Mflifi This is to certify that the thesis entitled Epidemiology of Loose Smut, Caused by Ustilago tritici presented by BOTANY AND PLANT PATHOLOGY ROS EMARY MOONEY; LORI A has been accepted towards fulfillment of the requirements for Ph.D. deg-cam Plant Pathology 4mm r ’ Majoé‘femr DateSMZX; [9Y0 0-7639 OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to remove charge from circulation records EPIDEMIOLOGY OF LOOSE SMUT OF WHEAT, CAUSED BY USTILAGO TRITICI By Rosemary Loria A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology ABSTRACT EPIDEMIOLOGY OF LOOSE SMUT OF WHEAT, CAUSED BY USTILAGO TRITICI By Rosemary Loria Loose smut of wheat (Triticum aestivum L.), caused by Ustilago tritici (Pers.) Rostr., reduces yields an estimated l to 2% in Michigan. This two-part study of the epidemiology of loose smut in Michigan in- cluded (l) investigating spore dispersal and infection in infected fields and around a point source of inoculum, and (2) determining the effect of wetness period, wheat head development, and embryo accessibility on in- fection efficiency. 1. Spore dispersal was positively correlated with wind speed, solar radiation, and temperature and negatively related to relative humidity; and leaf wetness. There was no detectable correlation between spore numbers and rain. Infection in seed samples taken at random from infected fields during the three years of the study was consistently low (<0.3%), and only slightly higher near individual smutted (50.53%) heads in those fields. Infection could not be related to the incidence of smutted heads, leaf wetness, rainfall, relative humidity, or wind speed during flowering in these fields, though cool temperatures seemed to be favor- able for infection. Spore catches at 0.9 and 2.7 m from a point source of inoculum were usually related to wind speed and direction. Infection occurred only O-O.9 m from the inoculum source and usually near the Rotorod traps with the highest spore catches. Infection of healthy potted plants exposed to inoculum for short periods of time occurred during two out of a total of nine exposure periods during l978 and 1979. Maximum infection (0.7%) occurred when: the average air temperature was 16 C; leaf wetness duration was 20 hr; average wind speed was 10.2 km/hr; rainfall was 0.77 cm; plants were flowering; and the spore catch was 2.8 x 103. II. Varying the inoculum concentration by a factor of lo3 did not effect infection of heads, though those inoculated during anthesis (2.5%) were more susceptible than heads inoculated before (0.80%) or after (0.66%) anthesis. Infection was somewhat higher with longer wetting periods up to 32 hr (22.9%) in length, but was relatively high with no wetting period (12.3%). Infection of florets in which the glumes had been clipped to improve accessibility (5.7-40%) was higher than for non- clipped florets (0-l4.3%) at all stages of flowering. Spore germination was increased with vapor pressure inside florets and was higher inside than outside florets at the two highest vapor pressures. To Kathy ii ACKNOWLEDGEMENTS I would like to express my appreciation to Drs. Alan Jones and Maury Wiese for their guidance during the course of my graduate education. Dr. Jones' assistance in the preparation of this dissertation was especially valuable. I also thank Drs. E. D. Goodman, J. L. Lockwood, D. C. Ramsdell and C. Stevens for serving on my graduate committee. Appreciation is expressed to Scott Eisensmith for statistical and computer consultation, and to Anne Mills for technical assistance. Most importantly, I am very grateful to Lenny for his unfailing love and support. TABLE OF CONTENTS LIST OF TABLES ......................... LIST OF FIGURES ........................ GENERAL INTRODUCTION AND LITERATURE REVIEW ........... Literature Cited ...................... PART I SPORE DISPERSAL AND INFECTION OF GENESSEE WHEAT BY USTILAGO TRITICI IN NATURALLY INFECTED FIELDS AND AROUND A POINT SOURCE OF INOCULUM INTRODUCTION .......................... MATERIALS AND METHODS ..................... RESULTS ............................ DISCUSSION ........................... LITERATURE CITED ........................ PART II EFFECTS OF WETNESS PERIODS, GROWTH STAGE AND EMBRYO ACCESSIBILITY ON INFECTION OF WHEAT BY USTILAGO TRITICI INTRODUCTION .......................... MATERIALS AND METHODS ..................... RESULTS AND DISCUSSION ..................... LITERATURE CITED ........................ APPENDICES ........................... APPENDIX A: INCIDENCE 0F LOOSE SMUTTED HEADS AND SUBSEQUENT INFECTION OF USTILAGO TRITICI IN COMMERCIAL WHEAT FIELDS IN MICHIGAN ................ iv Page Introduction ........................ 5l Methods .......................... 5l Results and Discussion ................... 52 Literature Cited ...................... 55 APPENDIX B: EFFORTS TO PRODUCE ANTISERA TO USTILAGO TRITICI Introduction ........................ 57 Materials and Methods ................... 57 Results and Discussion ................... 59 Literature Cited ...................... 