Reprinted from P HYTOPATHOLOGY, Vol. 55, No. 10, 11 32-1134, October 1965 Printed in U. S. A. Parasitic Aspects of a , oreades T. H . Filer, Jr. Former Research Assistant, Department of Plant Pathology, Washington State University, Pullman ; now Plant Pathologist, Southern Hardwoods Laboratory, Southern Forest Experiment. Station, Forest Service, USDA, Stoneville, Mississippi. Portion of a Ph.D. thesis, Washington State University. Scientific Paper No. 2636 , Washington Agricultural Experiment Stations, Pullman, Project No. 1322. Accepted for publication 2 April 1965. ABSTRACT Marasmius oreades parasitizes Poa pratensis, not require natural openings or wounds. The myce­ Festuca rubra, and Agrostis 'tenuis. The fungus pene­ lium ramifies in the cortical cells and destroys the trates the root directly in all three species and does cell contents.

Many hymenomycetous fungi are associated with the approximately four times as fast as the Washington disease "Fairy Ring" of turfgrasses. M arasmitts oreades isolates. (Bolt. ex Fr.) Fr. is one of the more common species. Host species used in this study were Paa pratensis Withering (6) was the first to associate this fungus L., Festuca rubra L. , and Agrastis tenuis Sibth. Seeds with the di sease and to report that it killed grass. were surface sterilized with 50% ethyl alcohol for 30 Ballion ( 1) and Bayliss (2 , 3) attributed the death of sec, followed by soaking in 5.28 % sodium hypochlorite vegetation to parasitism by the fungus. Shantz and (Clorox) for 10 min, and then placed on moist filter Piemeisel (5) reported that death of vegetation was papers in sterilized petri plates. Nine days after ger­ due to the impermeable nature of the infested soil re­ mination, five seedlings were transferred to each of sulting from physical changes brought about by the 22 5 test tubes (29 X 200 mm ) containing 6 g of Per­ fungus. They suggested that the parasitic attack was lite saturated with 20 ml of modified Murray's medium secondary. Since controversies still exist as to the para­ as desc ribed by Zscheile (7). sitic nature of the fungus, the purpose of this inves­ Forty-eight hr later the tubes were inoculated with tigation was to study the possible parasitism of the 9-mm di scs of inoculum taken from petri plate cul­ fungus on three turfgrass species. tures of M. areades. Three tubes of each grass species Examination of hand and microtome sections from were inoculated with each fungus isolate. grass roots growing in M. oreades-infested soil was Initial studies of seedlings inoculated in vitro had made to determine if the fungus could be observed. indicated that the time from inoculation to infection Although it was possible to demonstrate the presence was less than 7 days. Starting 2 days after inoculations, of hyphae in the roots of field-grown grass species, it a set of tubes containing each host-fungus combination was impossible to prove by this method that the hyphae was removed at 24-hr intervals for 5 days. A sample of present were produced by this fungus. each root was used to reiso late the fungus and another The pathological histology of the host was studied in portion of the same root was retained for sectioning. the laboratory using surface-sterilized seedlings in vitro Root segments, 0.5 em long, were fixed and killed in with pure cultures of M. oreades . formalin, acetic acid, and ethyl alcohol solution. The MATERIALS AND METHODS.-Four isolates of Maras­ material was taken through a tertiary butyl alcohol mitts oreades were used through this study. Isolate 1 series and embedded in paraffin as described by Sass was obtained from sporophores collected near Tacoma, ( 4). Wash. , by C. J. Gould, Western Washington Experi­ Longitudinal and cross sections of embedded material ment Station, Puyallup. were cut 8 f.t thick with a rotary microtome. The best Of approximately 200 isolations made from sporo­ differential staining was obtained with the safranin-fast phores of M. areades collected in Everett, Olympia, green combination (4). Seattle, and Tacoma, Wash., only two were retained In addition to microtome sections, freehand sections (isolates 2 and 3). These two were cultured from were made of different-aged seedlings grown in the stipes and differed in their growth rates and cultural laboratory and grass plants taken from a fairy ring. characteristics. Isolates 1 and 3 were similar ; 75 % of Freehand sections were made for microscopic examina­ the isolations made from sporophores in Washington tion of large quantities of roots suspected of having resembled isolates 1 and 3. mycelia present. Isolate 4, cultured from a , was obtained from RESULTS.