A S T U D Y O F H E L M I N T H O S P O R I U M S A T I V U M P . K . & B . AS A N U N R E P O R T E D P A R A S I T E O F A G R O S T IS P A L U S T R IS HUDS. By WILLIAM KLOMPARENS A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1953 A STUD Y O F H E L M I N T H O S P O R I U M UN REPO RTED P A R A S IT E O F S A T IV U M P . K . & B . AS AN A G R O S T IS P A L U S T R IS HUDS. By William Klomparens AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology Year 1953 A pproved 1 WILLIAM KLOMPARENS ABSTRACT Symptoms are described for an extremely destructive disease of creeping bent grass, Agrostis palustris Huds. Large areas of turf may turn smoky blue, then chlorotic, followed by the complete destruction of the affected grass plants. He1minthosporium sativum P. K. & B. is the fungus constantly associated with the disease symp­ toms. Fifty-eight isolations and identifications are listed, with sources ranging from Texas to Ohio. H. sativum, isolated from creeping bent grass, and an isolate from J. J. Christensen, of Minnesota, both were found to cause complete death of established creeping bent grass. These same isolates were compared for pathogenicity to bent grass seedlings and to wheat; the damage caused by all isolates was the same on any one host. Four attempts to produce the perithecial stage were unsuc­ cessful, using twelve media, ultraviolet light, and varying environ­ mental conditions. The variability of bent grass isolates of PL sativum grown on artificial media corresponded with the variability of this species as reported in the literature. H. sativum isolates from creep­ ing bent grass were found to be stimulated when grown in medium containing small amounts of mercuric and mercurous chloride (10 and 50 ^g /m l), while the known isolate from Minnesota was not 2 WILLIAM KLOMPARENS stimulated. Nematodes were tested alone and in combination with fungi for pathogenic effects. of the tests. ABSTRACT No effect was found under the conditions Dr. G. Thorne, Nematologist, however, felt that Tylen- chorhynchus sp. was parasitizing a sample of creeping bent grass sent to him for identification and observation. Curvularia sp. was found to be mildly parasitic to creeping bent grass. Pleosphaerulina sp. was described and shown to be parasitic on creeping bent grass. ACKNOW LEDGM ENTS The author wishes to express his gratitude to Dr. J. R. Vaughn who, in the capacity of major advisor, was constantly available for dis cussion of the various phases of this investigation. Thanks are also due to Dr. Vaughn, Dr. E. S. Beneke, Dr. G. P. Steinbauer, and Dr. W. B. Drew for critically reading this manu­ script and providing helpful suggestions as to organization and clarity. The suggestions and interest shown by Dr. E. A. Andrews, Dr. D. J. DeZeeuw, and Dr. J. Tyson contributed materially to the various phases of this research. Their interest and aid are sincerely appre­ ciated. The advice and aid so freely given by other members of this department, too numerous to mention, was of considerable help. Sin­ cere thanks are extended to these people. Last, but far from least, are the thanks due to Dr. Wehmeyer and Dr. Thorne for their valuable contributions to sections of this manuscript. Sections of this work could not have been completed without their assistance. TABLE OF CONTENTS Page INTRODUCTION.................................................................................................................. 1 LITERATURE R E V IE W ............................................................................................... 3 THE DISEASE ...................................................................................................................... 8 MATERIALS AND METHODS................................................................................ 13 Fungus M orphology................................................................................................... 13 ........................................................................................... 13 Pathogenicity Studies Perithecial Production Trials .................................................................... 24 EXPERIMENTAL R ESU LTS................................................................................... 28 Fungus M orphology................................................................................................... 28 Pathogenicity Studies ........................................................................................... 30 ....................................................................................... 30 ................................................................................... 34 Cultural S t u d i e s .......................................................................................................... 42 Established grasses Bent grass seedlings Perithecial production trials ................................................................ 42 Variation due to m e d iu m ........................................................................... 42 .................................................................... 46 ............................................................................................................................. 52 BIBLIOGRAPHY................................................................................................................. 54 APPENDIX (Plates) 57 DISCUSSION AND CONCLUSIONS SUMMARY ............................................................ INTRODUCTION There are approximately 15,000 acres of Agrostis palustris Huds. (creeping bent grass) maintained as putting greens on golf courses in the United States, and although figures are lacking, obser­ vation leads the author to estimate that there are at least another 15,000 acres in golf course fairways, parks, and lawns. Thirty thousand acres is not in itself an imposing figure, but when the value per acre is set at $10,000 (12), the 300 million dollar estimated value becomes a considerable investment. The very high standards for fine turf set up by golf club members, park superintendents, and individual home owners has led, understandably, to conditions favoring diseases caused by fungi. Creeping bent g ra ss, Agrostis p a lu stris, probably has had more tinwanted nurturing and care lavished upon it than any of the other turf grasses, such as the colonial and velvet bents, the bluegrasses, and fescues. The high fertility and water levels, close clipping (down to 3/16 inch), and the crushing and scuffing by feet, mowers, tracto rs, golf clubs, and croquet mallets must contribute to the constant p re s ­ ence of disease problems. 2 Parasitic organisms affecting turf grasses have, of course, been recognized for many years. The common Sclerotinia homoeocarpa (dollar spot), Pellicularia filamentosa or Rhizoctonia (brown patch), Gloeocercospora sorghi (copper spot), and Fusarium and Typhula sp. causing snow mold have been treated with hundreds of compounds in attempts to eliminate unsightly spots and blotches in order to attain eventually the unmarred green carpet so desired. In 1949, the failure of these compounds to control an appar­ ently new and extremely destructive disease on the creeping bent grasses stimulated this investigation. Demonstration of the constant association of a species of Helminthosporium with the diseased condition was relatively easy, but the w riter then was beset with the difficulties that have confronted all researchers who have worked with root rots of established grasses. Working with individual seedlings or individual established plants leaves much to be desired, since their natural environment includes large numbers of individuals in close quarters. There is also the necessity of re-creating the predisposing factors of soil and moisture conditions, fertilization, presence of other fungi, close clipping, bruising, and weekly applications of fungicides. These are but a few of the recognized complicating factors that must be evaluated in the following discussion. L IT E R A T U R E R E V IE W In 1923, Drechsler, in an excellent, comprehensive publication, beautifully illustrated twenty-four species of Helminthosporium on various grasses (5). and named. Many of these species were newly described Of this group, only one, H. stenacrum Drechs., was r e ­ ported to be associated with a withering of creeping bent grass blades of Agrostis stolonifera L. Also in 1923, Drechsler reported that Helminthosporium giganteum Heald and Wolf had been observed as a parasite of creeping bent grass (6). Bent grass parasitism by H. giganteum was again referred to in 1928 on A. stolonifera L. and on A. canina L. (velvet bent) (7), and was further confirmed in 1929 (8). In 1931, in Connecticut, a species of Helminthosporium was reported to have caused severe damage to creeping bent grass (21). The recently seeded areas were the m o s t severely affected, with the symptoms appearing as if the grass had been