6O LIST OF TABLES Table l. PART I Correlation of log (aerial concentrations of Ustilago tritici + l) with environmental parameters in naturally infected Genessee wheat fields during periods of peak spore dispersal in 1977, 1978, and 1979 ..... Summary of environmental conditions during flowering, percentage of loose smutted heads available as inoculum, and subsequent infection of Genessee wheat by Ustilago tritici in naturally infected fields during I977, 1978, and 1979 .................. Infection of Genessee wheat by Ustilago tritici at 0-15, 15-30, and 30-45 cm from individual smutted heads in naturally infected Genessee wheat fields during 1977 and 1979 ........................ Rotorod spore trap catches of Ustilago tritici telio- spores and subsequent loose smut infection around a point source of inoculum in a Genessee wheat field during 1978 ....................... Rotorod spore trap catches of Ustilago tritici telio- spores and subsequent loose smut infection around a point source of inoculum in a Genessee wheat field during 1979 ....................... . Summary of environmental conditions, plant growth stage, and Rotorod trap catches of Ustilago tritici telio- spores during exposure of potted Genessee wheat plants to an inoculum source during 1978 ............ Summary of environmental conditions, plant growth stage, and Rotorod trap catches of Ustilago tritici telio- spores during exposure of potted Genessee wheat plants to an inoculum source during 1979 ............ PART II Effect of the stage of wheat head development and exposure of the embryos to inoculum by clipping the glumes, on infection of Genessee wheat by Ustilago tritici ......................... vi Page 17 22 23 25 26 30 31 45 TABLE Page 2. Germination of teliospores of Ustilago tritici on millipore filter squares inside and outside of Genessee wheat florets in response to ambient vapor pressure ........................ 47 APPENDIX A 1. Incidence of loose smutted heads and subsequent head and plant infection in 17 commercial wheat fields in Michigan in 1977 and 1978 .............. 53 vii LIST OF FIGURES Figure 1. PART I Field plot diagram for investigation of teliospore dispersal and infection of Ustilago tritici around a point source of inoculum in a smut-free wheat field during 1978 and 1979 ........... Aerial concentration of Ustilago tritici teliospores in a naturally infected wheat field in East Lansing, MI, during 1979. Arrow indicates the period of peak flowering of healthy wheat heads ......... Hourly concentrations of teliospores of Ustilago tritici per cubic meter of air, with corresponding wind speed, air temperature, relative humidity, dew, and solar radiation at Kawkawlin, MI, 4 June 1977 . . . . Hourly concentrations of teliospores of Ustilago tritici per cubic meter of air, with corresponding wind speed, air temperature, relative humidity, dew, and solar radiation at Kawkawlin, MI, 2 June 1977 . . . . Rotorod catches of teliospores of Ustilago tritici at eight compass directions at 0.9 and 2.7 m from an inoculum source and corresponding running distance of wind for 16 June 1978 ........... Rotorod catches of teliospores of Ustilago tritici at eight compass directions at 0.9 and 2.77m from an inoculum source and corresponding running distance of wind for 15 June 1978 ........... PART II Infection of clipped and nonclipped florets of Genessee wheat heads by Ustilago tritici in relation to the stage of head development ....... viii Page 14 18 I9 20 27 28 44 GENERAL INTRODUCTION AND LITERATURE REVIEW Loose smut of wheat (Triticum aestivum L.), caused by Ustilago tritici (Pers.) Rostr., has been a sporadic problem in Michigan. The incidence of loose smutted heads has been observed to range from O to 5% in individual fields, and is estimated to average between 1 and 2% for the state (35). An incidence of 1% loose smutted heads results in approximately a 1 million dollar loss in yield to Michigan wheat growers. The current control practices are elimination of infected seed from production through seed certification, and/or eradication of the fungus from infected seedlings with a systemic fungicide seed treatment. Unfor- tunately, there are difficulties with both of these control practices. A maximum of 0.5% loose smutted heads is allowed in foundation or certified seed fields. Since little is known about the relationship between inoculum density, environmental conditions, and subsequent loose smut infection, present certification procedures potentially allow acceptance of.infected and rejection of healthy seed lots. Carboxin is the only fungicide registered for the control of loose smut of wheat. Since there is no economically feasible means of dis- tinguishing between infected and healthy seed lots, this fungicide is applied to seed regardless of whether loose smut control is necessary. This policy results in excessive pesticide usage and encourages the development of resistant strains of the fungus. Resistance to other 2 oxathion fungicides by U, mgygig has already been demonstrated under laboratory conditions (33). The purpose of this research project was to investigate the epidemiology of loose smut of wheat, with the intent of obtaining some of the information necessary to develop a mathematical model for loose smut infection. Research concerning spore dispersal and infection in the field and infection under controlled conditions is described in parts one and