Perennation of Sclerotium rolfsii var. delphinii in Iowa

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1 2003 Plant Management Network. Accepted for publication 26 October Published 1 December Perennation of Sclerotium rolfsii var. delphinii in Iowa Brooke A. Edmunds, Graduate student, and Mark L. Gleason, Professor, Department of Plant Pathology, Iowa State University, Ames Corresponding author: Mark L. Gleason. mgleason@iastate.edu Edmunds, B. A., and Gleason, M. L Perennation of Sclerotium rolfsii var. delphinii in Iowa. Online. Plant Health Progress doi: /php rs. Abstract Stalk and petiole rot of herbaceous ornamental perennials, caused by S. rolfsii var. delphinii, is a widespread disease in landscape plantings and nursery production fields in the Midwest United States. A better understanding of perennation of the pathogen could aid in development of more effective management recommendations. Sclerotia of S. rolfsii var. delphinii were produced on strips of inoculated cotton batting placed on moistened sand in a plastic crisper. After one week at 27 C, sclerotia were removed, sealed in nylon mesh bags, and the bags were buried immediately below the soil surface and at 15-cm and 30-cm depths at two locations in Iowa in September Germination of sclerotia was quantified in October and November 2001 and in April, May, June, and July 2002 by plating recovered, surface-sterilized sclerotia on carrot agar amended with antibiotics. Survival was highly variable but in June and July was greatest at the soil surface (~50%). This is the first documented evidence that S. rolfsii var. delphinii will overwinter in the Midwest and survive a sufficient length of time to cause disease in the next growing season. Introduction The soilborne fungus Sclerotium rolfsii Sacc. var. delphinii (Welch) infects at least 19 herbaceous ornamental species in the Midwest (4,7,8). However, S. rolfsii var. delphinii is closely related to S. rolfsii, a pathogen causing similar symptoms in regions with warm, temperate winters (1), including the southern United States. S. rolfsii has an exceptionally broad host range, attacking over 200 genera of vegetable, grain, and ornamental crops (7). The economic importance of S. rolfsii has spurred extensive research on its life cycle, etiology, and management. S. rolfsii var. delphinii has received far less study, although its impact appears to be increasing, especially in the Midwest. Because the two pathogens share many morphological traits and are closely related genetically (8,12), results from epidemiological studies with S. rolfsii have been extrapolated to S. rolfsii var. delphinii. Comparative studies, however, have shown that the two pathogens differ in optimal temperature, host range, colony morphology, and size of sclerotia (14). S. rolfsii var. delphinii is active in warm, humid conditions, and the diseases that it causes generally appear in midsummer in the Midwest. Diagnostic symptoms on hosta (Hosta spp.) include yellow, wilted leaves (Fig. 1) that soften and turn brown near the soil line (Fig. 2). Severe infections may result in the death of entire crowns or plants (6). Peony and other ornamentals with less succulent tissue than hosta do not exhibit stalk collapse, but may exhibit girdling lesions at the soil line and wilting of foliage (6).

