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Isolation of Fungi Associated with Hemlock Woolly Adelgid (Homoptera: Adelgidae)
JOURNAL OF INVERTEBRATE PATHOLOGY ARTICLE NO.
70, 76–77 (1997)
IN974668
NOTE Isolation of Fungi Associated with Hemlock Woolly Adelgid (Homoptera: Adelgidae) The hemlock woolly adelgid, Adelges tsugae Annand (HWA), originated in Asia and was introduced into the United States in 1924 (McClure, 1987). It is causing major damage to eastern hemlock, Tsuga canadensis (L.) Carr., in New England. A search for parasites and predators of this forest pest in the Northeast was conducted recently (Montgomery and Lyon, 1995) but a survey of associated entomopathogenic fungi has not been done. Five dominant eastern hemlock trees with visible heavy infestations of HWA located in Holyoke, Massachusetts, were selected for sampling. Ten 2-m-long branch samples were taken from the middle and upper canopy of each tree. From these branches we cut 10-cm sections of apical portions containing an abundance of HWA. Two hundred apical samples were taken on each of the following dates: January 29, 1996; February 14, 1996; and March 14, 1996. Prior to examination the apical samples were stored individually in glass test tubes at 1°C. Six thousand HWA sistens (10 per apical section) were examined, within 2 weeks of sampling, under 403 magnification, 2882 of which showed symptoms of fungal infection, i.e., off-color, mummified, or with external fungal growth. Two methods were used to recover fungi from these specimens. Obligatory parasitic fungi were prepared as wet mounts on slides using glycerol and water and identified at 4003 using phasecontrast microscopy. Other specimens were surface sterilized for 1–2 min with 2.5% NaOCl (using commercial bleach) placed on sterile hygroscopic paper under humid conditions or plated directly on potato dextrose agar, potato dextrose egg agar, or Czapek’s medium (Anonymous, 1982; Smith and Onions, 1983). Of the 6000 HWA examined, fungi were found associated with approximately 37%. Seventeen different fungal genera were recovered (Table 1). Three genera (Verticillium, Beauveria, and Paecilomyces) contain species known to be major entomopathogens. Some species of Acremonium are entomopathogens. The genera Trichoderma and Gliocladium contain species that are recognized antagonists of plant pathogens. The remain-
0022-2011/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
ing genera contain species that are primarily saprophytic. Identifications were verified by R. Humber (USDA, ARS, Ithaca, NY) and will be identified to species at a later date. All isolates have been placed in long-term storage at the Entomology Research Laboratory, University of Vermont, and the USDA, Agricultural Research Service Collection of Entomopathogenic Fungi at Ithaca, New York. Given the approximately 50% mortality we observed in the field population in a cold-hardiness study done by the authors in 1996 (unpublished data), it is possible that fungi contribute to the natural regulation of HWA populations. Fungal bioassays were conducted using field-collected HWA (dosage: 1 3 108 spores/ml) and lab-reared green peach aphid (GPA), Myzus persicae (Sulzer) (dosage: 5 3 106 spores/ml), to determine the relative pathogenicity of several isolates that we recovered (Table 2). Mortality rates of 42–90% among the treated HWA, and 25–90% among the GPA, compared to 10– 11% among the controls were obtained (Table 2). We also conducted bioassays with representatives from all other genera found in association with HWA that are not known to be entomogenous (those listed in Table 1 but not in Table 2). GPA was used as the test organism (dosage: 5 3 106 spores/ml) because the trials were conducted several months after the last sampling