two of the dissertation, respectively. Antisera to U, tritici were prepared in an attempt to evaluate the potential of a serological seed indexing technique for loose smut of wheat. This work is described in Appendix B of the dissertation. Liter- ature pertinent to the etiology, epidemiology, and seed indexing of loose smut of wheat, and related diseases, are reviewed in the remainder of this introduction. U, tritici survives as dormant mycelium primarily within the scutellum and the growing point region of the infected wheat seed (2, ll, 12). When an infected seed germinates, the pathogen is carried upwards in the growing point by hypocotyl elongation. The node of the first foliage leaf, which becomes the first crown node, contains mycelium of the fungus which colonizes the rest of the crown as it develops. By the time tiller growth begins, the fungus is already established in the immature ear, and is carried upwards with it. Spore formation occurs sometime before the infected ear emerges from the uppermost leaf. Ears about 2.5 cm in length are brown in color and contain some spores, along with large amounts of mycelium. As the ear expands spores are formed in large numbers and, upon emergence, it appears as the typical black spore mass (2). The spores quickly become powdery, and are available for dispersal. 3 Teliospores of loose smut rely completely on external conditions for dispersal, and inoculation depends on the chance deposition of one or more spores onto the ovary of a healthy floret, which is exposed when the glumes open during flowering. The time required for a wheat flower to open was found to be about 3 min, and the total time that elapsed between opening and closing averaged 26 min and 30 sec (14). Individual flowers are therefore exposed to inoculum for only a very short period of time. However, the duration of blooming of the flowers of an individual wheat head mayrange from 2 to 7 days, averaging about 3 days (1A).. Other Ustilago species have been shown to have diurnal spore dis- persal patterns with maximum release occurring in the afternoon. This pattern has been attributed to increased wind speed during this period (17, 27, 28, 29). Impaction of raindrops On smutted heads disperse Ustilago spores by the "tap" and "puffi mechanisms described by Gregory (7), but rainfall also washes spores from smutted heads and from the air (10, 17, 28). Very little information is available on the effects of environmental conditions on natural infection by U, tritici. Humid conditions at the time of flowering have been shown to be favorable to infection when the spores are introduced into the wheat flowers (30, 31), and it is generally known that loose smut of-wheat is more severe in humid than in arid climates (31). The effect of temperature on infection efficiency has not been examined thoroughly. However, when the germination of teliospores of U, tritici was investigated in relation to temperature ig_!jtrg, the minimum was below 5 C, the optimum was about 20 to 25 C, and the maximum was between 25 and 30 C (19). The relative importance of infection of the wheat embryo directly through the ovary wall or via the stigma has been investigated by several workers (5, ll, 16, 24, 34). Though direct penetration of the ovary wall by mycelium present on its surface has been documented (1), there is no histological evidence that penetration through the stigma results in infection of the ovary (25). Upon penetration of the ovary wall, the mycelium traverses the parenchyma of the pericarp; and after about 11 days, has entered the integuments (testa) and nucellus region. After permeating this region, it passes towards the base of the grain and enters the aleurone layer. The fungus then enters the scutellum where it grows profusely and enters the growing point of the embryo (15). Dormant mycelium with thickened cell walls is formed as the infected grain matures, and is able to survive for long periods of time in the dry seed (25). There are conflicting reports on the importance of the environment between planting and heading on subsequent incidence of loose smutted heads. While Neill (18) found that different preemergence soil temp- eratures had little effect on the infection levels of g, tritici, Tieman (32) stated that a late seeding of spring wheat at relatively high temperatures increased disease incidence. Oort (20) also found that a warm soil promoted loose smut in wheat. Soil fertilization has been reported to increase (9), decrease (6), or have no effect (32) on disease incidence. Vernalization has been shown to either reduce loose smut (13) or have no effect (8). Seeds infected with U, tritici survived soaking and freezing as well as noninfected seeds, and plants produced from them had similar levels of infection as those from non- treated seeds (3). However, more winter killing occurred in wheat plants grown from infected seed under winter conditions at Ithaca, NY 5 than under milder conditions at Rosslyn, VA (30). A seed indexing technique has been developed to detect embryo in- fection of wheat and barley with U, tritici and U, nude, respectively (26). This technique involves isolating the embryos from the grains, then staining and clearing them for