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A selective medium for the isolation of Beauveria tenella and of Metarrhizium anisopliae
268
NOTES
A Selective tenella
Medium for the Isolation of Beauveria and of Metarrhizium anisopliae
Various methods have been suggested to isolate entomopathogenic fungi from mycelium-covered insect carrions. The main difficulty is to eliminate the contaminating agents which start developing within and on the insect body immediately after its death. Surface disinfection of the integument is usually sufficient to isolate fungi successfully from the spores formed on the dead insect when placed within a damp environment. The simultaneous use of an isolation culture medium detrimental to bacterial growth ensures success in most cases (E. Miiller-Kogler, “Pilzkrankheiten bei Insekten,” pp. 7.578, P. Parey, Berlin, 1965). It is more difficult, however, to obtain a fungus isolation from an insect carrion already much pervaded by contaminants, i.e. old insect carrions found in a forest or entomological samples received from faraway countries. In such cases, the insect carrion is sort of mummified in a thick cover of mycelium, spores, bacteria, and various fungi. A surface disinfection of the integument remains inefficient because it does not preclude of saprophytic microthe quick “regrowth” organisms. This makes it necessary to use selective culture media that act against bacterial growth and that minimize (the development of such “over-running” fungi as species of Mucor. The cherry-extract culture medium (G. H. Boerema, Neth. J. Plant Pathol., 69, 76-103, 1963), often used in our laboratory as an isolating medium, is insufficient to preclude an excessive growth of these contaminants. Satisfactory results are not even always attained with such media as those derived from ox-gall and rose bengale, whether used with streptomycin or not (T. P. Martin, Soil Sci., 69, 215-232, 1950). Starting from Martin’s basic medium (glucose = 10 g; peptone = 10 g; dried ox-gall
= 1.5 g; rose bengale = l/15,000; agar = 30 g; distilled water = 1000 ml), we have added various antibiotic substances acting as bacteria and fungi inhibitors, excluding two entomopathogenic fungus species which are now under laboratory examination: Beauveria tenella and Metarrhizium anisopliae. After testing various products, we have chosen Chloramphenicol ( Roussel-Uclaf Laborator. ies) as lthe bactericidal agent because of it5 wide spectrum of use, and Actidione or Cycloheximide (Upjohn Laboratories) as the antifungal antibiotic. Actidione is specially active on most fungus species except those pathogenic for vertebrates (A. 0. Whiffen, J. Bacterial., 56, 283, 1948). The activity of these products on ‘the Beauveria or Metawhizium conidiospores was tested by the slide method and in petri dishes. Concentrations of 250 ug of Actidione and of 500 pg of Chloramphenicol/ml of medium could be used without greatly reducing the germinating power of spores. The culturemedium preparation requires special safety precautions because the products are noxious for the experimentator. Chloramphenicol may be added to the medium prior to sterilization, but in return Actidione should be added by sterile methods after an autoclave treatment starting from a sterile solution (aqueous mother solution with a 10 mg/ml concentration, sterilized in the autoclave for 15 minutes at 120°C and kept in a refrigerator). Using such a method we have successfully isolated Beauveria tenella and Metarrhizium anisopliae strains by starting from heavily contaminated insect carrions, and thus we have been able to compare the virulences of strains from various origins. Owing to the importance of selecting highly virulent fungal strains in such a way, we now contemplate extending the scope of this study to other entomopathogenic fungus species. Preliminary
NOTES
269
tests have shown that this method may also Biological Control and Biocenosis Research be used successfully for isolations from the Station I(. H. VEEN P. FERRON
A Microsporidian,
Thelohania