matted down with oil. This condition, which occurred again the following year, was favored by hot, damp weather. Dahl, in 1932, again reported parasitism of H. giganteum on creeping and velvet bent grass. Helminthosporium was reported to have caused damage to creeping bent grass in the 4 Netherlands in 1934 (26). borne. The parasite was also reported to be seed- Infections of lawn grasses by a Helminthosporium which caused a condition described as "black blotch" were reported from South Africa in 1934 (20). This seems to be sim ilar to the condition reported from Connecticut in 1931. A withering effect was reported in 1935 to be due to infection of creeping bent grass by H. erythrospilum Drechs. when this new species was described (9). In 1941, Helminthosporium sp. were reported on both velvet and creeping bent grass (18). In this general listing, as in most others, no species were noted. Wernham and Kirby, in 1943 (28), described a Helminthosporium on creeping bent grasses. The disease occurred in warm, humid weather and attacked all grasses under observation. exhibited were as follows: bluish cast, darkening, and withering, with the tops dying down to the soil line. the undamaged stolons. The symptoms Recovery was slow from The general symptoms and the indistinct margins of the diseased areas led to the application of the term "m elting-out." Struble (24), in 1943, in describing techniques for disease-control studies, included Colletotrichum in the "melting-out" complex. Review of literature failed to reveal whether or not this organism was ever proven to have been involve!d in the disease process. At any rate, nothing further has been mentioned with 5 regard to the Colletotrichum. The M m elting-out'1 was mentioned again in 1949 (16) in a discussion of turf disease control measures. In conversation with C. C. Wemham in 1951 it was brought out that the disease, as it was described in 1943, more than likely was caused by Curvularia sp. plained question. This, however, results in one unex­ The Pennsylvania workers stated that velvet bent was the least susceptible to melting-out, in contrast to Howard (14), 1952, who described C urvularia-Incited "fading-out" as being very prevalent on velvet bent grass. Observations in the Midwest have shown the Curvularia to be very widespread on the creeping bent grass, but seldom causing extensive damage. Velvet bent is not used to any extent in the Midwest; thus, a comparison of susceptibility with the creeping bents was not possible. Although Helminthosporium is often mentioned in association with disease of the fine turf g rasses, only D rechsler--and Dahl, in one instance--mentioned the species involved. The literature, there­ fore, loses some of its value, since the exact organisms causing the various symptoms are a m atter of conjecture. It is believed that the fungus involved in the following discus­ sion is Helminthosporium sativum P. K. & B., and the possibility exists that nematodes may also be a factor. Review of the literature here will be limited to that pertinent to this problem. H. sativum has been investigated more thoroughly than any other species of this genus. Christensen (1, 2, 3) showed that the fungus was extremely variable in morphology, cultural growth, and pathogenicity. His inoculations of three- to four-month-old plants of Agrostis alba L., A. palustris Huds., and A. stolonifera were without success. It has also been shown that although H. sativum is active under semiarid conditions, high humidity and temperature often favor expression of symptoms (4). Elliot, in 1949 (10), pre­ sented data indicating a possible reason for the great variation in conidial measurements often present in the literature. Among others, the length of 11. sativum spores was shown to be influenced by sugar concentration in the medium. The lower concentrations of sugar in the medium produced the longer spores. Stevens (23), in 1922, and Sprague (22), in 1950, very com­ pletely covered the characteristics of the genus. Stevens' very com­ plete early work was the basis for many subsequent studies, while the latter briefly reviewed literature to date. The role of nematodes in a root-rot complex was determined by Jenkins in 1948 (15). Four genera of meadow nematodes were found to be furnishing entry points for subsequent infection by fungi and bacteria. There are, of course, well-known examples of nema­ todes being the prim ary cause of root lesions and plant damage (11). 7 The correlation between numbers of nematodes present in the soil in relation to damage was determined for celery in 1952 (19). As ex­ pected, the larger numbers caused the most extensive damage. T H E D IS E A S E In the summer of 1949, Dr s. J. R. Vaughn and E. A. Andrews were asked to investigate an apparently new and destructive disease of creeping bent grass present on several golf courses in Michigan (see Table I). Observation and isolation by Andrews resulted in cul­ tures of Helminthosporium sp. which were turned over to the author for identification and detailed investigations. The symptoms present in 1949 were apparently dependent upon the extremely hot, humid conditions that were present that particular summer, since subsequent observations indicated that a change in environmental conditions arrested the disease progress. The visible symptoms were a smoky blue cast on areas varying from one to three or four feet in diameter, followed by yellowing and complete killing of grass plants in the affected areas. The irregular, dead areas had m ore-or-less definite margins, and the appearance of being watersoaked and matted down. There was no new growth from within the most severely affected spots, since root rot is present in the blue and yellow stages. The earliest symptom is a m ore-or-less oval spot on the leaves, 3 to 6 mm. long and half as wide. The spots, beginning as 9 TABLE I DETERMINATIONS OF THE PRESENCE OF H. SATIVUM ON ATTACHED, DYING, OR DEAD CREEPING BENT GRASS BLADES1 Summer Locality Occurrences 1949 East Lansing, Michigan 1 1950 East Lansing, Michigan 7 Brighton, Michigan 5 Grand Rapids, Michigan 2 Warren, Michigan 2 Ann Arbor, Michigan 1 Detroit, Michigan 4 East Lansing, Michigan 2 Brighton, Michigan 1 Ann Arbor, Michigan 1 Detroit, Michigan 5 Dallas, Texas 3 Franklin, Michigan 1 Lansing, Michigan 1 Chicago, Illinois 2 Cincinnati, Ohio 1 Westerville, Ohio 1 1951 - 1952 10 TABLE I (Continued) Summer 1952 (Cont.) Locality Occurrences Birmingham, Michigan 1 Cleveland, Ohio 1 Madison, Wisconsin 1 Franklin, Michigan Detroit, Michigan Total Note: Medinah, Illinois 1 Park Ridge, Illinois 1 Niles, Illinois 1 Worth, Illinois 1 Orland Park, Illinois 1 Lisle, Illinois 1 Rocky River, Ohio 1 Pontiac, Michigan 1 Willoughby, Ohio 1 58 In approximately 90% of these determinations, Curvularia sp. was present, while in 30 to 40% Pleosphaerulina sp. was also present. 1 Many of the samples were received by mail; thus, correlation of disease severity and spore concentration on the sample was not pos­ sible. A large number of these are not in culture at present. ^ 11 minute yellow flecks, soon p ro g ress through the spot stage to a gen­ eral w atersoaked-appearing blotch (Plate 4, Appendix). General yel­ lowing is often evident at this point, and roots may be damaged. Platings of roots when the g ra ss is in this yellow stage have led to successful recovery of the organism. Roots, stolons, and aerial parts may all show loss of vigor at the same tim e, indicating that infection may be general and not n ecessarily progressing from one organ to another. The re v erse may also be tru e --s lig h t symptoms on aerial p a rts with root-rotting causing the chlorosis, wilting, and death of a e ria l p a rts. Observations by the author over a period of four summers have indicated that the smoky blue color does not always precede the yellow stage. Slides and tissue platings of dead and dying grass almost invariably show an organism , which subsequently will be iden­ tified as Helminthosporium sativum, to be the m ost prevalent organ­ ism. The fungus spores, although p resent in creeping bent turf at all tim es, are so numerous in affected areas as to account fo r some of the dark color. Other organism s commonly isolated and observed in associa­ tion with Helminthosporium were Curvularia sp. and Pieosphaerulina sp. Nematodes were also observed in many turf samples. In some instances the nematodes were feeding on blades near o r at the soil line 12 Howard (14) has shown the Curvularia to be parasitic in its own right, and subsequent discussion will show parasitism by Pleosphaerulina, but the exact role of the nematodes in the disease proc­ ess has not been fully determined. M A T E R IA L S AND M E T H O D S Fungus Morphology Although many fungi were isolated infrequently, Helminthosporium was consistently found, and its identity was determined by comparative measurements, and observations as to color, method of germination, and general morphological characteristics. One hundred spores and sporophores from culture and from nature were measured and ob­ served in order to arrive at the identification described in the sec­ tion on results. Limited numbers of measurements of perithecia and ascospores of Pleosphaerulina sp. were made; however, its pathogenicity was well established following Koch's postulates. Pathogenicity Studies In January of 1950, spore suspensions derived from the original isolate furnished by E. A. Andrews were sprayed on twoand-one-half-week-old seaside creeping bent plants. at 24-hour intervals were made on this seeded grass. Three inoculations The deep crocks 1 In the following discussion, where grass from seed was used, all soil for planting was steam sterilized. 