-All isolates of M . oreades were reisolated A. H. McCain, University of California, Berkeley. The from roots of P. pratensis, F. rubra, and A. tenuis. California isolate was similar to isolate 2. Microtome sections showed hyphae in the grass roots Mycelium of M. areades is snow white. The culture 3 days after the inoculum was placed in the tubes adja­ characteristics varied, depending on the particular iso­ cent to the roots. The time required for the fungus to late involved and the media used. Generally, cultures invade the roots was limited by the rate of growth of of isolates 1 and 3 showed a white, cottony appearance the fungus which was 1-5 mm/ 24 hr. caused by numerous aerial hyphae, whereas isolates 2 After 30 days, hyphae were fo und in all tissues of the and 4 showed a white, resupinate growth with sparse roots except the phloem and xylem. The hyphae rami­ aerial mycelium. At 25 C the California isolate grew fied through cells with no consistent pattern of growth 1132 October 1965] F I LER, J R. : MARASMIUS 11 33

Fig. 1. A) The hyphae of Marasmius oreades (isolate 3) in cortical tissue of Agro stis tenuis. B) Clamp conn ecti ons on hyphae of M arasmius oreades (isolate 4) in co rtical tissue of Poa pratensis . C) P enetration of an epidermal cell near a root hair of Agros tis tenuis by hypha of M arasmius oreades (isolate 4). D) Mantle of mycelium covering root of Agrostis tenuis 3 months after inoculation. (Fig. I-A). Hyphal morphology changed somewhat M . areades can penetrate the roots of Agrastis, Fes­ after hyphae entered plant ti ssue. The hyphae increased tuca, and Paa direc tly, and does not require a natural in diameter and branched mo re than hyphae grown on opening or wound (Fig. I-C ) . In sections of in fected artific ial media. The diameter of the hyphae ranged roots, hyphae we re observed entering the epidermal from 1. 6 to 3. 2 f.l . Although clamp connections were ob­ cells directly. served in the plant tissues (Fig. I-B) , they were pro­ In roots of all three grass species, the hyphae were duced less frequently in the host than on artificial found penetrating cells in whi ch the nuclei appeared media. intact. In contrast, the nucleus of the host cell from 1134 PHYTOPATHOLOGY [Vol. SS which the hyphae subsequently emerged was disrupted. evidence from microtome sections of grass roots grown The roots of grass taken from infested field soil with the fungus in vitro showed that the fungus had contained basidiomycetous hyphae that were similar to penetrated the roots. The mycelium ramified in the those of M. oreades observed in roots of grass seed­ cortical cells and destroyed the cell contents. By com­ lings grown in vitro. In the field or laboratory, the paring healthy with diseased roots, it appeared that the fungus formed a dense mantle of mycelium on the fungus induced increased branching of roots, but the outside of the root as shown in Fig. i-D. new roots were also destroyed. DISCUSsION.-Grass seedlings grown in large test tubes with pure cultures of the fungus showed fungal LITERATURE CITED infection in the roots. The seedlings received sufficient 1. BALLION, P. 1906. Recherches sur les cercles myceliens moisture to eliminate the possibility that moisture (ronds de fees) . Actes Soc. Linn. Bordeaux 61 :62 -88. deficiency was necessary for fungal infection. Roots of 2. BAYLISS, J. S. 1911. Observations on Marasmius the seedlings were killed or severely damaged by the oreades and Clitocybe gigantea, as parasitic fungi causing "fairy ring". J. Econ. BioI. 6:111-132. fungus. 3. BAYLISS, J. S. 1926. Concerning "Fairy rings" in pas­ Comparison of the staining characteristics of sec­ tures. Ann. Appl. BioI. 13:277-288. tioned healthy and diseased root tissue indicates that 4. SASS, J. E. 1958. Botanical microtechnique. 3rd ed. Iowa the fungus can penetrate living cells. Sections of in­ State ColI. Press, Ames. 228 p. 5. SHANTZ, H., and R. PIEMEISEL. 1917. Fungus fairy rings oculated grass roots showed that mycelium had pene­ in eastern Colorado and their effect on vegetation. J. trated living cells, and judging by their staining Agr. Res. 11:191-246. properties, invaded cells were apparently killed by the 6. WITHERING, W. 1796. An arrangement of British plants. fungus. Vol. 4. 3rd ed. G. G. and J. Robinson, London. 7. ZSCHEILE , F. P . 1951. Nutrient studies with the wheat Microtome sections of infected grass roots growing bunt fungus Tilletia caries. Phytopathology 41:1115- in fairy rings in the field combined with supporting 1124.