2 Fig. 1. Wilted leaves and collapsed petioles on hosta infected by S. rolfsii var. delphinii. Fig. 2. Browning and softening of petiole bases of hosta caused by petiole rot, and sclerotia of S. rolfsii var. delphinii. White mycelium and sclerotia are usually present on the surface of infected plant tissue and surrounding soil. Sclerotia, the diagnostic sign of this fungus, are small (1.1 to 2 mm in diameter), reddish brown spheres that are typically present in large numbers (Fig. 2). Sclerotia are the primary means of longdistance dissemination of S. rolfsii var. delphinii, since the fungus produces no spores. Sclerotia also serve as the survival structures of the fungus, enabling it to persist in an inactive state during winter or other adverse periods. Previous studies suggested that the viability of buried sclerotia of S. rolfsii var. delphinii declined over time, and that only a small percentage of sclerotia survived for 6 months to 2 years (10,18). These studies, however, utilized sclerotia that had either been produced on agar media or were dried before being exposed to weathering. Sclerotia of S. rolfsii produced on agar are morphologically and physiologically distinct from sclerotia from those that develop on host plant tissue (11,15). Drying sclerotia of S. rolfsii prior to burial can reduce survival rates by inducing the sclerotia to produce a nutrient exudate that stimulates colonization by opportunistic soil-borne microorganisms (3,16). It is reasonable to assume that culturing on agar and drying similarly affect sclerotia of S. rolfsii var. delphinii. To clearly understand the ecology of this disease, therefore, sclerotia used in field studies should be produced under conditions that resemble the natural environment as closely as possible. An understanding of S. rolfsii var. delphinii s propensity to survive winters in the Midwest would help gauge the risk of this pathogen becoming a long-term threat and could provide insights for improving disease management. The objectives of this study were to (i) confirm observations suggesting that S. rolfsii var. delphinii is able to overwinter in the Midwest and (ii) clarify the impact of burial on the survival of sclerotia. A preliminary report has been published (5). Experiments on the Perrenation of S. rolfsii var. delphinii During the winter and spring of , the experiment was conducted at the Iowa State University Horticulture Research Station near Gilbert, IA (Clarion-Webster-Nicollet loam soil) and the Muscatine Island Research Farm located near Fruitland, IA (Fruitfield-Elrick-Toolesboro sand-loam soil) (Fig. 3). Both fields had been in a grass fallow during the previous year and were tilled to a depth of 30 cm approximately one month before the start of the experiment.

3 Fig. 3. Map of locations in Iowa where the study of survival of S. rolfsii var. delphinii sclerotia was conducted. Muscatine is in southeast Iowa and Gilbert is in central Iowa. Air temperature was collected by automated weather stations located in Ames (approximately 5 miles south of Gilbert) and Muscatine. Monthly averages were compiled by and downloaded from the ISU Environmental Mesonet website (9). Thirty-year averages of air temperatures for each site were computed from historical data also downloaded from this online database. An isolate (B4) of S. rolfsii var. delphinii obtained from an infected hosta plant in Ames, IA was used throughout the study. A method was devised to produce sclerotia (in vitro) similar in size and shape to those resulting from natural infections in the field. Plastic crispers (30 22 cm) were filled with nonsterile sand to a depth of 2.5 cm. Three cm strips of sterile cotton batting were then placed on the sand surface. Thirty mycelial plugs, taken from the edge of an actively growing culture of S. rolfsii var. delphinii on potato dextrose agar, were distributed approximately 2.5 cm apart on the strips. Sterile distilled water was added to moisten, but not saturate, the cotton and sand. The crispers were then placed in a dew chamber at 27 C and 100% relative humidity with a 12-hour photoperiod. At 7 days, numerous sclerotia of S. rolfsii var. delphinii had formed on the cotton strips (Fig. 4). The crispers were then placed in a growth chamber at 27 C and 40 to 50% relative humidity with a 12 hour light/dark regime to allow the sclerotia to mature. After 2 days, the sclerotia were brushed from the cotton strips into a glass petri dish. Fig. 4. Numerous sclerotia of S. rolfsii var. delphinii were produced on cotton balls after incubation on moist sand. These sclerotia were used in field studies. Nylon screen bags (0.5-mm mesh) were used to retain sclerotia during the study. The bags were constructed by stitching around the edge of two cm layers of nylon screen fabric with polyester thread. Twenty-five sclerotia were added to each bag through an open end, which was then sewn shut. A 1-m length of nylon string was tied through a plastic grommet placed in the corner of each bag. To prevent drying of sclerotia, screen bags containing sclerotia were stored in plastic bags in a crisper for 12 hours at 27 C in the dark prior to burial. The screen bags were placed at Gilbert site and Muscatine site on September 13 and 18, 2001, respectively (Fig. 5). Bags were arranged according to a randomized complete block design with six replications. Each replication

