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A technique for monospore cultures and the determination of nucleus numbers in Metarrhizium anisopliae
276
NOTES
Diseased larvae change uniformly from a normal green to yellowish-green as the mycosis progresses. Under field conditions infected larvae retain their turgidity up to 3 hours after death. The liquid contents of the body diminish rapidly following this stage and the larvae soon become flaccid and laterally compressed (Fig. 1). Larvae dying in the field usually seek an elevated position on the mint plant and remain suspended and attached to the plant after death. The forcibly discharged conidia are elongate and elliptical to nearly cylindrical in form with a rounded apex and papillated base (Fig. 2). Newly formed conidia are without vacuoles, but soon develop them after being discharged. Conidial dimensions averaging 19.7 & 2.44 p in length to 8.1 % 0.13 p wide were similar to those obtained by W. H. Sawyer (Mycologia, 23, 411-432, 1931). The primary conidia may often germinate with a stout germ tube and, in the presence of moisture, function as a conidiophore with the secondary conidium forming at its apex. Histological observations indicated the pathogenesis of E. sphaerosperma in infected loopers to be similar to that described in larvae of Rhopobata vacciniana (IV. H. Sawyer, Annuls of Botany, 47, 799-811, 1933). Following penetration, the
A Technique for Monospore of Nucleus Numbers
mycelium grows abundantly throughout the hemocoel ( Fig. 3), penetrating the fat body and musculature. As the infected larvae approach death the mycelial elements become irregularly shaped and eventually issue tubes (Fig. 4) which grow outward toward the cuticle. In the later stages of infection the mycelial apex pressing against the cuticle form large bulbous structures (Fig. 5). The cuticle is soon broken down and penetrated by fascicles of mycelium (Fig. 6) which later function as conidiophores. When infected loopers are maintained under high humidity sporulation usually occurs within 6 hours after death. G.
WILLIAM
YENDOL
Pesticide Research Laboratory and Graduate Study Center Department of Entomology The Pennsylvania State University University Park, Pennsylvania 16802 J. D.
PASCHKE
Department of Entomology Purdue University Lafayette, Indiana 47907 Received March 17, 1967
Cultures and the Determination in Metarrhizium anisopliae
For biological control purposes it is important to select strains of entomogenous fungi that are as virulent as possible. However, genetic differences within a strain could cause differences in virulence. Selection of a particular strain can be made by using monospore cultures. Heterokaryosis is a well-known phenomenon in Fungi Imperfecti and plays a role
in increasing variability. Variability in cultures of entomogenous Fungi Imperfecti could be reduced by monospore cultures, but if there is more than one nucleus per spore, recombinations of the nuclei could decrease virulence. A strain of Metarrhizium anisopliae, a forma minor (J. R. Johnston, Puerto Rico Board of Commissioners of Agr., Bull., 10,
FK;. 1~imy1
FIG. 1. Spore of Metarrhizium anisopliue. Coloration: 2. Central part of spore of Meturrhizium anisopliue, with acetate. 42,000 X.
~6-28, 1915) was isolated from Schistocerca ~~~c~~:cI~.~‘cI adults six years after they had h been killed. by the fungus in Eritrea. The dry locust carrions were obtained from the British Museum [see F. L. Balfour-Browne> Proc. R. Entomol. Sot. London (A), 35, 65-74. 19601. Nuclei could not be observed with phasecontrast or with interference microscopy. (:onvmtional histological techniques (diffrrent fixations, and coloration bv iron hematoxylin, gentian violet, aceto-carmine, aceto-orceine and )lay-Griinwald Giemsa ) also gave negative results, even when pregerminated spores were used. Examination of the nuclei by electron microscopy1 was successful, using the following technique: Spores were suspended ’ The electron microscopy was accomplished the Service Institute for Applied hfechanics Technical Physics in Agriculture, Wageningen. Netherlands.
at and The
uranyl acetate. double-walled
10,000 X. nucleus. Coloration:
in a 3% agar medium just before it hardened. Pieces of this agar were prefixed in a solution of glutaraldehyde (6% ) in Siirensen phosphate buffer (4-5 hours), washed with Sijrensen buffer pH 6, 8 and postfixed in osmium tetroxide ( 1% ) in Siirensen buffer for 42 hours. Spores were washed again in the same buffer, dehydrated in ethanol, and embedded in Vestopal W via styrene (48 hours 60°C) following the method used by S. M. Kurtx i J. Ultrastr. Res., 5, 468, 1961) . Clearly differentiated nuclei could 1,~ distinguished in only a few of the electron micrographs (e.g., Figs. 1 and 2), and only one nucleus per spore was observed. Similar results were obtained with another strain of the fungus, a forma major, isolated from Oryctes nasicornis and observed by aid of an interference microscope. Since the spores of these two strains of Metarrhixium anisopliae have only one nucleus
NOTES
278
per spore, monospore cultures of them should have a minimum of variability. Monospore cultures were prepared as follows: Small circles (diam i 2 mm) were engraved on the bottom lid of petri dishes with the aid of a Leitz marker objective
(Type MAKKI, photograph 3). Sterile, filtered, water agar was poured into warm, sterile petri dishes to give a very thin film of agar. The center of the circle was marked with an ink dot on the underside of the petri dish. Small droplets containing, on the average, one Metarrhizium-spore were delivered on the agar above the ink dots by means of a sterile Pasteur pipette. The dots were then removed and the number of spores in each circle mounted under phase contrast with a magnification of 16x (objective), 8X (ocular). The field corresponded with the field of the engraved circles. Circles with only one spore were marked again with an ink dot to aid in their transfer to Sabouraud maltose agar with a sterile loop. K. H. VEEN Agricultural University Department of Entomology Wageningen, The Netherlands
FIG. n. Device dish with engraved
for monospore circles. Photo
Multiplication
cultures; petri by G. Eimers.