During attempts to establish laboratory cultures of field-collected larvae of the imported cabbageworm, Pieris rapae (July to October, 1965), caterpillars infected with a speciesof Thelohania were obtained. Infected individuals displayed definite mottling, sluggishness,reduced feeding, smaller size, and emitted both oral and diarrhetic discharges. Caterpillars infected at any particular instar failed to molt further, and thus failed to pupate. They remained in the larval stage 12 to 14 days longer than the apparently uninfected individuals of the samegroup. When Brussels sprout leaves were inoculated with a massive suspensionof spores obtained from infected insects and fed to second- and third-instar uninfected caterpillars, successfulinfection to all 45 test hosts was obtained. Death occurred 6 to 13 days after treatment. None of the control insects showed signs of the disease. Observations on Giemsa-staineddry smears show that schizonts are generally spherical, have deep red nuclei, and a dense, blue cytoplasm. Sporogonic stages are distinguished from schizonts by their larger size and weak staining characteristics. The nuclei stain pink to reddish blue, sometimes surrounded first by an unstained area, then by a light blue cytoplasm. Formation of eight sporoblasts follow the third division of a sporont. Sporoblasts (Fig. IA) eventually give rise to a group of eight spores (Fig. 1B). Length of fresh mature spores has a range of 5.62 to 6.85> the mean being 6.17 f 0.01 u. The spore width has a 3.12 to 3.75 range with a mean of 3.42 _t 0.02 u (stained spore size in 11, length: 5.00-5.62, 5 = 5.20 * 0.07;
La Mini&e par Versa&s, France Accepted February 14, 1966
sp.,
in
Pieris
rapae
FIG. 1. Distinctive stages of Thelohanin sp. among dislodged spores (Giemsa stain, 2250 x). A. .4n octosporous sporont. B. A group of eight spores.
width: 2.50-3.75, ,r-= 2.98 If- 0.01). Polar filaments extruded under pressure have a mean length of 56.15 + 4.27 n. Based on the observations that (a) each sporont produces eight sporoblasts and ultimately eight spores, and (b) the length of the spores is less than four times their breadth, the pathogen belongs to the genus Thelohania (Nosematidae: Microsporidia) . Examination of histological sections indicated that all major tissuesof the host were infected. In some infected tissues, as with
NOTES
A Selective tenella
Medium for the Isolation of Beauveria and of Metarrhizium anisopliae
Various methods have been suggested to isolate entomopathogenic fungi from mycelium-covered insect carrions. The main difficulty is to eliminate the contaminating agents which start developing within and on the insect body immediately after its death. Surface disinfection of the integument is usually sufficient to isolate fungi successfully from the spores formed on the dead insect when placed within a damp environment. The simultaneous use of an isolation culture medium detrimental to bacterial growth ensures success in most cases (E. Miiller-Kogler, “Pilzkrankheiten bei Insekten,” pp. 7.578, P. Parey, Berlin, 1965). It is more difficult, however, to obtain a fungus isolation from an insect carrion already much pervaded by contaminants, i.e. old insect carrions found in a forest or entomological samples received from faraway countries. In such cases, the insect carrion is sort of mummified in a thick cover of mycelium, spores, bacteria, and various fungi. A surface disinfection of the integument remains inefficient because it does not preclude of saprophytic microthe quick “regrowth” organisms. This makes it necessary to use selective culture media that act against bacterial growth and that minimize (the development of such “over-running” fungi as species of Mucor. The cherry-extract culture medium (G. H. Boerema, Neth. J. Plant Pathol., 69, 76-103, 1963), often used in our laboratory as an isolating medium, is insufficient to preclude an excessive growth of these contaminants. Satisfactory results are not even always attained with such media as those derived from ox-gall and rose bengale, whether used with streptomycin or not (T. P. Martin, Soil Sci., 69, 215-232, 1950). Starting from Martin’s basic medium (glucose = 10 g; peptone = 10 g; dried ox-gall
= 1.5 g; rose bengale = l/15,000; agar = 30 g; distilled water = 1000 ml), we have added various antibiotic substances acting as bacteria and fungi inhibitors, excluding two entomopathogenic fungus species which are now under laboratory examination: Beauveria tenella and Metarrhizium anisopliae. After testing various products, we have chosen Chloramphenicol ( Roussel-Uclaf Laborator. ies) as lthe bactericidal agent because of it5 wide spectrum of use, and Actidione or Cycloheximide (Upjohn Laboratories) as the antifungal antibiotic. Actidione is specially active on most fungus species except those pathogenic for vertebrates (A. 0. Whiffen, J. Bacterial., 56, 283, 1948). The activity of these products on ‘the Beauveria or Metawhizium conidiospores was tested by the slide method and in petri dishes. Concentrations of 250 ug of Actidione and of 500 pg of Chloramphenicol/ml of medium could be used without greatly reducing the germinating power of spores. The culturemedium preparation requires special safety precautions because the products are noxious for the experimentator. Chloramphenicol may be added to the medium prior to sterilization, but in return Actidione should be added by sterile methods after an autoclave treatment starting from a sterile solution (aqueous mother solution with a 10 mg/ml concentration, sterilized in the autoclave for 15 minutes at 120°C and kept in a refrigerator). Using such a method we have successfully isolated Beauveria tenella and Metarrhizium anisopliae strains by starting from heavily contaminated insect carrions, and thus we have been able to compare the virulences of strains from various origins. Owing to the importance of selecting highly virulent fungal strains in such a way, we now contemplate extending the scope of this study to other entomopathogenic fungus species. Preliminary
NOTES
269
tests have shown that this method may also Biological Control and Biocenosis Research be used successfully for isolations from the Station I(. H. VEEN P. FERRON
A Microsporidian,
Thelohania
During attempts to establish laboratory cultures of field-collected larvae of the imported cabbageworm, Pieris rapae (July to October, 1965), caterpillars infected with a speciesof Thelohania were obtained. Infected individuals displayed definite mottling, sluggishness,reduced feeding, smaller size, and emitted both oral and diarrhetic discharges. Caterpillars infected at any particular instar failed to molt further, and thus failed to pupate. They remained in the larval stage 12 to 14 days longer than the apparently uninfected individuals of the samegroup. When Brussels sprout leaves were inoculated with a massive suspensionof spores obtained from infected insects and fed to second- and third-instar uninfected caterpillars, successfulinfection to all 45 test hosts was obtained. Death occurred 6 to 13 days after treatment. None of the control insects showed signs of the disease. Observations on Giemsa-staineddry smears show that schizonts are generally spherical, have deep red nuclei, and a dense, blue cytoplasm. Sporogonic stages are distinguished from schizonts by their larger size and weak staining characteristics. The nuclei stain pink to reddish blue, sometimes surrounded first by an unstained area, then by a light blue cytoplasm. Formation of eight sporoblasts follow the third division of a sporont. Sporoblasts (Fig. IA) eventually give rise to a group of eight spores (Fig. 1B). Length of fresh mature spores has a range of 5.62 to 6.85> the mean being 6.17 f 0.01 u. The spore width has a 3.12 to 3.75 range with a mean of 3.42 _t 0.02 u (stained spore size in 11, length: 5.00-5.62, 5 = 5.20 * 0.07;
La Mini&e par Versa&s, France Accepted February 14, 1966
sp.,
in
Pieris
rapae
FIG. 1. Distinctive stages of Thelohanin sp. among dislodged spores (Giemsa stain, 2250 x). A. .4n octosporous sporont. B. A group of eight spores.
width: 2.50-3.75, ,r-= 2.98 If- 0.01). Polar filaments extruded under pressure have a mean length of 56.15 + 4.27 n. Based on the observations that (a) each sporont produces eight sporoblasts and ultimately eight spores, and (b) the length of the spores is less than four times their breadth, the pathogen belongs to the genus Thelohania (Nosematidae: Microsporidia) . Examination of histological sections indicated that all major tissuesof the host were infected. In some infected tissues, as with