4 consisted of a plot divided into six subplots; each subplot corresponded to a month during which sampling was to be when that bag was collected. In each subplot, a 20-cm-wide, 30-cm-deep hole was dug using a posthole digger. In each hole, one bag was buried at 30 cm, another was buried at 15 cm, and a third bag was secured to the soil surface using two metal landscape staples. Bags were attached to a post with the nylon string. Each bag was considered an experimental unit. Fig. 5. Placement of nylon bags, each containing 25 sclerotia, at Muscatine field site. At each location, three bags (30-cm depth, 15-cm depth, and surface) were sampled from each of the six replications in mid-october and mid-november 2001, and monthly from April to July Nylon bags were placed in labeled plastic bags in a cooler for transport, and then transferred to a refrigerator for 4 to 6 hours before plating. Unopened bags were rinsed under tap water to remove adhering soil. In a laminar-flow hood, the washed bags and contents were surface sterilized in a 10% NaOCl solution for 60 seconds. The bags were rinsed twice in sterile distilled water (SDW), blotted dry between paper towels, cut open, and sclerotia were removed individually with sterile forceps. Germination was tested by plating the sclerotia on Bacto-agar (Difco Laboratories; Sparks, MD) amended with carrot puree and the antibiotics tetracycline and streptomycin. Carrot puree was prepared in September 2001 by pureeing 13.5 kg of twice-autoclaved, mature carrots in a blender, dividing the puree into 200-ml aliquots in plastic beakers, and freezing them. Media was prepared 24 hour before plating. One aliquot of carrot puree was thawed in a microwave oven and added to 25 g of bacto-agar in 800 ml of SDW. The mixture was autoclaved, and 0.15 g each of the antibiotics tetracycline hydrochloride and streptomycin sulfate were added after the mixture cooled to touch. Five sclerotia were distributed evenly on the surface of each carrot agar plate. The plates were placed in plastic crispers and incubated under continuous light at 25 C for 48 hours (Fig. 6). Sclerotia were considered to have germinated if the white, threadlike mycelium characteristic of S. rolfsii var. delphinii was present when the plates were observed under a dissecting microscope after 48 hours. The number of sclerotia recovered and the number germinated were recorded for each replication at each depth. Data were subjected to analysis of variance using the GLM procedure of SAS (SAS Institute, Cary, NC). Means were compared by the least significant difference (LSD) at P = S. rolfsii var. delphinii Overwinters in Iowa Viable sclerotia persisted into July (Figs. 7a and 7b), when ambient temperatures in the Midwest rise to levels that are optimal for germination of sclerotia and pathogenesis (6). This is the first documented evidence that S. rolfsii var. delphinii can survive the winter in the Midwest and persist long enough into the summer to cause disease.

5 Fig. 6. Sclerotia and mycelium of S. rolfsii var. delphinii on carrot agar plate. Fig. 7. (a) Survival of sclerotia of S. rolfsii var. delphinii at Muscatine, IA. The bar indicates the least significant difference (LSD) between points. * = month in which the incidence of surival of sclerotia differed significantly (P < 0.05) among the three burial depths. (b) Survival of sclerotia of S. rolfsii var. delphinii at Gilbert, IA. The bar indicates the least significant difference (LSD) between points. * = month in which the incidence of survival of sclerotia differed significantly (P < 0.05) among the three burial depths. (c) Average monthly temperatures at Gilbert and Muscatine, IA for and the 30-year average for each location.

