- Email: [email protected]
Persistence of Resting Spores ofEntomophaga maimaiga,a Fungal Pathogen of the Gypsy Moth,Lymantria dispar
JOURNAL OF INVERTEBRATE PATHOLOGY ARTICLE NO.
69, 195–196 (1997)
IN964645
NOTE Persistence of Resting Spores of Entomophaga maimaiga, a Fungal Pathogen of the Gypsy Moth, Lymantria dispar Entomophaga maimaiga is an important fungal pathogen of the gypsy moth, Lymantria dispar. It was first discovered in North America in 1989 (Andereadis and Weseloh, 1990; Hajek et al., 1990) and has rapidly spread throughout gypsy moth populations in the northeastern and mid-Atlantic United States (Elkinton et al., 1991; Reardon and Hajek, 1993). E. maimaiga has a life cycle that is typical of most Entomophthorales and produces thick-walled resting spores (azygospores) which are the normal overwintering stage (Andreadis and Weseloh, 1990). Resting spores, found in leaf litter and soil, germinate throughout the larval period of the gypsy moth and are responsible for initiating epizootics (Hajek and Roberts, 1991; Weseloh and Andreadis, 1992). Very little is known about the long-term persistence of the resting spore stage of E. maimaiga. Shimazu et al. (1986) reported that resting spores undergo an obligate dormant period and remain viable in the soil for 1 year only. However, Hajek et al. (1993) have suggested that resting spores may survive longer. Because the gypsy moth is episodic and typically exists at very low population densities, the survival and effectiveness of E. maimiaga would clearly be enhanced if its resting spores persisted between pest outbreaks. Accordingly, the present study was conducted to determine the long-term persistence of resting spores of E. maimaiga in the forest. Gypsy moth larval cadavers containing E. maimaiga resting spores were collected at Sleeping Giant State Park in Hamden, Connecticut, on 16 July 1990. The procedures for storage were as follows. Approximately 500 cc of infected cadavers was placed atop a 2-cm layer of sand, which overlay a piece of burlap on a 30 3 40 cm piece of aluminum window screen on the forest floor. A 14 3 18 3 28 cm wooden box (‘‘infection box’’) with an open bottom and a top covered with a fine nylon mesh was placed over the cadavers to isolate them from other potential sources of contamination. Four of these boxes were prepared and held under natural conditions until the next spring (April/May 1991) and were then treated in two different ways. (1) A large plastic container (32 3 25 3 10 cm) was inverted on top of two of these boxes on 3 April 1991 to eliminate rainfall. Inverted enamel trays were additionally placed under the screens
to isolate the boxes from soil moisture and extraneous fungal contamination in the soil (dry treatment). (2) Noninverted enamel trays were placed under the screens of the other two boxes and were filled with water twice weekly, so that the water was in direct contact with the sand under the cadavers. No plastic containers were placed over these boxes so that they would receive normal precipitation (wet treatment). These treatments were left in place for all other years of the study, except that extra water was not provided for the ‘‘wet’’ treatment in any year other than 1991. In particular, the enamel trays were left under the boxes in order to isolate the boxes from extraneous fungi in the soil, and the plastic containers were left on the ‘‘dry’’ treatment boxes. Bioassays were begun on 17 April 1991. Twenty-five third-instar gypsy moths were placed in 19 3 7 cm diameter cylindrical window-screen cages that also contained a 36-ml plastic cup 1⁄4 full of artificial diet (Yamamoto, 1969, or Southland Products, Inc., AR). One cage was placed in each of the four boxes. After 1 week, larvae were exchanged for fresh ones. Exposed larvae were reared individually in the laboratory at 25°C, approximately 50% RH, and 18 hr light/ day in 36-ml plastic cups about 1⁄4 full of artificial diet and checked every 2–3 days for 2 weeks for infection by E. maimaiga. All larvae were microscopically examined for the presence of E. maimaiga hyphal bodies and conidial spores. Three consecutive weekly assays were done. After bioassays were completed, the boxes were left in the forest for the duration of the study. Additional gypsy moth cadavers (1991 spores) were collected at the same location on 15 July 1991. These were stored in two boxes located directly on the forest floor (i.e., no sand, burlap, or screen) on 23 July 1991. Gypsy moth larvae were exposed in each infection box as described previously for each of the next 5 years starting on 30 April 1992, 3 May 1993, 25 April 1994, 28 April 1995, and 23 April 1996. In these years, no extra water was provided for the 1990 spores that had received extra water in 1991. Starting in 1992, two control boxes placed over caged larvae on enameled trays were established to monitor extraneous sources of contamination. No attempts were made to monitor soil moisture during this study.
195
0022-2011/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
196
NOTE
FIG. 1. Yearly infection of gypsy moth larvae exposed in cages to E. maimaiga resting spores collected in 1990 (top graph) and 1991 (bottom graph). In the controls (not shown because no infection occurred) cages were placed directly on clean enamel trays. The bars are averages for each week’s exposure in each year, and the vertical brackets are standard errors.