14 were kept covered to. raise the humidity. house ranged from 68° F. to 76° F. Temperature in the green­ Platings of the blades, roots, and crowns were made at five 3-day intervals one month later. Sur­ face sterilization of the plant parts used for isolation was with 1-1000 mercuric chloride and 0.008 percent sodium hypochlorite. On March 10, 1950, 24-day-old seeded seaside creeping bent grass in flats was given a spore suspension spray on approximately one-third of the area in three of four flats. The flats were placed in a humidity chamber where a relatively constant environment of 88 percent relative humidity and a temperature of 77° F. was main­ tained. On March 21 and 27, 1950, the above test was repeated, with the only apparent differences being the slight variation in temperature, which, during these trials, was 79° to 84° F.; the humidity, which ranged from 91 to 94 percent; and the fact that two inoculations three days apart were employed. Fight flats of seaside creeping bent which had been established for two and one-half months were treated as pairs in the following manner on April 4, 1950: 1. Had been seeded in steamed soil and received a spore suspension. 2. suspension. Had been seeded in inoculated soil and received no spore 3. Had. been set from stolons in sterile soil and were given a spore suspension. 4. One stolon-set and one seeded into steamed soil and no spore suspension. One of each of the flats was placed in a separate humidity chamber and held at 86° F. and 90 to 92 percent relative humidity. On August 22 and on August 26, 1950, six flats of well-established Washington creeping bent grass were given spore suspension sprays as follows: one flat, not inoculated; one inoculated with Helmintho- sporium from bent grass; one received Curvularia sp.; and the last was sprayed with a mixture of the Helminthosporium and Curvularia. In both instances the flats were held in humidity chambers for five days at 90 percent relative humidity and 80° F. On September 7, 1951, well-established Washington creeping bent was removed from the grass plot on the college farm and brought into the laboratory in flats. Three of five flats were inoculated with a single-spore isolate derived from a previously successful inoculation test; and one flat, with Curvularia sp. Six-inch bell jars were placed over areas of these flats to increase the relative humidity. After three days, when leaf spotting first appeared, individual blades were marked with fragments of thread and left beneath the bell jars for further development of the disease. 16 On April 1, 1952, seven flats of closely knit turf of the Wash­ ington variety were taken from the field and inoculated with a known Helminthosporium sativum received from Dr. J. J. Christensen, three Helminthosporium isolates from creeping bent grass, Pleosphaerulina sp., and an unidentified pycnidial-producing isolate from bent grass. Subsequent to inoculation at opposite ends of the flats, all were held at 90 percent relative humidity and 80° F. for 48 hours. The flats were then held under greenhouse environmental conditions which were approximately 70° F. and 65 percent relative humidity. From June 30 to July 25, 1952, pairs of flats of a mixture of the Arlington and Congressional varieties of creeping bent grass were given spore suspensions derived from an isolate of Helminthosporium obtained in Indiana. These flats, being out of doors, were subject to the prevailing weather, but were covered the first nine nights of this period by wet paper toweling, which served to increase the relative humidity. The flats were reinoculated every five days during the period of the test. Since it had often been observed that regrowth into an area of dead bent grass was extremely slow and difficult, the following test was devised to determine possible residual action by toxic met­ abolic products given off by the causal organism(s). On July 15, 1952, mycelial and spore suspensions, sterile extracts, and sterile 17 medium control solutions were applied to plots of Wasliington creep­ ing bent, replicated three tim es. The treatm ent m aterials were all from 2 percent m alt extract solution (Difco). One lite r of each of the following was prepared: 1. Mycelium and spore suspension. 2. Sterile* extract of No. 1, 3. P u re unused medium. 4. Sterile distilled water. The replicated plots were marked with golf tees pressed below the surface of the g rass, and received the regular mowing and w ater­ ing of the plot proper. The undiluted solutions were distributed oyer as small an a re a in the plot as possible by very slow, direct pouring. A second treatm ent was applied twelve days la ter, on July 27. On August 20, 1952, a te st was set up to determine possible correlation between nitrogen fertilization and source of nitrogen with the disease initiation and progress. Four grass varieties--A rlington, 1 The extract was obtained by filtering and treating the ex­ tra c t for 24 hours with 1 cc. propylene oxide per lite r of volume. The m aterials are f ir s t enclosed in a flask with a rubber cork above the cotton plug; the cork is then removed, and the solution allowed to air for 48 hours (with periodic shaking). Treatments 2, 3, and 4 were treated with the propylene oxide to offset any effect that it might have. 18 Toronto, Congressional, and Washington creeping bents were used. * Three fertilizers--ammonium sulfate, Nu Green (urea), Milorganite (organic nitrogen)--and no fertilization were the four treatments. Based upon actual nitrogen content, each of the twelve fertilizer plots received 12 ounces of nitrogen per 1000 square feet from each of the sources, applied in one-foot-wide strips lengthwise on the plot. This was lightly watered in to prevent burning by the inorganic forms of nitrogen. A spore suspension originally isolated from diseased grass from Illinois was applied across the four treatments on each grass variety. A reinoculation across the fertilizer strips was c a r­ ried out one week later. The second inoculation was with an isolate obtained from diseased grass from Dallas, Texas. The entire plot was watered with a fine mist three or four times a day for the three-week observation period. Seedling pathogenicity of an isolate (H. 10) derived from the original isolation, H. sativum, and of Curvularia sp. was determined by pi a n t i n g seeds of seaside creeping bent and Yorkwin wheat treated with mercuric chloride and sodium hypochlorite in steamed inoculated soil on November 16, 1952. Rows of twenty-five seeds each, replicated 1 The author is grateful to Oakland Hills Country Club, Bir mingham, Michigan, for use of the plot containing the four grass varieties side by side. 19 four times, were seeded one day after inoculation of the steamed soil with spores and mycelium of the respective fungi. To facilitate counting of the bent grass seedlings, the following technique suggested by Dr. D. J. deZeeuw was used. The twenty-five bent grass seeds were dipped in 10 percent solution of Methocel (Dow) and placed at intervals on one-fourth-inch strips of personal tissue. seeds were firmly fixed and spaced. Thus, the Upon turning the paper seeds down in the soil, no inhibitory effects on germination were noted, and counts were easily obtained. This test, as well as the next to be described, were preceded by unreplicated preliminary trials which indicated the value of repeating the tests. Seedling susceptibility was further tested in petri dishes in the laboratory, beginning December 30, 1952, Four turf grasses— Poa annua L. (annual bluegrass); two varieties of Agrostis tenuis Sibth. (colonial bent), Highland and Astoria; and Agrostis palustris Huds. (creeping bent) of seaside variety—were used. used for inoculation were: The organisms H. sativum, isolate H. 10; Curvularia sp.; Pieosphaerulina sp.; Aspergillus nlger; and Penicillium notatum. The isolates, singly and in combination, were atomized onto seeds placed in sterile petri dishes containing filter paper. Each dish, containing twenty-five seeds, was replicated four times. Seed-borne contamina­ tions interfered with an earlier test, so all seeds except one series 20 of the seaside variety were surface disinfested. Seed