microscopic examination. The presence of mycelium in the embryo has been shown to be highly correlated with field and greenhouse indexes for disease in barley (21, 22). Unfor- tunately, this technique has limited applicability to wheat, since the numbers of loose smutted plants are affected not only by the percentage of infection in the seed, but also by the host variety and physiological race of the fungus. Popp has shown that the infection of the plumular- bud tissue of embryos is directly related to the development of smut in adult wheat plants, and that the presence of the fungus in this area of the embryo is a reliable indicator of adult plant infection. However, since up to 1000 embryos must be examined to accurately determine the level of loose smut in a single seed sample (23), this method of seed indexing is economically feasible only when loose smut is a limiting factor in wheat production. 10. 11. 12. LITERATURE CITED Batts, C. C. V. 1955. Observations on the infection of wheat by loose smut (Ustilago tritici (Pers.) Rostr.). Trans. Brit. Mycol. Soc. 38:465-475. Batts, C. C. V., and Ann Jeater. 1958. The development of loose smut (Ustilago tritici) in susceptible varieties of wheat, and some observations on field infection. Trans. Brit. Mycol. Soc. 41:115-125. Buchheim, A. N. 1935. [Effect of freezing on germinability of wheat seeds infected with loose smut and on the development of the plants raised from them]. Pl. Prot. Sta., Leningrad, Bul. 6:134-137. Doling, D. A. 1965. Some ecological factors influencing the proportion of loose smut (Ustila o nuda (Jens.) Rostr.) in wheat crops. Ann. Appl. Biol. 55:303-306. Freeman, E. M., and E. C. Johnson. 1909. The loose smut of wheat and barley. Bull. U. S. Pl. Ind. No. 152, 48 pp. Gassner, G., and H. Kirchhoff. 1934. Zur Frage der Beeinflussung des Flugbrandbefalls durch Umweltfactoren und Chemische Beizmitt Beizmittel. Phytopath. Zeitschr. 7:487-503. Gregory, P. H. 1973. The Microbiology of the Atmosphere. Leonard Hill, Aylesbury. 377 pp. Hanna, W. F. 1936. Effect of vernalization on the incidence of loose smut of wheat. Sci. Agr. 16:404-407. Hiltner, L., and F. Land. 1922. Ueber den Einfluss der Dungun insebesan dere mit Kalkstickstoff, auf die Stdrke des Brand- befalls des Getreides. Mitt. Deut. Landw. Ges. 37:353-357. Hirst, J. M., and 0. J. Stedman. 1963. Dry liberation of spores ‘ by raindrops. J. Gen. Microbiol 33:335-344. Lang, W. 1910. Die Bluteninfektion biem Weizenflugbrand. Zentalbl. f. Bakt. Abt. 2, 25:86-101. Lang, W. 1917. Zur Ansteckung der gerstche durch Ustilago nuda. Ber. Deuts. Bot. Ges. 35:4-20. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. Lasser, E. 1938. Der Einfluss von Licht and Jarowisation auf den Befall von Weizen, Hafer und Gerste durch Tilletia, Ustilago, und Helminthosporium. Kuhn Arch. 44:161-210. Leighty, C. E., and T. B. Hutchenson. 1919. On the blooming and fertilization of wheat. J. Amer. Soc. Agron. 11:143-162. Leighty, C. E., and W. J. Sando. 1924. On blooming of wheat flowers. J. Agr. Res. 27:231-244. McAlpine, D. 1910. The smuts of Australia. 288 pp. Melbourne. Mills, J. T. 1967. Spore dispersal and natural infection in oat loose smut (Ustilago avenae). Trans. Br. Mycol. Soc. 50:403- 412. Neill, J. C._ 1925. Loose Smut of Wheat II. Field experiments in seed disinfestation with hot water. New Zealand J. Agr. 30:167-174. Novopokrovsky, I. V., and Skaslcin, F. D. 1925. [Effect of temperature on the germination of the chlamydospores of cereal smuts (genus Ustilago).] Pamphlet of the North Caucasus Region Land Administration, Rostoff-on-Don. 28 pp. Oort, A. J. P. 1940. De vat baar heid voor stuiflorand van in Nederland verboulude of beprofde rassen van Tarwe en Gerst. Meded. Landb. Hoogesch. Wageningen 44. Russel, R. C. 1950. The whole embryo method of testing barley for loose smut as a routine test. Sci. Agr. 30:361-366. Russel, R. C., and W. Popp. 1951. The embryo test as a method of forecasting loose smut infection in barley. Sci. Agr. 31:559-565. Russel, R. C., B. J. Sallans, and H. B. Cannon. 1961. The use of sequential sampling in embryo test for loose smut in barley seed. Can. J. Plt. Sci. 41:772-779. Ruttle, M. L. 1934. Studies on barley smuts and on loose smuts of wheat. Tech. Bull. N. Y. Agric. Exp. Sta. No. 221, 39 pp. Shinohara, M. 1976. Behavior of Ustilago nuda (Jens.) Rostrup and U, tritici (Pers.) Rostrup in their host tissues. Rev. Plant Protection Research 9:124-142. Simmonds, P. M. 1946. Detection of the loose smut fungi in embryos of barley and wheat. Sci. Agr. 26:51-58. Sreeramulu, T., and B. P. R. Vittal. 1972. Spore dispersal of the sugar cane smut (Ustilago scitaminea). Trans. Brit. Mycol. Soc. 58:301-312. 28. 29. 30. 31. 32. 33. 34. 35. 8 Sreeramulu, T. 1962. Aerial dissemination of barley loose smut (Ustilago nuda). Trans. Brit. Mycol. Soc. 45:373-384. Sreeramulu, T. 1959. The diurnal and seasonal periodicity of spores of certain plant pathogens in the air. Trans. Brit. Mycol. Soc. 42:177-184. Tapke, V. F. 1929. Influence of varietal resistance, sap acidity, and certain environmental factors on the occurrence of loose smut in wheat. J. Agr. Res. 39:313-339. Tapke, V. F. 1931. Influence of humidity on floral infection of wheat and barley by loose smut. J. Agr. Res. 43:503-516. Tieman, A. 1925. Untersuchungen uber die Empfanglichkeit des Sommerweizens fur Ustilago tritici und den Einflus der ausseren Bedingungen dieser Krankeit. Kuhn. Arch. 9:405-475. Van Tuyl, J. M. 1977. Genetics of fungal resistance to systemic fungicides. Meded. Landbouwhogeschool Wageningen, 77-2. Vanderwalle, R.