6 A significant decline in survival over time at both locations was noted. Survival was not significantly affected by depth of burial until June and July at the Gilbert site and July at the Muscatine site. At the Muscatine site, sclerotia did not survive at either the 15-cm or 30-cm depths on the final sampling date, whereas the survival rate at the soil surface was 60%. Sclerotia buried the Gilbert site showed a somewhat different trend for the July assessment; survival at 15 cm and the surface was 53% and 58%, respectively, whereas survival at 30 cm declined to 9.3%. Our finding that burial increased mortality of S. rolfsii var. delphinii sclerotia is consistent with results of survival studies with S. rolfsii sclerotia (17). Survival was highly variable among replications, however, suggesting that deep burial may not be considered a reliable management technique. Many environmental and biological factors that affect sclerotia survival, including soil moisture levels, aeration, organic matter content, and parasitic microorganisms (2), exhibit a high degree of spatial variability, which may have contributed to the spatial variability in survival rates among replications and locations in this study. Some sclerotia recovered intact from the soil were black, shriveled, and showed signs of bacterial contamination after culturing (Edmunds, unpublished data), and failed to germinate. Occasionally, empty sclerotial rinds or rind fragments were also found in the recovered bags. Although air temperatures from November through January were slightly higher than the 30-yr average (Fig. 7c), rising soil temperatures as mid-summer approached may have been the limiting factor in sclerotial survival. Higher temperatures would increase soil microbial activity (13), which may have accelerated sclerotial mortality. Management Implications Results of this study advance our understanding of the ecology of S. rolfsii var. delphinii. This basic knowledge will be helpful in devising effective management techniques for commercial producers and homeowners. At this point, recommendations cannot be made for deep burial as a management technique, since our results show that effectiveness in reducing sclerotial survival may depend on soil type and geographic location. Further research is needed to clarify the effects of depth of burial, soil moisture, and microbial activity on survival of S. rolfsii var. delphinii and resulting potential for future epidemics in the northern U.S. It would also be interesting to complete a longerterm study to determine the impacts of fallowing on survival of sclerotia in the soil. Our results support the view that S. rolfsii var. delphinii can survive from year to year and poses a perennial threat to susceptible species in northern landscapes. This finding emphasizes the importance of carefully inspecting susceptible planting stock, diligent scouting for disease symptoms, and quarantining symptomatic plants in order to avoid establishment of S. rolfsii var. delphinii (6). Literature Cited 1. Aycock, R Stem rot and other diseases caused by Sclerotium rolfsii. N. Dak. Agric. Exp. Sta. Bull Coley-Smith, J. R., and Cooke, R. C Survival and germination of fungal sclerotia. Annu. Rev. Phytopathol. 9: Coley-Smith, J. R., Ghaffar, A., and Javed, Z. U. R The effect of dry conditions on subsequent leakage and rotting of fungal sclerotia. Soil Biol. Biochem. 6: Edmunds, B. A., and Gleason, M. L First report of petiole rot of Pulmonaria longifolia caused by Sclerotium rolfsii var. delphinii. Plant Dis. 87: Edmunds, B. A., and Gleason, M. L Overwinter survival of S. rolfsii var. delphinii in Iowa. Phytopathology 93:S Edmunds, B., Gleason, M., and Schuch, U Crown rot: A serious disease of hosta and other ornamentals. Online. Iowa State Univ. Ext. Pub. SUL May Farr, D. F., Bills, G. F., Chamuris, G. P., and Rossman, A. Y Fungi on plant and plant products in the United States. American Phytopathological Society, St. Paul. 8. Harlton, C. E., Levesque, C. A., and Punja, Z. K Genetic diversity in Sclerotium (Athelia) rolfsii and related species. Phytopathology 85: Iowa State University Iowa Environmental Mesonet. Online. ISU Department of Agronomy.

7 10. Javed, Z. U. R., and Coley-Smith, J. R Studies on the germination of sclerotia of Sclerotium delphinii. Trans. Brit. Mycol. Soc. 60: Linderman, R. G., and Gilbert, R. G Behavior of sclerotia of Sclerotium rolfsii produced in soil or in culture regarding germination stimulation by volatiles, fungistatis, and sodium hypochlorite treatment. Phytopathology 63: Okabe, I., Morikawa, C., Matsumoto, N., and Yokoyama, K Variation in Sclerotium rolfsii isolates in Japan. Mycoscience 39: Paul, E. A., and Clark, F. E Soil microbiology and biochemistry. Academic Press, Inc., San Diego. 14. Punja, Z. K., and Damiani, A Comparative growth, morphology, and physiology of three Sclerotium species. Mycologia 88: Punja, Z. K., Jenkins, S. F., and Grogan, R. G Effect of volatile compounds, nutrients, and source of sclerotia on eruptive germination of Sclerotium rolfsii. Phytopathology 74: Smith, A. M Biological control of fungal sclerotia in soil. Soil Biol. Biochem. 4: Smith, V. L., Jenkins, S. F., Punja, Z. K., and Benson, D. M Survival of sclerotia of Sclerotium rolfsii: Influence of sclerotial treatment and depth of burial. Soil Biol. Biochem. 21: Williams, G. H., and Western, J. H The biology of Sclerotinia trifolium Erikss. and other species of sclerotia-forming fungi. II. The survival of sclerotia in the soil. Annu. Rev. Appl. Biol. 56: