and no surface sclerotia. The CAG 1 and 3 testers produced a buff colored mycelium and brown sclerotia. Testers from CAG 2, 4, 6 and 7 all produced mycelium closer to white in color. The CAG 5 tester produced mycelium that was darker in color, more Similar in pigmentation to that of the R, éplppi_isolate. A similarity was apparent between the mycelial pigmentation of the CAG 1 and 3 testers and Michigan isolates 1, 2, 3, 5 and 6. Burpee (6) reported that the pigmentation of 3, cerealis isolates from turfgrasses was buff to light brown in color after eight weekS of growth at 23°C in the absence of light. -— Temperature-Growth Relations. The influence of temperature on the lfl.!i££2 mycelial growth rate of the Michigan isolates and the CAG testers is shown in Figure 10. The data are presented in Appendices 1 and 2. There was limited variation in the growth rates of Michigan isolates 1, 2, 3,_5 and 6 at all temperatures. Compared to those isolates, Michigan isolate 4 exhibited significantly different growth rates at all temperatures tested. The CAG 1 tester and Michigan isolates 1, 2, 3, 5 and 6 exhibited Similar growth relations at all of 29 Table 3. Mycelial pigmentation of several Rhizoctonia isolates on PDA after 40 days at 23°C in the absence of light. Dirty White Buff Light Brown CAG 2, 4, 6 and 7 Michigan 1, 2, 3, 5 and 6 CAG 5 tester Michigan 4 CAG 1 and 3 testers R. solani 3O 7???? 2% 25b 7%,, 7/2 2% Sea 73%??? 263/662 Balm E 55%. 85-5 742%; 23%: 25$ 2??? ZZZ $01.55. 2??? 262,2 :75 2 So; m a m m m EL 0 505 5050550 5 21.]. 5mm: VN\:uH rezomo 4¢H4m0>z MI-I is an average of the growth rates of Michigan The mycelial growth rates of several binucleate Rhizoctonia isolates 1, 2, 3, 5 and 6. isolates incubated at different temperatures in the absence of light. Figure 10. 31 the temperatures tested. Compared to the other Rhizoctonia isolates, Michigan isolates 1, 2, 3, 5 and 6 and the CAG 1 tester grew more rapidly at 10°C and more Slowly at 28°C than any of the other isolates with the exception of CA0 6. The average optimal growth rate of Michigan isolates 1, 2, 3, 5 and 6 was 0.78 cm/24 hours at 23°C. This is close to the growth rate of the CAG 1 tester, 0.80 cm/24 hours at 23°C. These results compare favorably with those of other researchers. Burpee (6) reported the optimal ipflyltgp growth rate of several 3. cerealis isolates from turfgrasses occurs at 23°C. Lipps and Herr (21) reported the optimal growth rate of an R. cereal1§_type culture to be 0.72 cm/24 hours at 23°C. Nuclear Staining. Based on the nuclear staining, Michigan isolates 1 through 6 were all found to have binucleate vegetative hyphal cells (Figure 11a) and by comparison, the R. solani isolate appeared to be multinucleate (Figure 11b). 0f the Rhizoctonia spp. recognized as pathogens of turfgrasses, R, cerealis is the only one with predominantly binucleate hyphal cells (32). Hyphal Anastamosis. The vegetative hyphae of five Michigan isolates (isolates 1, 2, 3, 5 and 6) anastamosed with the CAG 1 tester isolate and failed to anastamose with the CAG 2 through CAG 7 testers (Table 4). Michigan isolates 1, 2, 3, 5 and 6 anastamosed with each other in all possible combinations. Cell wall and cytoplasmic fusion as described by Parmeter (25) indicate the occurrence of hyphal anastamosis (Figure 12). Michigan isolate 4 failed to anastamose with the CAG 1 tester or any of the other Michigan isolates. The CAG 1 is known to be made up of R. cerealis and is distinct from 3, solani and other binucleate Rhizoctonia spp. (6). Based on the Figure 11. 