treatment was the use of mercuric chloride and sodium hypochlorite, as mentioned previously. There was, however, a final rinse in sterile distilled water before seeding. The spore suspensions were standardised to approximately fifty spores per drop and atomized into the dishes at the time of seeding. Counts of seedlings (based upon attainment of one-fourth-inch height) were made for nine consecutive days. When the control plants showed loss of vigor and chlorosis, the ex­ periment was concluded. The plates were held at room temperature near a window for the entire period. Due to the numerous instances in which nematodes were ob­ served in many of the previously described experiments, the follow­ ing test was set up with hopes that their role in the disease process could be determined. One source of nematodes was obtained from Dr. R. Kiesling, who found them in isolations made from the crown of diseased bean plants. This will be referred to as Nematode 2. A second selection was made from soil in flats of creeping bent which had been held in the greenhouse for four months. ferred to as Nematode 1. This will be re­ An attempt was made to isolate in the genus Tylenchorhynchus, since a prepared, identified slide was avail­ able for comparison. Dr. Gerald Thorne had previously made the identification from samples of damaged grass sent by the author. In 21 this selection, the soil in a beaker was washed with slowly running water; the beaker was then tilted for a period of time, after which the supernatant liquid was poured off. The residue was placed in small drops on plates containing 3 percent agar. With the add of a dissecting microscope, the nematodes could be seen to move well out into clear areas. The individuals were isolated by removing the small circle of the agar on which the nematode was present. This was transferred to either 2 percent malt extract agar plates containing grass clippings or to a rabbit dung-malt extract medium. Either medium was found to be suitable for increasing the numbers of the nematodes. to as Nematode 1. The latter, from the bent grass flats, is referred The identity of these was to be ignored until it was determined whether they caused damage when alone, or if they contributed to the root rotting when placed in combination with various fungi. Treated seaside creeping bent was seeded into 2 x 2 x 4 plant bands or into 2 x 2 x 2 inch inch plant bands for subsequent trans­ planting into inoculated soil or for spore suspension inoculation. The two band types were used to furnish stands of established grasses of different ages--the deep-band series to be inoculated three weeks 1 The author is deeply indebted to Dr. E. A. Andrews for suggesting the use of bands as a means of obtaining easily separated units of established grass. 22 later than, the other. The first series was set up on February 1, 1953, and the second on February 20. At the time of inoculation, these grass stands were twenty-four and forty-three days old, re ­ spectively. and tillered. The grass, even in the first series, was well established The fungi used alone and in combinations are listed in the following table, which indicates the twenty-two treatments used with each series. 1. Absolute check. 2. Nematode 1. 3. Nematode 2. 4. Nematode 1 + H. sativum. 5. Nematode 2 + H. sativum. 6. Nematode 1 + H. 10. 7. Nematode 2 + H. 10. 8. H. sativum. 9. H. 10. 10. H. sativum + H. 10. 11. Nematode 1 + spore suspension of H. 1 0 . 12. Nematode 2 + spore suspension of H. 1 0 . 13. Nematode 1 + spore suspension of H. sativum. 14. Nematode 2 + spore suspension of H. sativum. 15. Nematode 1 + Nematode 2. 16. Nematode 1 + Nematode 2 + H. sativum + H. 10. 17. Pleosphaerulina sp. spore suspension. 18. Curvularia sp. spore suspension. 19. H. sativum + Pleosphaerulina sp. spore suspensions. 20. H. 10 + Pleosphaerulina sp. spore suspensions. 21. H. 10 spore suspension. 22. H. sativum spore suspension. In the first ten treatments the fungi and the nematodes were incorporated in the soil previous to transplanting the grass. In the last twelve treatments spore suspensions 6f the fungi were used, while in the last six, no nematodes were involved. Each of the treatments was replicated three times with each series. The green­ house temperature and relative humidity were raised by means of escaping steam in a far comer. A hygrothermograph was used to record the temperature and humidity at all times. The high tempera ture and humidity were maintained for six days, after which cooler, drier conditions were used to determine regrowth from affected grass in the first series. At the time of Initiating the second series the temperature and humidity were again raised for a thirteen-day period. removed. The grass was cut at irregular intervals and the clippings Microscopic examinations and tissue platings of blades and roots were made periodically. 24 Perithecial Production Trials / Transfers from three single-spore Helminthosporium cultures, H. 7, H. 9, and H. 24, were placed in petri dishes containing 15 cc. each of nine media. Each treatment was replicated four times to furnish one plate for each of the four conditions under which they were held. 1. The conditions used were: Humidity chamber 26° C., 65 percent relative humidity, and constant darkness. 2. As (1), but constant low light (7-1/2 watt bulb at 12 inches). 3. 22° C. incubator (dark). 4. Room temperature and light conditions. To each of nine lots of grass clippings sterilized by propylene oxide (using the previously described technique) was added 15 cc. of one of the following media: 1. Czapek's ^ sucrose nitrate solution with 30 gm. sucrose and 2 percent agar. 2. Czapek's as above, but only 15 gms. sucrose. 1 Czapek's sucrose nitrate solution: sodium nitrate, 2 gms.; potassium dibasic phosphate, 1 gm.; potassium chloride, 0.5 gm.; magnesium sulfate, 0.5 gm.; ferrous sulfate, 0.5 gm.; sucrose, 30.0 gms.; 1000 cc. distilled water (plus 17 gms. agar). 3. Medium from M. R. Hatfield composed of 2.0 percent dextrose, 0.5 percent yeast extract, 0.25 percent Bacto-peptone, 1000 cc. distilled water. 4. 2 percent malt extract agar (Difco). 5. Pure 2 percent agar. 6. 2 percent 7. Coon's synthetic broth* + agar. 8. 2 percent Bacto nutrient agar. 9. 2 percent prune agar (Difco). corn meal agar (Difco). The three isolates on the nine media were held under the four conditions for two weeks. violet light and replaced. They were then treated with ultra­ The ultraviolet light treatment was one Slimline lamp (2537 A) at 8 inches for 40 seconds. A second attempt to produce the perfect stage was initiated on February 5, 1951. Twenty-four 200 cc. screw-cap bottles were used in place of the petri dishes. The bottles were used so that one-half of the series could be held for a long period of time with little likelihood of contamination. The promising isolate H. 7 was chosen for seeding, since it repeatedly produced sclerotial-like bodie in various media. 1 Coon's synthetic broth: sucrose, 7.2 gms.; dextrose, 3.6 gms.; potassium nitrate, 2.02 gms.; magnesium sulfate, 1.23 gms.; potassium dibasic phosphate, 2.72 gms.; 1000 cc. distilled water. 26 The medium used was the dextrose, yeast extract, Bactopeptone medium (No. 3 of the previous test), with the following addi­ tions: To one set was added 0.5 gm. of glycine and 0.5 gm. of asparagine; to a second set was added 0.5 gm. of glycine; while the third set received 0.5 gm. of asparagine as an addition. bottle, 30 cc. of medium were used. F o r each The warm medium was allowed to solidify with the bottles on their sides in order that a large area wotild be available for growth. After seeding, two bottles of each of the three media were held in the four environmental conditions mentioned in the previous test. These were held for two months, after which one-half were transferred to a refrigerator. The bottles in the refrigerator were held for an additional ten months and then returned to the original environments for three weeks. Those not refrigerated were observed periodically, but received no further treatment. The cultural growth of eleven single-spore isolates was ob­ served on 2 percent malt extract agar. The comparative growth of these eleven, plus six additional isolates, was also observed on Coon's synthetic medium (with 2 percent agar). Each of the isolates was seeded into the petri dishes, and after seven days the type of cultural growth was recorded. The purpose was to determine if cultural— growth groups established on a natural medium would be 27 identical or similar to subsequent artificial grouping when grown on a synthetic medium. Since an earlier in vitro experiment (17) demonstrated that there was an actual stimulation of one Helmlnthosporium isolate from creeping bent grass by low concentrations of mercurous and mercuric chloride, this test was repeated with two Helmlnthosporium sp. from bent grass and the H. sativum isolate obtained from Min­ nesota. The three fungi