‘ 1942. Note sur la biologie d' Ustilago nuda tritici. Schaf. Bull. Inst. Agron. Glembloux. 11:103. Wiese, M. V. Personal communication. PART I SPORE DISPERSAL AND INFECTION OF WHEAT BY USTILAGO TRITICI IN NATURALLY INFECTED FIELDS AND AROUND A POINT SOURCE OF INOCULUM INTRODUCTION Loose smut of wheat (Triticum aestivum L.), caused by Ustilago tritici (Pers.) Rostr., occurs sporadically in Michigan and reduces yield and estimated 1 to 2% annually. Infection occurs during the flowering stage of plant development when teliospores are dispersed from smutted heads to neighboring healthy heads, and infect through the ovary wall of individual florets. Knowledge of the factors that affect U. tritici teliospores dispersal is very limited, but spore dispersal in other Ustilago species have been studied (6, 9, 10). Wind and rainfall have the greatest influence on dispersal, and maximum dispersal was often observed when wind speeds were highest (9). Rainfall increases the aerial concentration of teliospores during the beginning of rain by impaction of raindrOps on smutted heads, but then washes Spores from the heads and the air resulting in a decrease in spore numbers (4, 6). Field infection of wheat by U. tritici teliospores is thought to be favored by high humidity and wetness (12), but the relationship between spore dispersal, host susceptibility, and environmental factors is unclear. The purpose of this study was to investigate spore dispersal and infection of wheat PY.Q° tritici in relation to environmental factors, under Michigan conditions. 10 MATERIALS AND METHODS Spore dispersal and infection in naturally-infected fields. Dispersal of teliospores of U. tritici was studied by continually monitoring aerial spore concentrations in naturally infected wheat fields from the beginning of heading until the grains were three-quarters formed, stages 11 through 22 on the Romig numerical scale [RNS, 11-12] for assessment of wheat growth (1), respectively. Fields were located in Grand Rapids (GR77) and Kawkawlin (KAW77), MI, in 1977; at East Lansing (EL78) and Ithaca (IT78), MI in 1978; and at East Lansing (EL79), MI, in 1979. All fields were planted to Genessee wheat and were between 0.8 and 1.6 ha. A Burkard 7-day volumetric spore trap (Burkard Manufacturing Co., Ltd., Rickmansworth, Hertfordshire, England) was centrally located in each field with the orifice at head height, and was adjusted to sample 8 L of air per minute. Teliospores on the Melinex tapes were idenitifed by their characteristic size, shape, and color. Hourly spore counts were obtained by counting the spores in each hourly (2 mm) band when spore numbers were low ((100 teliospores/hr), and by traversing the tape once at 2 mn intervals and correcting for the area sampled when spore numbers were high (>100 teliospores/hr). At location GR77, temperature and relative humidity were measured with a 7-day recording hygrothermograph placed 1 m above ground level in a standard instrument shelter. Leaf wetness was recorded with a deWitt 11 12 7-day recording leaf wetness meter (Valley Stream Farms, Orono, Ontario, Canada) at 2 m above ground level. Wind speed and rainfall data were obtained from the Kent County Airport 6.4 km away. At KAW77, temperature, relative humidity, and leaf wetness were measured as at GR77. Wind speed was measured in the field at 3 m above ground level using a contact anemometer attached to a 7-day recorder. A 7-day recording mechanical pyranograph was used to measure solar radiation. At EL78 and IT78, temperature, relative humidity, and leaf wetness were monitored as in 1977. Rainfall and solar radiation were also measured at EL78 using a tipping-bucket rain gauge attached to a 7-day recorder and a 7-day recording mechanical pyranograph, respectively. At EL79, temperature, relative humidity, leaf wetness, solar radiation and rainfall were measured as in 1978. A contact anemometer was used to record wind speed. Weather observations were recorded continually during the period of spore dispersal at all locations, and were manually transcribed into hourly values. Plant growth was monitored at 3-7 day intervals during the period of Spore trapping with the RNS method. The percentage of smutted tillers was determined by counting 100 tillers in each of ten randomly selected areas in the field. After the grain matured, seeds were collected randomly in the field and in a circular pattern at 0-15, 15-30, and in some cases, 30-45 cm from 10 to 20 individual smutted heads per field. This seed was planted at East Lansing in 1977, 1978, and 1979, grown to heading, and the numbers of smutted and healthy heads produced fron each seed lot, consisting of 800-1200 seeds, was determined. Correlation coefficients between hourly spore counts and weather data were calculated for periods of peak spore diSpersal at all 13 year-locations and were tested for