Results for the wet and dry treatments for each year were similar, so these data have been pooled. Spores from cadavers remained infective for the full 6 years of the study. High levels of infection (up to 80%) occurred most years (Fig. 1). No infections occurred in the controls, showing that younger, extraneous resting spores were not involved in causing infections in larvae in the treatment groups. For each year, the weekly infection patterns in the bioassays conducted with the 1991 spores were similar to those conducted with the 1990 spores. Results show that a high degree of genetic variability occurs in the induction of resting spore germination, so that some spores germinate each year over a period of at least 6 years. However, the factor(s) responsible is unknown. This variability is clearly advantageous for long-term survival of the fungus because of the episodic nature of gypsy moth populations (typically, 8–10 years occur between pest outbreaks). If resting spores remain viable significantly longer than 6 years, the fungus could initiate an epizootic very early in an outbreak and prevent significant defoliation. Thus, we expect E. maimaiga to persist as an effective natural enemy of the gypsy moth in North America. KEY WORDS: Entomophaga maimaiga: Entomophthorales; resting spores; persistence; Lymantria dispar; gypsy moth.
The authors thank Morgan Lowry and Colleen Moser for their help in carrying out this study.
REFERENCES Andreadis, T. G., and Weseloh, R. M. 1990. Proc. Natl. Acad. Sci. USA 87, 2461–2465. Elkinton, J. S., Hajek, A. E., Boettner, G. H., and Simons, E. E. 1991. Environ. Entomol. 20, 1601–1605. Hajek, A. E., and Roberts, D. W. 1991. Biol. Contol. 1, 29–34. Hajek, A. E., Humber, R. A., Elkintion, J. S., May, B., Walsh, S. R. A., and Silver, J. C. 1990. Proc. Natl. Acad. Sci. USA 87, 6979–6982. Hajek, A. E., Larkin, T. S., Carruthers, R. I., and Soper, R. S. 1993. Environ. Entomol. 22, 1172–1187. Reardon, R., and Hajek, A. E. 1993. AIPM Technology Transfer Bulletin, U.S. Dept. Agric. Forest Service. Shimazu, M., Koizumi, C., Kushida, T., and Mitsuhashi, J. 1986. Appl. Entomol. Zool. 22, 216–221. Weseloh, R. M., and Andreadis, T. G. 1992. Environ. Entomol. 21, 901–906. Yamamoto, R. T. 1969. J. Econ. Entomol. 62, 1427–1431.
R. M. WESELOH T. G. ANDREADIS Connecticut Agricultural Experiment Station P.O. Box 1106 New Haven, Connecticut 06504 Received February 21, 1996; accepted December 6, 1996
69, 195–196 (1997)
IN964645
NOTE Persistence of Resting Spores of Entomophaga maimaiga, a Fungal Pathogen of the Gypsy Moth, Lymantria dispar Entomophaga maimaiga is an important fungal pathogen of the gypsy moth, Lymantria dispar. It was first discovered in North America in 1989 (Andereadis and Weseloh, 1990; Hajek et al., 1990) and has rapidly spread throughout gypsy moth populations in the northeastern and mid-Atlantic United States (Elkinton et al., 1991; Reardon and Hajek, 1993). E. maimaiga has a life cycle that is typical of most Entomophthorales and produces thick-walled resting spores (azygospores) which are the normal overwintering stage (Andreadis and Weseloh, 1990). Resting spores, found in leaf litter and soil, germinate throughout the larval period of the gypsy moth and are responsible for initiating epizootics (Hajek and Roberts, 1991; Weseloh and Andreadis, 1992). Very little is known about the long-term persistence of the resting spore stage of E. maimaiga. Shimazu et al. (1986) reported that resting spores undergo an obligate dormant period and remain viable in the soil for 1 year only. However, Hajek et al. (1993) have suggested that resting spores may survive longer. Because the gypsy moth is episodic and typically exists at very low population densities, the survival and effectiveness of E. maimiaga would clearly be enhanced if its resting spores persisted between pest outbreaks. Accordingly, the present study was conducted to determine the long-term persistence of resting spores of E. maimaiga in the forest. Gypsy moth larval cadavers containing E. maimaiga resting spores were collected at Sleeping Giant State Park in Hamden, Connecticut, on 16 July 1990. The procedures for storage were as follows. Approximately 500 cc of infected cadavers was placed atop a 2-cm layer of sand, which overlay a piece of burlap on a 30 3 40 cm piece of aluminum window screen on the forest floor. A 14 3 18 3 28 cm wooden box (‘‘infection box’’) with an open bottom and a top covered with a fine nylon mesh was placed over the cadavers to isolate them from other potential sources of contamination. Four of these boxes were prepared and held under natural conditions until the next spring (April/May 1991) and were then treated in two different ways. (1) A large plastic container (32 3 25 3 10 cm) was inverted on top of two of these boxes on 3 April 1991 to eliminate rainfall. Inverted enamel trays were additionally placed under the screens