were seeded into 2 percent malt extract agar containing a mixture of mercurous and mercuric chloride con­ centrations of 0, 10, 50, 100, 250, and 500 ^ / m l and incorporated into the medium by serial dilution. autoclaved with the medium. The mercury mixture was not The replicates of four plates were seeded with 5 mm. disks of five-day-old inoculum and daily growth increments recorded in millimeters. The purpose was to determine whether more of the isolates than the one previously tested would show this stimulation and whether a known isolate of H. sativum would show stimulated growth if small amounts of mercury were in­ corporated in the medium. EXPERIMENTAL. RESULTS Fungus Morphology The measurements of the structures of Helminthosporium that are considered to be of diagnostic value are listed in Table II. Other characteristics, such as spore color and variability, brittle epispore, and germination from one or both terminal cells, were observed to fit very well the description of H. sativum (see Plate I). The spores were found to be slightly wider than H. sativum, which were listed by Sprague (22) as ranging from 15 to 20 yu. of the one hundred measured was 20.50 ^tc. The mean of the width Variation among spores from the H. sativum isolate from Minnesota and within one culture originally from a single spore are shown in Plate II* Spores on sporophores are pictured in Plate VI. Of the Pleo sphae rulina, Dr. L. E. Wehmeyer, of the Univer­ sity of Michigan, wrote the author as follows: . . . i t has hyaline spores, in saccate asci in small, membranous perithecia, and is found on living leaves, all of which are characteristic of this genus. Last summer, Dr. E. G. Simmons, of Dartmouth College sent me a fragment of Agrostis with, apparently, the same fungus, and just recently Dr. R. P. Korf of Cornell sent me a very similar species on Liingustrum leaves, collected by Dr. Williamson. All three collections have 4-septate spores, running 28-35 x 10-13^. 29 T A B L E II MEASUREMENTS OF ONE HUNDRED CONIDLA OBTAINED FROM HELMINTHOSPORIUM ISOLATED FROM CREEPING BENT GRASS Spore Sporophore Source Width Length Grass 2% malt Extract agar Saptations 46-101 y< 17-25^ avg. 76.15^ avg. 20.50yc{ 27-63 yA 17-25y n avg. 52.38^*4 avg. 20.43yn 2- 10 Length Width 80-191/u 6-8y* 2-9 Helminthosporlum satlvum: spores, 60-120 x 15-20^< ; septations, 3— 10. Sporophores, 110-150 x 6-8 up to 8 septate (22). 30 There have been a number of species described with very similar spores; i.e., P. briosiana, P. oryzeae, I?. phase oil, etc. The genus needs revision, for the species have been described mostly on host differences. The author's observations as to spore size, color, and septation coincide with those mentioned above. Infection of the creeping bent grass was observed to be either as direct penetration or through stomata. See Plate VII for pictures and camera lucida drawings of perithecia, asci, spores, and infection by germinating ascospores. Plate VII includes a picture of this organism. Seventeen single as co- spore isolations resulted in cultures that were apparently identical, and later produced perithecia. Pathogenicity Studies Established g rasses. The first pathogenicity test in January, 1950, run under ordinary greenhouse conditions, resulted in-no ap­ parent damage or symptoms. There was, however, successful r e ­ covery of the organism from the various plant parts when surf ace sterilized plant parts were plated in nutrient medium. The flats inoculated on the tenth of March, 1950, but held at an elevated humidity and tempferature, resulted in severe killing of large areas of the grass within 96 hours (see Plate III). The symp­ tomatic "blue-stage' 1 was present as the killing progressed. Dead 31 and. dying blades wera literally covered with spores and sporophores of Helminthosporlum sativum. In the tests repeated on March 21 and 27, however, there were no symptoms, and the organism was not recovered. The pathogenicity test of April 4, 1950, which was on two-andone-half-months-old, well-established, stolon- and seed-set plants in sterile and nonsterile soil resulted in slight symptoms. Leaf spotting was evident at various times, but tissue platings showed recovery of the organism from the blades of Treatment Number 1 only. This treatment was inoculation of grass from seed sown in sterile soil. The seeded turf which had been established in infested soil (Treat­ ment 2) and the stolon-set turf (in sterile soil) indicated no disease, and recovery of the organism was not possible. a spore suspension sim ilar to Treatment 1. The la tter received Nematodes were ob­ served in slides of root parts of the plants that had been stolonset in the steamed soil. The inoculations in August, 1950, resulted in very slight leaf spot or blotch symptoms. Since this turf had been stolon-set, it was not surprising to find nematodes in the slides. Since symptoms were so slight, tissue platings were not attempted. Although the temperature and humidity were high for five days, the test of patho genicity was considered negative. 32 The flats of grass brought in from the grass plot on Sep­ tember* 1951, which were inoculated with Helminthosporium and Curvularia, and random areas covered with bell jars showed infec­ tion from ascospores of Pleosphaerulina. These spores apparently were present in the field, and under the moist conditions were able to penetrate and infect the Washington variety blades. Watersoaked blotches on leaves marked with thread fragments proved to be ne­ crotic areas in which germinating, penetrating ascospores were ob­ served (see Plate VTI). Ten platings of leaf blades resulted in r e ­ covery of Pleo sphae rulina eight times, Helmintho sp orium three times, and Curvularia once. The results on the seven flats brought in from the field in April, 1952, and inoculated with Pleosphaerulina, H. sativum, three Helminthosporium isolates from bent grass, and the unidentified pycnidial form are as follows: The grass was damaged most severely by nematodes, although limited penetration by germinated ascospores of the Pleo sphae rulina and Helmintho spo rium conidla was observed. Thousands of nematodes were found on and in all plant parts. Sam­ ples of turf from this same source were later sent to Dr. Gerald Thome, Senior Nematologist, USDA, who in part replied as follows: The creeping bent turf sample contained hundreds of nemas belonging to the genus Tylenchorhynchus. The species seems to be most closely related to T. magnicaudatus. . . . 33 if they ere feeding on the graes roots —as I am sure they are doing. . . . This type of nema is ecto parasitic anil does not usually enter the root tissues, although I have occasionally found them there. . . . Little is known of this group and I can cite no references on them. . . . [See Plate VIII.] The flats of a mixture of Arlington and Congressional bent grass varieties that were treated out of doors in midsummer (June 30 to July 25, 1952) showed only slight symptoms at any time during this twenty-five-day period, despite the fact that they had been cov­ ered with wet paper toweling for nine consecutive nights and inocu­ lated five times during this period. Germinating Helminthosporium conidia produced a slight leaf spotting on July 2, but there was no progression or severe killing at any time. The extremely heavy inoculation carried out in the test of July 15, 1952, which was set up to determine possible action by toxic metabolic products produced by H. sativum, indicated that mass of inoculum may be a factor in this disease. Although the sterile extracts produced no apparent damage, all of the replicates receiving the mycelium-spore suspension were of a definite yellow color three days after the inoculation. The blades were observed to have been directly penetrated by Helminthosporium germ tubes and apparently were chlorotic due to the mass infections (see Plate IV). Unfortu­ nately, a drop in temperature occurred which probably prevented 34 disease progression since the yellowing disappeared in 36 hours did not recur after a second inoculation was made. The test to determine whether or not the source of nitrogen, or its immediate presence, affected the disease on established grass showed no results. There was a 48-hour period of warm humid weather during the time that the four varieties were being tested* but the re st of the three-week period apparently was not favorable / for disease development. The plot had received the same mowing and care as surrounding greens on the Oakland Hills Country Club, but did not have the traffic of players and bruising to which the others were subject. It is of interest to note that, two weeks p re ­ vious to this trial, sixteen of eighteen greens on the golf course had been attacked by the fungus under discussion. Bent grass seedlings. The pathogenicity of H. 10, H. sativum, and Curvularia sp. to Yorkwin wheat and to seaside bent in infested soil is indicated in Table HI. The actual