significance with the Student's t-test using the Statistical Package for the Social Sciences (7). Weather data were summarized for each year-location for the time period when wheat was at or near flowering [RNS, 15-19] with the Epidemiological Information Storage, Retrieval and Analysis System (2) and, along with inoculum levels, was compared with infection at those locations. Spore dispersal and infection around agpoint source of inoculum. In 1978 and 1979 at East Lansing, teliospore dispersal and infection were monitored at two distances from a point source of inoculum. Eight infected plants, with a total of 12 smutted tillers, were transplanted into a 16 L metal container. Smutted heads were covered with plastic bags to prevent dispersal of the spores, and the container was placed in a wheat field just prior to head emergence. The field had been planted to carboxin-treated Genessee wheat seed the previous fall, was free of loose smut, and was at the same growth stage as the introduced smutted plants. Sixteen rotorod samplers (Ted Brown Associates, Los Altos Hills, CA 94022) were positioned at head height at eight compass points (N, NE, E, SE, S, SW, W, NW) on concentric circles 0.9 and 2.7 m from the inoculum source (Figure 1). Spore traps were simultaneously turned on fer 30 min every 2 hr with a time clock. Rods were thinly coated with a silicone compound (General Electric, Waterford, NY 12188), and spore catches were microscopically determined by traversing the width (2 mm) of the 64 mm I rods at 10 randomly selected locations at a magnification of 400 X, and correcting for the area sampled. Smutted heads were removed from the bags and spore sampling was initiated when the heads on the plants in the field were 25% emerged [RNS, 11]. Temperature, dew point, rainfall, wind speed, and wind direction were monitored continuously during the period 14 INOCULUM DISPERSAL FROM A POINT SOURCE - FIELD PLOT DIAGRAM NE / 2.7 Meters NW / .9 Meters @ INOCULUM souace . ROTOROD SPORE TRAPS Figure 1. Field plot diagram for investigation of teliospore dispersal and infection of Ustilago tritici around a point source of inoculum in a smut-free wheat field during 1978 and 1979. 15 of spore collection using a Climatronics Electronic Weather Station and 30-day recorder (Climatronics, Hauppage, NY 11787). Field-grown potted wheat plants, at the same growth stage as the field plants, were placed in close proximity to the smutted plants and, along with the rods on the rotorod samplers, were changed daily until the kernels on the plants were three-quarters filled [RNS, 22]. Potted plants were removed from the field to a greenhouse. Mature seeds were harvested from the potted plants, and from plants in the field located between the eight compass points and 0-3 and 3-9 m from the inoculum source. Seed samples from potted and field grown plants were planted in the greenhouse and field, respectively, and the number of smutted and healthy tillers at heading was determined. Weather data were summarized for the time periods when potted plants were exposed and spore dispersal was monitored. Infection of potted plants was compared with summarized weather and spore catch data during the exposure period. Directional spore trap catches were compared with wind speed and direction, and correlation coefficients were calculated. Directional spore trap catches were also compared with the incidence of infection in seed samples collected around the inoculum source. RESULTS Spore dispersal and infection in infected fields. Teliospores of .U. tritici were first detected in air as the heads of healthy wheat emerged, and increased in numbers during flowering (Figure 2). Peak concentrations occurred during or soon after flowering was complete, and concentrations often remained high until most heads had passed anthesis. This pattern was typical of spore dispersal at all locations. Spore catches were positively associated with air temperature, solar radiation, and wind speed at all locations for which data were available, but only the correlation coefficients for spore catches and wind speed were always statistically different from zero (Table 1). Spore catches were always inversely related with relative humidity and leaf wetness, and correlation coefficients were significanlty different from zero for two of the five locations for leaf wetness and for three of the five locations for relative humidity, respectively. There was no detectable correlation between spore numbers and rainfall. Daily spore catch was often diurnal with peak catch occurring in the afternoon (Figure 3). Wind speed, temperature, and solar radiation were highest, and relative humidity and leaf wetness lowest during this afternoon period. Diurnal patterns of spore catch were lacking on days with low afternoon wind speeds (Figure 4) or on days with rain in the late afternoon. 16 17 TABLE 1. Correlation of log (aerial concentrations of Ustilago tritici +1) with environmental parameters in naturally infected Genessee Wheat fields during periods of peak spore dispersal in 1977, 1978, and 1979. Correlation Coefficientsa Location Air [eafi Rain Relative Solar *Wind Temperature Wetness Humidity Radiation Speed GR77 0.055 -0.508 0.317 -0.125 -- 0.646* KAW77 0.609** -0.462** -- -0.731** 0.197 0.761** EL78 0.162 -0.112 -0.039 -0.016 0.166 0.316** IT78 0.687** -0.449* -- -0.626** -- -- EL79 0.698** 0.075 -- -0.702** 0.654** 0.650** aAsterisks indicate significant correlation coefficients at 3:0.01 (**) or 3=o.os (*). 