period, and HWA were not available. GPA mortality after 6 days ranged from 13 to 47%, compared to 11% among the controls. This suggests that, though less pathogenic than the known entomopathogens, these fungi exhibit some activity against an insect host and should not be discounted when evaluating potential biological control agents. Our results indicate that several entomopathogenic fungi exist naturally in wild HWA populations in New England, some with a demonstrated potential for management of this introduced pest. Further detailed studies are needed to determine their pathogenicity at the range of temperatures and humidities that naturally occurs in the forest. In addition, evaluation of the growth and infection characteristics, such as rate of germination and spore production, would facilitate
76
77
NOTE
TABLE 1 Fungi Isolated from Hemlock Woolly Adelgid (HWA) Collected in Holyoke, Massachusetts, January–March 1996
Fungal species Alternaria sp. Cladosporium sp. Trichoderma lignorum (Tode) Harz Trichoderma sp. Rhinocladiella sp. Fusarium sp. Epicoccum sp. Gliocladium sp. Chlamydomyces sp. Paecilomyces sp. Beauveria bassiana (Balsamo) Vuillemin Pestalotia sp. Verticillium lecanii (Zimmerman) Viegas Embellisia (allii) (Companile) E. Simmons Acremonium sp. Scopulariopsis sp. Cylindrocarpon sp. Geotrichum (candidum) Link emend J. Carmieh
% of Number total of HWAa collecteda 978 534 289 157 88 49 36 27 15 11 6 5 5 3 1 1 1 1
33.93 18.53 10.30 5.45 3.05 1.70 1.25 0.94 0.52 0.38 0.21 0.17 0.17 0.10 0.04 0.04 0.04 0.04
a No fungi or other pathogenic microorganisms were isolated from 675 (23.42%) of the 2882 symptomatic HWA evaluated.
their further development into viable biological control agents. KEY WORDS: Entomopathogenic fungi; Adelges tsugae; fungal management of Adelgidae; biological control. Many thanks are extended to Richard A. Humber, USDA, ARS (Ithaca, NY) for verifying identifications of our entomopathogenic fungi and reviewing earlier versions of the manuscript. We also appreciate the technical assistance of William Reid. This work would not have been possible without the cooperative efforts of numerous personnel in the Vermont Department of Forests, Parks and Recreation and the Massachusetts Department of Environmental Conservation. Funding for this work was received in part from the USDA, Forest Service, NE Area State and Private Forestry, Coop. Agreement NA-96-158. REFERENCES Anonymous. 1982. ‘‘The Oxoid Manual,’’ 5th ed., Oxoid Ltd., Basingstoke, England.
TABLE 2 Bioassay of Select Isolates against Hemlock Woolly Adelgid (HWA) and Green Peach Aphid (GPA)a Identification No.b 5165 5167 5164 5170 5171 5178 5180 5177 Not assigned None
% Mortality Fungal species
HWA
GPA
V. lecanii V. lecanii Scopulariopsis sp. B. bassiana B. bassiana Paecilomyces farinosus (Holm ex SF Gray) Brown & Smith P. farinosus Gliocladium sp. Fusarium sp. None (Control)
82 72 90 58 NDc
80 90 48 60 52
64 ND ND 42 10
52 44 27 25 11
a Bioassays were conducted separately: 50 HWA and 60 GPA were used for each bioassay. HWA were drenched by spraying with an aqueous solution of 1 3 108 spores/ml and GPA with 5 3 106 spores/ml. Mortality was assessed after 6 and 10 days for GPA and HWA, respectively. b Accession number for the ARS Collection of Entomopathogenic Fungi where the isolates are stored at USDA, ARS Plant, Soil and Nutrition Laboratory, Tower Road, Ithaca, NY 14853. c ND, no data; bioassays using these fungi against HWA were not conducted.
McClure, M. S. 1987. Connecticut Agricultural Experiment Station Bulletin 851. Montgomery, M., and Lyon, S. 1995. Final Report Coop. Agreement 42-778, USDA, FS, Hamden, CT. Smith, D., and Onions, A. H. S. 1983. ‘‘The Preservation and Maintenance of Living Fungi.’’ Commonwealth Mycological Institute, Kew, Richmond, England.