stand of the wheat was significantly reduced only by the H. 10, but the infection of the wheat pi a n ts was high. Based upon evidence of coleoptile and crown lesions, the infection of the wheat by H. 10 and H. sativum was 80 and 65 percent, respectively (see Plate IV). The creeping bent grass stands were significantly reduced by both H. sativum and H. 10. Statistically, 35 T A B L E HI STANDS1 OF YORKWIN WHEAT AND SEASIDE CREEPING BENT THIRTEEN DAYS AFTER SEEDING INTO STEAMED, INFESTED SOIL (average of four replicates of twenty-five seeds each) Treatment Yorkwin Wheat Severely Dwarfed Seaside Control 24.8 0 23.3 H. sativum 23.5 4 11.0** H. 10 22.0* 6 7.3** Curvularia sp. 25.0 0 21.0 L east Significant Difference: * L.S.D. 0.05 2.19 2.12 ** L.S.D. 0.01 3.15 3.05 The actual stand of plants is listed in the table. The in­ fection of the wheat seedlings based upon lesions caused by H. sativum and H. 10 was 65 and 80 percent, respectively (see Plate IV). / 36 there was also reduction of the bent g rass sp. stands by the Curvularia This was so slight, however, that it would bear repetition and substantiation. The seedling susceptibility of the four turf g rasses germ ­ inated in p e tri dishes is summarized in Table IY. Since germination of the annual bluegrass was so v ery low, counts of these seedings are not included. The significant reduction in stands on .Hie Astoria bent were caused by Hie H. sativum and by the H. 10. The stand of A storia seedlings in the P le osphae rulina sp. treatm ent was higher than the control at the conclusion of the treatm ent. This was not significant; however, it was observed that germination in these plates was considerably b e tter during the early days of Hie tria l. The High­ land bent g ra ss stand was significantly reduced by H. sativum and H. 10, while Pleosphaerulina sp. and Curvularia sp. significantly in­ creased the stand of this g rass. The seaside creeping bent was also significantly reduced in stand by H. sativum and by H. 10. It is of in terest to note, however, that the combinations of these fungi with Curvularia sp. gave a r e ­ verse effect. The combinations gave slightly higher but not signifi­ cantly b e tte r stands of seedlings. A significant increase in grass stand was observed with the combination of Curvularia sp. and Pleo sphae rulina sp. Neither of these gave this increase alone. 37 T A B L E IV STANDS OF BENT GRASS SEEDLINGS IN PETRI DISHES NINE DAYS AFTER SEEDING AND INOCULATION (average of four replicates of twenty-five seeds each) Grass Variety Treatment Astoria Control (surface disinfested) H. 10 ............... ............................................................................ H. sativum ............................................................. 21.3 11.8* 7.5** Highland 11.0 Seaside 17.3 5.3** 13.0** 1 12.5** stoic Curvularia sp........................................................... 20.0 14.0** 17.5 Pleosphaerulina sp.............................................. 22.5 13.0* 18.3 H. sativum + Curvularia sp...................... 18.5 H. 10 + Curvularia sp.................................... 16.0 Aspergillus n i g e r .............................................. 17.3 Penicillium notatum 16.8 ...................................... Curvularia + P leosph aerulina............... 20.0** Untreated s e e d ..................................................... 14.0** Least Significant Difference: ................... 9.8 1.99 1.76 ** L.S.D. 0 . 0 1 ............................... 13.8 2.79 2.21 * L.S.D. 0.05 38 There was also a significant decrease in stand with the untreated seaside seed. Many of the untreated seeds apparently were destroyed by an unidentified Phycomycete. The last test of pathogenicity of the several fungi and the nematodes is presented in Table V and Plate V. It is immediately apparent that the nematodes alone or together were not able to cause noticeable damage to the grass. evidence of dying grass. Treatments 2, 3, and 15 show no Furtherm ore, under the conditions of the test, they did not contribute to the disease when they were in com­ binations with the fungal organisms. Pleo sphae rulina sp. and Curvu­ laria sp. were but slightLy pathogenic to the bent grass. The H. 10 and II. sativum gave evidence of the most parasitic action. This parasitism was much more rapid and severe when spore suspensions were used in contrast to the soil infested with the organism. The one exception was Treatment 9, which resulted in a 60 percent kill in a period of twelve days. The combination of H. sativum and Pleosphaerulina sp. gave evidence of undiminished parasitic action, while this latter fungus in combination with H. 10 was only 50 per­ cent as lethal in Series A. In Series B, little difference was noted. The elevated relative humidity ranged from 63 percent during the day to 100 percent at night, with an average of 89 percent. The elevated temperatures ranged from 68° F. to 105° F . during the day, K E Y TO T R E A T M E N T S IN T A B L E V 1. Absolute check. 2. Nematode 1. 3. Nematode 2. 4. Nematode 1 + H. sativum. 5. Nematode 2 + H. sativum. 6. Nematode 1+ H. 10. 7. Nematode 2 + H. 10. 8. H. sativum. 9. H. 10. 10. H. sativum + H. 10. 11. Nematode 1 + spore suspension of H. 10. 12. Nematode 2 + spore suspension of H. 10. 13. Nematode 1 + spore suspension of H,. sativum. 14. Nematode 2 + spore suspension of H. sativum. 15. Nematode 1+ Nematode 2. 16. Nematode 1+ Nematode 2 + H. sativum + H. 10. 17. Pleosphaerulina sp. spore suspension. 18. Curvularia sp. spore suspension. 19- II. sativum + Pleo sphae rulina sp. spore 20. H. 10 + Pleosphaerulina sp. spore suspensions. 21. H. 10 spore suspension. 22. H. sativum spore suspension. suspensions. 40 TABLE Y DISEASE RATINGS OF ESTABLISHED SEASIDE BENT GRASS INOCULATED TWICE, THREE WEEKS APART Series Treatment A Feb. 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 0 0 0 0 0 0 1 1 3 1 2 2 2 0 0 3 0 0 2 2 3 3 Feb. 27 0 0 0 1 1 1 1+ 1+ 3 2 3 3 3+ 1 0 4 1 0 3+ 2 4 3 B Mar. 8 0 0 0 2+ 12+ 1+ 2 3 1+ 3 3 3 2 0 4 1+ 14 2 4 3 Mar. 8 0 0 0 10 0 0 0 0 0 1+ 2 0 10 10 0 2 1+ 1+ 1 A = inoculated Feb. 1; B = inoculated Feb. 30; 0 = no damage; 1 = 0-30% kill; 2 = 30-50% kill; 3 = 50-70% kill; 4 * 70-90% kill; 5 = 100% kill. 41 and averaged 87° F. After killing of grass parts was evident, the elevated temperatures and humidity were no longer used. The nor­ mal greenhouse temperature and humidity during this period of ob­ servation for recovery averaged 81° F. and 64 percent, respectively. No recovery was noted in any case. The fact that roots were destroyed was substantiated by ob­ servations of slides and comparison to roots of control plants. sue platings were also made periodically. Tis­ Recovery of the Curvularia sp. was unsuccessful, as was recovery of Pleosphaerulina sp. when it was in combination with H. sativum and H. 10—Treatments 19 and 20. It was recovered, however, from Treatment 17. the Helminthosporium was successful in each case. aerial parts were plated. Recovery of Both roots and In three isolations (Treatments 11, 12, 16), nematodes were found in the petri dishes. They apparently were in the dead and dying blades or roots, and were unaffected by the m er­ curic chloride-sodium hypochlorite surface sterilization. Since both blade and aerial parts were plated in the same petri dish and marked as such, the determination as to the source of the freely moving nematode was impossible. , 42 Cultural Studies P e ri thee ial production tr ia ls . The attempts to produce the sexual stage of the Helminthosporlum were unsuccessful. None of the twelve media or four conditions was promising enough to warrant further testing. There was no effect from the four environmental conditions nor from holding the flasks for ten months in a refrig­ erator. The ultraviolet light treatment and the use of nitrogen ad­ ditives were of no value. Variation due to medium. organism is shown in Plate VI. Evidence of the variability of the On the 2 percent malt extract agar, eleven original single-spore isolates were arbitrarily separated into eight cultural groups. These varied as to color,, amount of aerial growth, rate of growth, production of sclerotial-like bodies, and spore production. However, when these same eleven Isolates plus six additional isolates were put on Coon's synthetic agar, only six distinct groups could be distinguished. The comparative grouping in these last six as compared to the grouping on the malt extract is as follows: 1. 5of 6 together previously. 2.' 2of 3 together previously. 3. 1of 1 together previously. 43 4. 2 of 3 together previously. 5. 0 of 2 together previously. 6. 