1425 1375 1325 2755 2255 175 125 TELIDSPORES/M3 RIR 75 25 Figure 2. 18 ‘ EAST LANSING - 1979 : AN 1 1 I 1 1 I 1 1o 11 12 13 14 15 1e 9 JUNE Aerial concentration of Ustilago tritici teliospores in a naturally infected wheat field in East Lansing, MI, during 1979. Arrow indicates the period of peak flowering of healthy wheat heads. 19 Y Y T ‘7 V V W T r Y Y 7 V I V I T Y T j j V f Ill ‘ 5 xnwxnwuw "233 «yawn 8": 3 3'8 “a o 1 I an“ * 23% 532 3r AIR TEMP (CI :54: 3 ‘ i ( RELATIVE HUMIDITY ('76) 1 g as 3 u a- 5 33 “:3 1.0 340 35) o (f? ---L- I! :c:::r-......r..1-.-vvv~ I 2 4 G O 10 12 14 1616 20 22 24 0 HOUR (EST) Figure 3. Hourly concentrations of teliospores of Ustilago tritici per cubic meter of air, with corresponding wind speed, air temperature, relative humidty, dew, and solar radiation at Kawkawlin, MI, 4 June 1977. 20 u IIIIIIIIIIIIIIIIIIIIII I KAWKAWUNWWT’ 1.23 Our: 8:: 3 W ‘8 ‘a z 1 ? 7 7 7 9°? 9 23‘,{ 535 3 AIR rear (C) 3 1 1 1 1 1 1 1 1 } neumve Humour 1%) 8 8 8 DEW o d C . SOLAR RADIATION (OI-CAUCM’IIIN) f1‘2'114'1TG‘ HOUR (EST) Figure 4. Hourly concentrations of teliospores of Ustilago tritici per cubic meter of air, with corresponding wind speed, air temperature, relative humidity, dew, and solar radiation at Kawkawlin, MI, 2 June 1977. 21 Although field infection was low for all years and locations studied, some comparisons can be made between inoculum levels and environmental conditions present in the five study locations and subsequent disease development (Table 2). Infection percentage was not related to the percentage of smutted heads in the field during flowering. While 0.1 to 1.4% of the heads in the field were smutted, infection levels were 0.3% or less. Although the percent of smutted heads at KAW77 was nearly three times that at EL78, subsequent levels of infection were similar. Inoculum may have been limiting to infection at EL79, although infection did develop in samples taken within 30 cm of individual smutted heads (Table 3). Average temperature at flowering did not vary widely during the study. However, the temperatures at IT78 were relatively cool, with an average of 15 C, while at GR77 the average was 22 C (Table 2). Leaf wetness, relative humidity, and inoculum levels were similar at both locations, but infection was higher at IT78 (0.3%) than at GR77 (0.1%), suggesting that cool temperatures may be favorable for infection. Rainfall seemed to have little effect on disease severity, even though total rainfall amounts varied from 0.0 to 5.9 cm. Similarly, though widely different wind speeds were present, no significant effect of wind speed on disease severity was evident. The percentage of hours during flowering with leaf wetness or high relative humidity (>80%) tended to vary together, and ranged from 20 to 56% and 33 to 51%, respectively. However, neither leaf wetness nor high relative humidity had any detectable effect on the level of infection. Infection in wheat samples taken 0-15 and 15-30 cm from individual smutted heads were higher than in samples taken at random in the field, but differences in infection between the two distances were not 22 6.9....» .9. .33... :8. .32. 05 5o... c3828 3.3.59. comm :5... 33.5.... .63.. 332...... .8 .5983 .8325 Be. .2: Eu: 2.. 5 2.8; a... .o 28.5% 4.8 .m we... 3.3.5.. «>219. m5 um... 9:33... 95.5.. 2.5... .33 o... .8 23.83 68.38 32...! h2.... an... 9.202.... 9.7:... «.59. .33 9.. mo €8.82 .539... :2! me .5333“... .8 v.3... .3295: 3.8.. a... 2... 2.... 253mm o... .6 o.. m. .m mm o. 23-..? 2.: m... .6 m. I mm a m. 23-..). E: .6 m... N... . 9.. cm a. 23-33 a...“ N... e.. a... N 8 mm a. .313? 236. ..c a... a6 2 mm om NN «.2933 2.5 E .25.. :8 M .2 a. 2.. a. G. .5 33.. e8. .68... 3.35.... 325...: 8389.53 3.2. «.838»... c.5323 52. .x. .3 um; 3a .2. a. no. .. 2 em: :26 E :o . .33 .a.o. ecu .m... ...a. ac..=e me.u.. us.u~.=. 3.9.32. 5 .93.... 32.3.. .3 aces; 832.3 .o 5.33... 28:33:... new £5.38... 2. 032.26 3.3.. .5325. 38. .o $359.8 55.33.... 9.7:... 25.2.2.8 .3..o.....o...>=o .3 Pass... .~ 3.2. 23 TABLE 3. Infection of Genessee wheat by Ustilago tritici at 0-15, 15-30, and 30-45 cm from individual smutted heads in naturally infected Genessee wheat fields during 1977 and 1979. Distance from smutted head(cm)a Location 0-15 15-30 30-45 Samples5 Infectionc Samples Infection Samples Infection (No.) (No.) (No.) GR77 20 0.28 20 0.24 3 0.00 KAW77 10 0.28 10 0.53 2 0.00 EL79 15 0.05 15 0.04 - -- a Seed samples collected in circular patterns around individual smutted heads, with 800-1200 seeds per sample. bNumber of smutted heads around which samples were collected. cPercent of smutted heads produced from the seed sample. 