SVETLANA GOULI BRUCE L. PARKER MARGARET SKINNER Entomology Research Laboratory University of Vermont P.O. Box 53400 Burlington, Vermont 05405-3400 Received September 10, 1996; accepted February 24, 1997
70, 76–77 (1997)
IN974668
NOTE Isolation of Fungi Associated with Hemlock Woolly Adelgid (Homoptera: Adelgidae) The hemlock woolly adelgid, Adelges tsugae Annand (HWA), originated in Asia and was introduced into the United States in 1924 (McClure, 1987). It is causing major damage to eastern hemlock, Tsuga canadensis (L.) Carr., in New England. A search for parasites and predators of this forest pest in the Northeast was conducted recently (Montgomery and Lyon, 1995) but a survey of associated entomopathogenic fungi has not been done. Five dominant eastern hemlock trees with visible heavy infestations of HWA located in Holyoke, Massachusetts, were selected for sampling. Ten 2-m-long branch samples were taken from the middle and upper canopy of each tree. From these branches we cut 10-cm sections of apical portions containing an abundance of HWA. Two hundred apical samples were taken on each of the following dates: January 29, 1996; February 14, 1996; and March 14, 1996. Prior to examination the apical samples were stored individually in glass test tubes at 1°C. Six thousand HWA sistens (10 per apical section) were examined, within 2 weeks of sampling, under 403 magnification, 2882 of which showed symptoms of fungal infection, i.e., off-color, mummified, or with external fungal growth. Two methods were used to recover fungi from these specimens. Obligatory parasitic fungi were prepared as wet mounts on slides using glycerol and water and identified at 4003 using phasecontrast microscopy. Other specimens were surface sterilized for 1–2 min with 2.5% NaOCl (using commercial bleach) placed on sterile hygroscopic paper under humid conditions or plated directly on potato dextrose agar, potato dextrose egg agar, or Czapek’s medium (Anonymous, 1982; Smith and Onions, 1983). Of the 6000 HWA examined, fungi were found associated with approximately 37%. Seventeen different fungal genera were recovered (Table 1). Three genera (Verticillium, Beauveria, and Paecilomyces) contain species known to be major entomopathogens. Some species of Acremonium are entomopathogens. The genera Trichoderma and Gliocladium contain species that are recognized antagonists of plant pathogens. The remain-
0022-2011/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
ing genera contain species that are primarily saprophytic. Identifications were verified by R. Humber (USDA, ARS, Ithaca, NY) and will be identified to species at a later date. All isolates have been placed in long-term storage at the Entomology Research Laboratory, University of Vermont, and the USDA, Agricultural Research Service Collection of Entomopathogenic Fungi at Ithaca, New York. Given the approximately 50% mortality we observed in the field population in a cold-hardiness study done by the authors in 1996 (unpublished data), it is possible that fungi contribute to the natural regulation of HWA populations. Fungal bioassays were conducted using field-collected HWA (dosage: 1 3 108 spores/ml) and lab-reared green peach aphid (GPA), Myzus persicae (Sulzer) (dosage: 5 3 106 spores/ml), to determine the relative pathogenicity of several isolates that we recovered (Table 2). Mortality rates of 42–90% among the treated HWA, and 25–90% among the GPA, compared to 10– 11% among the controls were obtained (Table 2). We also conducted bioassays with representatives from all other genera found in association with HWA that are not known to be entomogenous (those listed in Table 1 but not in Table 2). GPA was used as the test organism (dosage: 5 3 106 spores/ml) because the trials were conducted several months after the last sampling period, and HWA were not available. GPA mortality after 6 days ranged from 13 to 47%, compared