0 of 2 together previously. Although there is a tendency toward grouping on both media, some isolates are apt to be culturally quite different when the medium is changed. At one time, eight single spores were taken from a single dish and allowed to grow separately. Seven of the eight were identical in cultural characteristics when grown on the same medium, while the other showed slight variation. Christensen (2, 3) and Stevens (23). This agrees with results of The entire test shows the ex­ treme variability which may be encountered when attempting to de­ scribe thisfungus on mentionedby Sprague an artificial medium. (22) The extremevariation and Christensen (2, 3) was easily repro­ duced in these studies. A difference in sensitivity to low concentrations of mercury was demonstrated by the repetition of an earlier test (17). Two isolates from bent grass H. 10 and H. 19 were stimulated by 10 and 50 ^ g/m l in the medium, while the H. sativum from Minnesota showed no stimulation at these concentrations (see Table VI). The H. 19, which was the test isolate used previously, was stimulated to the greatest extent, but not as markedly as in the previous trial. Plate 44 T A B L E VI DIAMETER OF FIVE-DAY-OLD COLONIES OF HELMINTHOSPORIUM SATIVUM AND OF TWO ISOLATES OBTAINED FROM CREEPIN'G BENT GRASS (measurements in mm. from replicated plates of 2 percent malt extract agar containing varying amounts of mercury) Isolate ^ g / m l of Mercurous and Mercuric Chloride Mixture^ Least Signif. Dif. 0.0 10.0 50.0 100.0 250.0 500.0 *.05 **.01 H. sativum 59.3 60.8 55.3 47.8 33.0 15.0 2 .0 6 2.86 H. 10 55.0 59.0* 57.8 36.8 14.8 11.5 2.88 3.98 H. 19 44.5 56.0* 55.8* 45.3 17.8 11.3 5.09 7.03 Based upon active ingredients contained in Calo-clor, Mallinckrodt Co. 45 X shows the growth on plates covering the range of mercury con­ centrations employed. D IS C U S S IO N A N D C O N C L U S IO N S Although the conidia of the isolates from creeping bent grass may be slightly wider than the figures given for H. sativum, and the stimulation by low concentrations of mercury occurred only with the bent grass isolates, there are important characteristics so nearly identical that the author believes the fungi are of' the same species. The reaction on the wheat was identical--both isolates caused the characteristic stem lesions, as shown in Plate IY. The parasitic action on bent grass seedlings and killing of established grass was nearly identical. Culturally, some of the bent grass isolates closely resemble the H. sativum growth on the same medium. the variation reported for H. sativum is easily obtained. And culturally, Cultures vary greatly in ability to produce conidia, rate of growth, color of the colony, and production of aerial mycelium. These variations may occur with changes in temperature and humidity, or change of medium. The color, method of germination of the spores, and mor­ phology of the sporophores is also the same for selected isolates of Helminthosporium when they are on the same medium and under identical environmental conditions. 47 The Helmlnthosporium from bent grass is therefore consid­ ered to be H. sativum, as previously stated. There are slight dif­ ferences in degree of severity of attack on the Colonial and creeping bents, and in tolerance to mercury, but these are not considered adequate for species differentiation. Plate II indicates the variability among spores from the same petri dish and similarities between the known H. sativum and an isolate from creeping bent grass. Also, Plate IX illustrates the growth in culture of three Helmlnthosporium isolates. The many failures in the attempts to produce severe killing on established stands of creeping bent grass may be partially ex­ plained to be due to the impossibility of controlling weather condi­ tions. The artificial inoculations under controlled environmental conditions did result in killing of established grass. However, some of these were not entirely satisfactory. The conclusion to be drawn is that H. sativum can be parasitic on creeping bent seedlings and on established stands of this grass. The rapidity of progression and severity of the disease as it occurs in nature, however, has only once been satisfactorily repeated (see Plate III). There is the possibility that large masses of inoculum are necessary. There is also the possibility that nematodes play an 48 important p art in furnishing entry points, if not actually contributing to the destruction of root and aerial parts by parasitic action. The possible action of Curvularia sp. and Pleosphaerulina sp. in conjunc­ tion with each other and with H. sativum should not be overlooked; however, in tests of these combinations, no evidence of combined parasitic action was noted. The possibility still exists that high nitrogen may be a factor in the disease progress. Fertilization of chlorotic areas has been a common practice on golf courses. An examination of these yellow areas has often indicated a high percentage of infection from H. sativum. There, of course, was no corrective reaction from the nitrogen, and the observations were that destruction of grass was more rapid following the fertilizer application. Although limited research was done on Pleosphaerulina and on nematodes, both were established as parasites of creeping bent grass in their own right. Plate VII indicates the morphology and method of penetration by the Pleosphaerulina, while the correspondence with Dr. Thome establishes the identity of the parasitic nematodes of the genus Tylenchorhynchus (see Plate VIII). Since no damage was caused by the nematodes in the one trial, either the wrong species was used, 49 the number introduced into the soil was too small, or the nematodes are not disease producers alone. The tolerance of the bent grass isolates of Helminthosporium to low concentrations of mercury may be a partial explanation as to the apparent sudden outbreak of this new disease on bent grass. Mercury compounds have for the last thirty years been the main components of standard fungicide sprays. The possibility arises that selection and mutation by the fungus may have resulted in these tolerant strains increasing in numbers to the point where sudden infections could occur. It has been observed that only extremely high concentrations of the inorganic mercuries have any noticeable effect on Helminthosporium. Such concentrations probably would cause as much damage to the grass by toxicity as the fungus would in its parasitism . However, it is probably nearer the truth to say that the disease has been present for years, but completely overlooked, due to the almost complete lack of work on fine-turf diseases by plant pathologists. Routine fungicide testing has been practiced to some extent, but thorough, complete investigations into the disease prob­ lems of ornamental grasses is nearly nonexistent. Within the last few years> considerable interest has been generated, and there are 50 now approximately ten plant pathologists in this country and abroad who are engaged in part-tim e turf disease control •studies. A present, the only control known is applications of the anti­ biotic fungicide Acti-dione (17, 27). In an unpublished report this m aterial was also shown to keep the numbers of conidia in estab­ lished grass at a minimum. Spore counts were made from samples obtained from plots of seaside bent grass receiving twelve different fungicidal sprays. Reduction in numbers of viable spores was ob­ served only in the Acti-dione-sprayed plots. From the observations so far made, the value of preventative spraying cannot be over­ emphasized, since mass establishment of the fungus seems to be a prerequisite to severe disease. Future work should include extensive tests on established field plots using H. sativum, alone and in combination with Curvularia sp., Pleosphaerulina sp., and Tylenchorhynchus sp. The various treatments should include a study of the masses of the different inocula necessary to incite disease. Preliminary sprays to reduce the amount of fungus already present in the soil should be employed. The spray residues could be leached with water just previous to the first inoculation. possible. The environment should be controlled insofar as 51 Hie various isolates should also be separated into biologic forxris for subsequent testing for pathogenicity. Christensen (1, 2) has shown that the biologic forms obtained from culture studies do vary greatly in pathogenicity. Since all isolates have not been tested, it is possible that the most pathogenic form was never used for inoculation. A testing program could be set up to determine if H. sativum can increase its tolerance to inorganic mercury, and if this toler­ ance is correlated with increased pathogenicity to creeping bent grass. The biologic forms could be transferred to medium contain­ ing increasing amounts of mercury and tested periodically on estab­ lished grass, or on the seedlings for a more rapid, analyzable dete r mination. A difficult, but perhaps necessary, study of the predisposing factors should be undertaken. Soil pH, flora, fauna, and fertility level may influence the disease. conditions may be necessary. Certain previous environmental Normal traffic and resulting injury to the grass may be a factor, as may be the insecticide applications previously used. SUMMARY 1. An apparently new disease of Agrostig palustris Huds. (creeping bent grass) is described. 