to isolate the boxes from soil moisture and extraneous fungal contamination in the soil (dry treatment). (2) Noninverted enamel trays were placed under the screens of the other two boxes and were filled with water twice weekly, so that the water was in direct contact with the sand under the cadavers. No plastic containers were placed over these boxes so that they would receive normal precipitation (wet treatment). These treatments were left in place for all other years of the study, except that extra water was not provided for the ‘‘wet’’ treatment in any year other than 1991. In particular, the enamel trays were left under the boxes in order to isolate the boxes from extraneous fungi in the soil, and the plastic containers were left on the ‘‘dry’’ treatment boxes. Bioassays were begun on 17 April 1991. Twenty-five third-instar gypsy moths were placed in 19 3 7 cm diameter cylindrical window-screen cages that also contained a 36-ml plastic cup 1⁄4 full of artificial diet (Yamamoto, 1969, or Southland Products, Inc., AR). One cage was placed in each of the four boxes. After 1 week, larvae were exchanged for fresh ones. Exposed larvae were reared individually in the laboratory at 25°C, approximately 50% RH, and 18 hr light/ day in 36-ml plastic cups about 1⁄4 full of artificial diet and checked every 2–3 days for 2 weeks for infection by E. maimaiga. All larvae were microscopically examined for the presence of E. maimaiga hyphal bodies and conidial spores. Three consecutive weekly assays were done. After bioassays were completed, the boxes were left in the forest for the duration of the study. Additional gypsy moth cadavers (1991 spores) were collected at the same location on 15 July 1991. These were stored in two boxes located directly on the forest floor (i.e., no sand, burlap, or screen) on 23 July 1991. Gypsy moth larvae were exposed in each infection box as described previously for each of the next 5 years starting on 30 April 1992, 3 May 1993, 25 April 1994, 28 April 1995, and 23 April 1996. In these years, no extra water was provided for the 1990 spores that had received extra water in 1991. Starting in 1992, two control boxes placed over caged larvae on enameled trays were established to monitor extraneous sources of contamination. No attempts were made to monitor soil moisture during this study.
195
0022-2011/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
196
NOTE
FIG. 1. Yearly infection of gypsy moth larvae exposed in cages to E. maimaiga resting spores collected in 1990 (top graph) and 1991 (bottom graph). In the controls (not shown because no infection occurred) cages were placed directly on clean enamel trays. The bars are averages for each week’s exposure in each year, and the vertical brackets are standard errors.
Results for the wet and dry treatments for each year were similar, so these data have been pooled. Spores from cadavers remained infective for the full 6 years of the study. High levels of infection (up to 80%) occurred most years (Fig. 1). No infections occurred in the controls, showing that younger, extraneous resting spores were not involved in causing infections in larvae in the treatment groups. For each year, the weekly infection patterns in the bioassays conducted with the 1991 spores were similar to those conducted with the 1990 spores. Results show that a high degree of genetic variability occurs in the induction of resting spore germination, so that some spores germinate each year over a period of at least 6 years. However, the factor(s) responsible is unknown. This variability is clearly advantageous for long-term survival of the fungus because of the episodic nature of gypsy moth populations (typically, 8–10 years occur between pest outbreaks). If resting spores remain viable significantly longer than 6 years, the fungus could initiate an epizootic very early in an outbreak and prevent significant defoliation. Thus, we expect E. maimaiga to persist as an effective natural enemy of the gypsy moth in North America. KEY WORDS: Entomophaga maimaiga: Entomophthorales; resting spores; persistence; Lymantria dispar; gypsy moth.
The authors thank Morgan Lowry and Colleen Moser for their help in carrying out this study.
REFERENCES Andreadis, T. G., and Weseloh, R. M. 1990. Proc. Natl. Acad. Sci. USA 87, 2461–2465. Elkinton, J. S., Hajek, A. E., Boettner, G. H., and Simons, E. E. 1991. Environ. Entomol. 20, 1601–1605. Hajek, A. E., and Roberts, D. W. 1991. Biol. Contol. 1, 29–34. Hajek, A. E., Humber, R. A., Elkintion, J. S., May, B., Walsh, S. R. A., and Silver, J. C. 1990. Proc. Natl. Acad. Sci. USA 87, 6979–6982. Hajek, A. E., Larkin, T. S., Carruthers, R. I., and Soper, R. S. 1993. Environ. Entomol. 22, 1172–1187. Reardon, R., and Hajek, A. E. 1993. AIPM Technology Transfer Bulletin, U.S. Dept. Agric. Forest Service. Shimazu, M., Koizumi, C., Kushida, T., and Mitsuhashi, J. 1986. Appl. Entomol. Zool. 22, 216–221. Weseloh, R. M., and Andreadis, T. G. 1992. Environ. Entomol. 21, 901–906. Yamamoto, R. T. 1969. J. Econ. Entomol. 62, 1427–1431.
R. M. WESELOH T. G. ANDREADIS Connecticut Agricultural Experiment Station P.O. Box 1106 New Haven, Connecticut 06504 Received February 21, 1996; accepted December 6, 1996