to 11% among the controls. This suggests that, though less pathogenic than the known entomopathogens, these fungi exhibit some activity against an insect host and should not be discounted when evaluating potential biological control agents. Our results indicate that several entomopathogenic fungi exist naturally in wild HWA populations in New England, some with a demonstrated potential for management of this introduced pest. Further detailed studies are needed to determine their pathogenicity at the range of temperatures and humidities that naturally occurs in the forest. In addition, evaluation of the growth and infection characteristics, such as rate of germination and spore production, would facilitate
76
77
NOTE
TABLE 1 Fungi Isolated from Hemlock Woolly Adelgid (HWA) Collected in Holyoke, Massachusetts, January–March 1996
Fungal species Alternaria sp. Cladosporium sp. Trichoderma lignorum (Tode) Harz Trichoderma sp. Rhinocladiella sp. Fusarium sp. Epicoccum sp. Gliocladium sp. Chlamydomyces sp. Paecilomyces sp. Beauveria bassiana (Balsamo) Vuillemin Pestalotia sp. Verticillium lecanii (Zimmerman) Viegas Embellisia (allii) (Companile) E. Simmons Acremonium sp. Scopulariopsis sp. Cylindrocarpon sp. Geotrichum (candidum) Link emend J. Carmieh
% of Number total of HWAa collecteda 978 534 289 157 88 49 36 27 15 11 6 5 5 3 1 1 1 1
33.93 18.53 10.30 5.45 3.05 1.70 1.25 0.94 0.52 0.38 0.21 0.17 0.17 0.10 0.04 0.04 0.04 0.04
a No fungi or other pathogenic microorganisms were isolated from 675 (23.42%) of the 2882 symptomatic HWA evaluated.
their further development into viable biological control agents. KEY WORDS: Entomopathogenic fungi; Adelges tsugae; fungal management of Adelgidae; biological control. Many thanks are extended to Richard A. Humber, USDA, ARS (Ithaca, NY) for verifying identifications of our entomopathogenic fungi and reviewing earlier versions of the manuscript. We also appreciate the technical assistance of William Reid. This work would not have been possible without the cooperative efforts of numerous personnel in the Vermont Department of Forests, Parks and Recreation and the Massachusetts Department of Environmental Conservation. Funding for this work was received in part from the USDA, Forest Service, NE Area State and Private Forestry, Coop. Agreement NA-96-158. REFERENCES Anonymous. 1982. ‘‘The Oxoid Manual,’’ 5th ed., Oxoid Ltd., Basingstoke, England.
TABLE 2 Bioassay of Select Isolates against Hemlock Woolly Adelgid (HWA) and Green Peach Aphid (GPA)a Identification No.b 5165 5167 5164 5170 5171 5178 5180 5177 Not assigned None
% Mortality Fungal species
HWA
GPA
V. lecanii V. lecanii Scopulariopsis sp. B. bassiana B. bassiana Paecilomyces farinosus (Holm ex SF Gray) Brown & Smith P. farinosus Gliocladium sp. Fusarium sp. None (Control)
82 72 90 58 NDc
80 90 48 60 52
64 ND ND 42 10
52 44 27 25 11
a Bioassays were conducted separately: 50 HWA and 60 GPA were used for each bioassay. HWA were drenched by spraying with an aqueous solution of 1 3 108 spores/ml and GPA with 5 3 106 spores/ml. Mortality was assessed after 6 and 10 days for GPA and HWA, respectively. b Accession number for the ARS Collection of Entomopathogenic Fungi where the isolates are stored at USDA, ARS Plant, Soil and Nutrition Laboratory, Tower Road, Ithaca, NY 14853. c ND, no data; bioassays using these fungi against HWA were not conducted.
McClure, M. S. 1987. Connecticut Agricultural Experiment Station Bulletin 851. Montgomery, M., and Lyon, S. 1995. Final Report Coop. Agreement 42-778, USDA, FS, Hamden, CT. Smith, D., and Onions, A. H. S. 1983. ‘‘The Preservation and Maintenance of Living Fungi.’’ Commonwealth Mycological Institute, Kew, Richmond, England.
SVETLANA GOULI BRUCE L. PARKER MARGARET SKINNER Entomology Research Laboratory University of Vermont P.O. Box 53400 Burlington, Vermont 05405-3400 Received September 10, 1996; accepted February 24, 1997