2. The fungal organism constantly associated with the dis­ ease is identified as Helminthosporium sativum P. K. & B. 3. Fifty-eight isolations and their areas of origin are listed. 4. The Helmintho sp orium is found to cause complete death of established creeping bent grass plants. 5. The bent grass isolates and a known H. sativum are shown to cause identical damage on established bent grass, bent grass seed­ lings, and Yorkwin wheat. 6. Twelve media and four environmental conditions are used in an attempt to induce production of the perithecial stage. No sexual stage was obtained. 7. The variability of seventeen bent grass isolates was ob­ served on two different media. Both medium and environment were found to contribute to this variation. 8. H. sativum and bent grass isolates are compared as to stimulation when grown in medium containing inorganic mercurials. 53 The Helminthosporium isolates from bent grass were found to be stimulated by low concentrations of mercury. 9. Nematodes {Tylenchorhynchus sp.) are found to be parasitic on creeping bent grass. 10. Curvularia sp. is found to be mildly parasitic on creep­ ing bent grass. 11. Pleosphaerulina sp. is shown to be an apparently unre­ ported parasite of creeping bent grass. B IB L IO G R A P H Y 1. Christensen, J. J. Studies on the parasitism, of Helmjr>t1iosporium sativum. Univ. of Minn. Ag. Expt. Sta. Tech. Bui. 11, 1922. 2. Christensen, J. J. Physiologic specialization and mutation in Helmintho sporlum sativum. Phytopath. XV, No. 12. Dec., 1925. 3. Christensen, J. J. The influence of temperature on the fre­ quency of mutation in Helminthosporium sativum. Phytopath. 19, No. 2. Feb., 1929. 4. Dosdall, Louise. Factors influencing the pathogenicity of Hel­ mlnthosporium sativum. Univ. of Minn. Ag. Expt. Sta. Tech. Bui. 17, 1923. 5. Drechsler, Chas. Some graminicolous species of Helmintho­ sporium. Jour. Ag. Res. 24: 641-740, 1923. 6. Drechsler, Chas. The occurrence of zonate eye-spot on various grasses and its mode of extension. Phytopath. 13: 59-60, 1923. 7. Drechsler, Chas. Zonate eye-spot of grasses caused by Hel­ minthosporium giganteum. Jour. Ag. Res. 37: 473-92, 1928. 8. Drechsler, Chas. Occurrence of the zonate eye-spot fungus Helminthosporium giganteum on some additional grasses. Jour. Ag. Res. 39: 129-35, 1929. 9. Drechsler, Chas. A leaf spot of bent grasses caused by Hel­ mlnthosporium erythrospilum N. sp. Phytopath. Vol. 25: 344, 1935. 10. Elliot, E. S. The effect of sugar concentration on conidial size of some spp. of Helminthosporium. Phytopath. 39: 95358. 1949. 55 11. Goodey, T. Plant Parasitic Nematodes and the Diseases They Cause. E. P. Dutton & Co., New York. 1933. 12. Grau, F. V., and O. J. Noer. Golf is played on grass. Year­ book of Agriculture, 1948, U. S. Gov't Printing Office, Washington, D. C. 13. Hitchcock, A. S. Manual of the Grasses of the United States. 2nd Ed. USDA Misc. publ. 200. 1951. 14. Howard, F. L. 1952: 3. 15. Jenkins, W. A. A root rot disease-complex of small grains in Virginia. Phytopath. 38, No. 7, pp. 519-527, 1948. 16 . Kirby, R. S., and H. W. Thurston. Prevention of turf diseases. Separate from The Penn. State College Agr. Ext. Service. May, 1943. 17. Klomparens, Wm., and J. R. Vaughn. The correlation of labora­ tory screening of turf fungicides with field results. Mich. State Col. Ag. Expt. Sta. Bui. Vol. 34, No. 4: 425-435, May, 1952. 18. Lefebre, C. L., and H. W. Johnson. Collection of fungi, bac­ teria and nematodes on grasses. PI. Dis. Reptr. 25: Turf fungicide studies, 1952. PI. Path. Report R. I. Expt. Sta., Kingston, R. I. 556-79, 1941. 19. Lownsberry, B. F., E. M. Stoddard, and J. W. Lownsberry. Paratylenchus hamatus pathogenic to celery. Phytopath. 42, No. 12, pp. 651-653, December, 1952. 20. Pole, Evans (I. B.). Aiming at better pastures and field crops. Ann. Rept. of Div. PI. Industry Fmg. So. Africa. 9: 539-68, 1934. 21. • Report of the director. 118, 1931. 22. Conn. Ag. Expt. Sta. Bui. 322: 117- Sprague, R. Diseases of cereals and grasses in North America. The Ronald Press Co., New York, 1950. 56 23. Stevens, F. L. The Helminthosporium root-rot of wheat, with observations on the morphology of Helminthosporium and on the occurrence of saltation in the genus. 111. Nat. Hist. Survey Bui. Vol. 14, Article 5, 1922. 24. Struble, F. B. Plot technique for disease control studies on fine turf. Phytopath, 33: 528-530, 1943. 25. Thome, 26. Van Luijk, A. Unterschungen uber Krankheiten der Graser. Willie Gommelin Scholten, Baarn (Holland) 13: 1-22, 1934. G. W. USDA Nematologist. Correspondence. 27. Vaughn, J. R., and Wm. Klomparens. Drugs on the green. Golf Course Reporter, Mar.-April, 1952. The 28. Wernham, C. C., and R. S. Kirby. Prevention of turf diseases under war conditions. Separate from The Penn. State College Agr. Ext. Service, Mar., 1949. APPENDIX 58 PLATE I Camera lucida drawings of Helminthosporium sativum conidia obtained from creeping bent grass. The shorter, germinating spores from artificial medium--the longer spores scraped from infected grass. See Table II for measurements. Approximately 500X. 59 PLATE I 60 PLATE II A, B,- C, D. Ej F. Conidia of H. sativum obtained from creeping bent grass. Conidia from II. sativum isolate received from J. J. Christensen, Minnesota. Note the lighter color of immature conidia and variability in spore shapes. agar. Photographs from spores produced on 2 percent malt extract Approximately 450X. 61 A B C D E F V PLATE II 62 PLATE HI A. Flats of Washington creeping bent grass grown in the greenhouse. Left flat inoculated with H. sativum conidia three days previously. Flat on the right sprayed with, sterile distilled water. Both held in humidity chamber (Plate VHI) at 77° C. and 90 percent rela­ tive humidity. B. Close-up of infected grass in Figure A three weeks later. plete killing was evident, since no regrowth occurred. Com­ 64 PLATE IV A. B. Yorkwin wheat seedlings from pathogenicity test. 8. From soil infested with H. 10 (creeping bent grass isolate). 7. Control. 6. From soil infested with H. sativum from Minnesota. Washington variety creeping bent grass with small oval spots and larger blotches from infection by H. sativum. 66 PLATE V Seaside creeping bent grass established for twenty-four days in plant bands, then transferred to pots for inoculation. sixteen days after inoculation. A. Photographed Selected treatments as follows: Spore suspension of H. 10 sprayed on grass growing in steamed soil. B. Spore suspension of If. sativum sprayed on grass growing in steamed soil. C. Spore suspensions of H. 10 plus H. sativum sprayed on grass established in steamed soil infested with Nematodes 1 and 2. D. Established grass growing in steamed soil infested with H. 10. E. Control. F. Established grass growing in steamed soil infested with Nema­ tode 1 and Isolate H. 10. P L A T E VI A. Helminthosporium isolates from creeping bent grass grown, on 2 percent malt extract agar. Eight artificial groupings from an original eleven isolates. B. Six artificial groupings of the isolates used in A grown on Goon's synthetic medium. The eleven isolates from A and six additional single-spore cultures resulted in only six groups on this medium. C. Immature Helminthosporium sativum conidium on a sporophore (1500X). D. E. Helminthosporium sativum sporophores and mature spore (260X ). 69 PLATE VI 70 PLA TE Vn A. Perithecia of Pleosphaerulina sp. on creeping bent grass (250X). B. Close-up of A (350X). C. Camera lucida drawings of germinating Pleosphaerulina sp. ascospores. Penetration of creeping bent grass leaf blades is shown by the arrows. Careful focusing showed direct penetration as well as entrance through stomata (750X). 71 PLATE VEt P L A T E V III A. Humidity chamber used for early pathogenicity tests containing plugs of infected grass for future isolation and identification work. B, C. Photographs of Tylenchorhynchus sp. from creeping bent grass. Mounts of the nemas prepared and identified by Dr. Gerald Thorne. 73 74 P L A T E IX A. Cultural characteristics of some of the fungi used in this studygrowing on Czapek's synthetic medium. B. 0. Pleosphaerulina sp. 1. Curyularia sp. 2. H. sativum. 3. H. 10 (creeping bent grass isolate). Cultural characteristics of Helminthosporlum sp. growing on 2 percent malt extract agar. Top left: H. 19 (creeping bent isolate). Top right: Bottom: H. 10 (creeping bent isolate). H. sativum, Minnesota. 75 PLATE IX 76 PLATE X Simulation of the growth of two Helminthosporium isolates from bent grass when grown in 2 percent malt extract agar contain­ ing small amounts of inorganic mercury. A. Isolate H. 10. To left: Control. Top right: 10 ^(g/ml mercury. Bottom left: 50 ^g/m l mercury. Bottom right: B. 100^g/ml mercury. Isolate H. 19. Top left: Control. Top right: 10 ^g /m l mercury. Bottom left: Bottom right: 50 ^ g /m l mercury. lOO^g/ml mercury. 77