32 Nuclear staining of several Rhizoctonia isolates employing the HCl-Giemsa technique. The cool season turfgrass pathogens were all found to be binucleate (a) and the R. solani isolate appeared multinucleate (b). 33 .m_moEmummce .ezaxz ucmceaam oz 1 .A=o_m:5 o_Emm_aouxu tee ..ez __mov wwmoseummce .ecaxc 5o :o_um>cmmao we» mwuocmo + 1 1 1 1 1 1 + + 1 + + + , m 1 1 1 1 1 1 + + 1 + + + m 1 1 1 1 1 1 a 1 1 1 1 1 1 + + + 1 + + m 1 1 1 1 1 1 + + + 1 + + N 1 1 1 1 1 1 + + + 1 + + H ~ocmmao cu mcmummu Ao a :_:u_: mm=_e> c a.m a a.m a a.m e a.m a a.m c a.m o e.m a e.m m m.m a a.e c a.e c a.e a e.m a 9.5 a 0.5 _otueoo a 5.o an e.o a 5.0 a ~.o a 5._ a “.4 ,5 5.o e m.5 a a.m a o.o a ~.~ 0 ”.5 a o.o a 0.5 e.~.e 0e555mmwm_wse 0 5.5 e e.~ a m.m u ~.~ e N." a «.5 a m.~ c ~.~ a 0.5 a ~.~ a n.m c Q.“ a m.“ a _.N a m.e =__5~oue_> 0 o.o a o.o a m.o a o.o a o.e a 5.0 a o.o a e.o a e.o a o.e . o.e a e.o a e.o a o.e 5 m.o aeo_eoLa_ o 0.0 a 0.0 a ~.H a 0.0 a ~.0 a n.— a 0.0 a _.0 u 0." o 0.0 a 0.0 u n.~ , a 0.0 a 0.0 n m.~ po~o=ou_qoca a m.0 u N.~ m N.m a ~.0 u 0.0 m 0.0 a m.0 a m.~ m N.m a m.0 u v.H u 5.0 . o ~.0 a 0.0 u #.e , oc_~m__c< a 0.0 a 0.0 u ~.¢ a 0.0 a 0.0 u m.¢ o 0.0 a 0.0 m ~.m a 0.0 a 0.0 u 0.0 a 0.0 a 0.0 u «.0 .bcocmm on ~.0 a «.0 u 0.m an “.0 u m.0 u 0.~ a ".0 a ~.0 u 0.~ a v.0 n 0.0 u 0.m . n 0.0 a 0.0 u v.~ commepoopcp nu m.0 on 0.0 a 0.“ on ~.0 u n.0 o 5.0 a m.0 u 0.0 a 0.~ a 0.0 a 0.0 a ¢.~ a 0.0 a 0.0 u 0.~ __:o_o:uoco—:0 500 000— Eng 00" 800 a 5001000" 500 00a Emu N 500 000“ =50100“ Emu n 500 000~ 500 00g Emmi” E001000“ 800 00— 500 ~ 0 mum—om_ m mum—om_ m wee—om_ N mun—om_ ~ mun—omm mu_o_me:d I .mmm.u.m=mh m=o_co> 0o mayo; omega =u.3 umuean :— .m o_ae5 42 At the 100 ppm a.i. concentration iprodione, propiconazol and benomyl produced the most significant and consistent growth inhibition of all isolates. Benomyl had produced little growth inhibition at the 1 ppm a.i. concentration, however, at this rate it prevented any fungal growth. Triadimefon and chlorothalonil provided significant growth inhibition, however, statistically they were not as efficacious as iprodione, propiconazol and benomyl (Figure 13). The degree of growth inhibition elicited at this rate by cyclohexamide + PCNB and anilazine was Significantly less than the other fungicides. The least effective compound was vindozolin. With the exception of vinclozolin all of the fungicides virtually prevented fungal growth at the 1000 ppm a.i. concentration. For some of the compounds, i.e., chlorothalonil and triadimefon, the increase in the a.i. concentration from 100 ppm to 1000 ppm incited litle additional growth suppression. Figure 13 illustrates the Similarity in the reactions of isolates 2 and 3 to each of the fungicides at the 100 ppm a.i. concentration. Isolates 1, 5 and 6 exhibited similar reactions. The repeat of thiS experiment produced the same results. In Vivo Fungicide Bioassay. All of the fungicides provided the inoculated plants with excellent protection from disease even after incubation for eight days in conditions which have been Shown to be favorable for disease development (Section I). The fungicide treatments had a significant effect on the development of foliar disease symptoms (Figure 14). In all cases where no disease symptoms developed there was little or no fungal mycelial growth away from the infested wheat Seeds. There was Figure 13. 43 The effect of fungicides on the in vitro growth of two R, cerealis isolates. Mycelial groWEh was measured after five days on PDA amended with 100 ug fungicide active ingredient/ml pDA. Within each block, bars topped with the same letter are not significantly different according to Duncan's Multiple Range Test, P = 0.05. 44 . .4 ISOLHTE 2 16358 N8; aenom mammo. 0