24 significant (Table 3). At KAw77 infection was higher in samples taken at 15-30 cm than at 0.0 to 15 cm of smutted heads. No infection was detected in samples taken 30-45 cm from smutted heads. Spore dispersal and infection near an inoculum source. Spore catches were often greater at 0.9 m than at 2.7 m from the inoculum source in any direction (Figures 5, 6), and were strongly associated with wind run and direction (Figure 5). Occasionally, the pattern of spore dispersal around the inoculum source was not directly related to the direction and speed of the wind coming from the inoculum source (Figure 6). Correlation coefficients calculated for directional spore trap catches and the amount of wind blowing across the inoculum source and towards a particular spore trap were 0.508 and 0.446 during 1978 and 0.723 and 0.665 during 1979 for 0.9 and 2.7 m, respectively. Only the correlation coefficient fOr spore catches at 0.9 m and wind run during 1979 was significantly different from zero (Ef0.05) according to the Student's t-test. Infection of field-grown plants around a point inoculum source was very low in 1978, occurring in only two of eight seed samles taken 0 to 0.9 m and in none of eight sampels taken 0.9 to 2.7 m from the inoculum source (Table 4). The two samples with infected seed were taken near the two Rotorod traps with the highest spore catches. In 1979, infection occurred in four of eight samples taken 0 to 0.9 m, but in none of the eight samples taken 0.9 to 2.7 m from the inoculum source (Table 5). Three of the four samples with infection were taken near the Rotorod traps with the highest spore catches. The four sets of potted wheat plants exposed near smutted heads for 23, 25, 26 and 36 hr during 1978 showed no infection, even though spores were present during each exposure period and the wheat heads were in a 25 TABLE 4. Rotorod spore trap catches of Ustilago tritici teliospores and subsequent loose smut infection around a point source of inoculum in a Genessee wheat field during 1978. Distance from inoculum source (m) Compass Direction 0.9 2.7 Rotoroda Infectionb Rotorod InfectionC catches (z) catches (%) N - NE 6.4 x 102 0.06 7.3 x103 0.00 NE - E 2.7 X 103 0.10 3.9 X 102 0.00 E - SE 3.1 X 102 0.00 6.4 X l02 0.00 SE - S 5.4 X l02 0.00 2.4 X l02 0.00 S - SW 3.l X l02 0.00 1.6 X l02 0.00 SW - N 4.4 X 103 0.00 2.0 X 103 0.00 H - NW 7.0 X 104 0.00 l.l X 103 0.00 NH - N l.4 X lo 0.00 4.8 X lo 0.00 a Average number of spores/rod/day. b Percent of smutted heads produced from seed collected at 0-0.9 m from the inoculum source, l200 seeds per sample. c Percent of smutted heads produced from seed collected at 0.9-2.7 m from the inoculum source, 1200 seeds per sample. 26 TABLE 5. Rotorod spore trap catches of Ustilago tritici teliospores and subsequent loose smut infection around a point source of inoculum in a Genessee wheat field during l979. Distance from inoculum source (m) Compass Direction 0.9 2.7 Rotoroda Infectionb Rotorod InfectionC catches (z) catches (z) N NE 41x104 009 47x103 000 ' . 4 . . 3 . NE - E 3.9 X l04 0.03 5.l X 103 0.00 E - SE 3.8 X lo3 0.07 5.3 X 102 0.00 SE - S 7.4 X 103 0.00 3.4 X 102 0.00 S - SW 2.2 X 103 0.00 5.1 X l0 0.00 SH - w 3.9 X 103 0.06 4.0 X lo2 0.00 w - NM 7.5 X 103 0.00 4.7 X l03 0.00 Nw - N 2.6 x lO 0.00 1.2 X 10 0.00 a Average number of spores/rod/day. Percent of smutted heads produced from seed collected at 0-0.9 m from the inoculum source, l200 seeds per sample. c Percent of smutted heads produced from seed collected at 0.9-2.7 m from the inoculum source, l200 seeds per sample. 27 :5 100 . East Lansing 1, . 5’15/78 Spores Trapped at g 801' El 0.9 M903“ 0 , 2.7 Meters i 30" trom Inoculum Source 3 I- 3 db h b '5 20 E % s a: ' § g§ 2” + - - Ell—1 S . 8 60" Wind from 5 Inoculum Source a D E a 40" c E p a 204!- a: E P e .— r I T I F N NE E SE 8 SW w ww Compass Direction Figure 5. Rotorod catches of teliospores of Ustilago tritici at eight compass directions at 0.9 and 2.7 m from an inoculum source and corresponding running distance of wind for 16 June 1978. 28 .0 100.. East Lansing r 3: _ 6/15/78 1“ 8 2 80" r- 3 Spores Trapped at E 50* .09 Meters 3 . .2] Meters E 40., trom Inoculum Source w LEE .2 555% a 20“ is? g . § e922 h- 60-» Wind from Inoculum Source Total Running Distance (K) . 1 T #p—J NE 32 3's 5 sw w ww Compass Direction Figure 6. Rotorod catches of teliospores of Ustilago tritici at eight compass directions at 0.9 and 2.7 m from an inoculum source and corresponding running distance of wind for 15 June 1978. 29 susceptible stage (Table 6). Although average temperatures were 15 to 23 C, relatively few hours of leaf wetness (2-8 hr) and little rain (0.00-0.21 cm) occurred during the four exposure periods. In 1979, infection occurred during two of five exposure periods (Table 7). Maximum infection (0.7%) occurred during exposure period 3 (29 hr) when: the average air temperature was 16 0; leaf wetness was 20 hr; average wind speed was 10.2 km/hr; rainfall was 0.77 cm; plants were flowering; and spore catch was 2.8 X 103. During exposure period 1 (27 hr), infection was 0.3% and the average temperature (24 C) was higher but leaf wetness (16 hr), rainfall (0.13 cm), wind speed (3.3 km/hr), and spore catch (1.8 X 103) were lower than for exposure period 3. Environmental conditions during exposure period 2 were intermediate to those of exposure periods 1 and 3, but most of the potted plants were blown down and may not have been suitable for assessing infection. 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