Received March 9, 1967
of
Tipula
and
Cells
of
Antheraea
The continuous line of Antheraea eucdypti ovarian cells which was established by T. D. C. Grace (Nature, 195, 788-789, 1962) is susceptible to infection by Sericesthb iridescent virus (A. J. D. Bellett and E. H. Mercer, Virology, 24, 645-653, 1964). This note reports the successful infection of this cell line with Tip&a iridescent virus (TIV), and with Chile iridescent virus (CIV) described by M. Fukaya and S. Nasu (Appl. Entomol. Zool., 1, 69-72, 1966). The suspensionsof the two viruses were prepared as follows: Larvae of the greater Gallerk mellonella, which had wax moth,
Chile
Iridescent
Viruses
in
eucalypti
been infected with either TIV or CIV, were surface-sterilized with 1% hyamine. Their abdominal legs were clipped and the oozing hemolymph, containing fragments of fat-body tissue, was collected in capillary tubes. The hemolymph was added to a modification of the Grace’s culture medium described previously (T. Hukuhara, J. Sericult. Sci. Japan, 35, 349-354, 1966). The cell cultures were incubated overnight with the virus suspension (1.5-2.0 ml) at 20-21°C. After this period the cultures were supplied with fresh culture medium. For light- and electron-microscope examinations the TIV-infected cells were fixed
NOTES
Diseased larvae change uniformly from a normal green to yellowish-green as the mycosis progresses. Under field conditions infected larvae retain their turgidity up to 3 hours after death. The liquid contents of the body diminish rapidly following this stage and the larvae soon become flaccid and laterally compressed (Fig. 1). Larvae dying in the field usually seek an elevated position on the mint plant and remain suspended and attached to the plant after death. The forcibly discharged conidia are elongate and elliptical to nearly cylindrical in form with a rounded apex and papillated base (Fig. 2). Newly formed conidia are without vacuoles, but soon develop them after being discharged. Conidial dimensions averaging 19.7 & 2.44 p in length to 8.1 % 0.13 p wide were similar to those obtained by W. H. Sawyer (Mycologia, 23, 411-432, 1931). The primary conidia may often germinate with a stout germ tube and, in the presence of moisture, function as a conidiophore with the secondary conidium forming at its apex. Histological observations indicated the pathogenesis of E. sphaerosperma in infected loopers to be similar to that described in larvae of Rhopobata vacciniana (IV. H. Sawyer, Annuls of Botany, 47, 799-811, 1933). Following penetration, the
A Technique for Monospore of Nucleus Numbers
mycelium grows abundantly throughout the hemocoel ( Fig. 3), penetrating the fat body and musculature. As the infected larvae approach death the mycelial elements become irregularly shaped and eventually issue tubes (Fig. 4) which grow outward toward the cuticle. In the later stages of infection the mycelial apex pressing against the cuticle form large bulbous structures (Fig. 5). The cuticle is soon broken down and penetrated by fascicles of mycelium (Fig. 6) which later function as conidiophores. When infected loopers are maintained under high humidity sporulation usually occurs within 6 hours after death. G.
WILLIAM
YENDOL
Pesticide Research Laboratory and Graduate Study Center Department of Entomology The Pennsylvania State University University Park, Pennsylvania 16802 J. D.
PASCHKE
Department of Entomology Purdue University Lafayette, Indiana 47907 Received March 17, 1967
Cultures and the Determination in Metarrhizium anisopliae
For biological control purposes it is important to select strains of entomogenous fungi that are as virulent as possible. However, genetic differences within a strain could cause differences in virulence. Selection of a particular strain can be made by using monospore cultures. Heterokaryosis is a well-known phenomenon in Fungi Imperfecti and plays a role
in increasing variability. Variability in cultures of entomogenous Fungi Imperfecti could be reduced by monospore cultures, but if there is more than one nucleus per spore, recombinations of the nuclei could decrease virulence. A strain of Metarrhizium anisopliae, a forma minor (J. R. Johnston, Puerto Rico Board of Commissioners of Agr., Bull., 10,
FK;. 1~imy1
FIG. 1. Spore of Metarrhizium anisopliue. Coloration: 2. Central part of spore of Meturrhizium anisopliue, with acetate. 42,000 X.
~6-28, 1915) was isolated from Schistocerca ~~~c~~:cI~.~‘cI adults six years after they had h been killed. by the fungus in Eritrea. The dry locust carrions were obtained from the British Museum [see F. L. Balfour-Browne> Proc. R. Entomol. Sot. London (A), 35, 65-74. 19601. Nuclei could not be observed with phasecontrast or with interference microscopy. (:onvmtional histological techniques (diffrrent fixations, and coloration bv iron hematoxylin, gentian violet, aceto-carmine, aceto-orceine and )lay-Griinwald Giemsa ) also gave negative results, even when pregerminated spores were used. Examination of the nuclei by electron microscopy1 was successful, using the following technique: Spores were suspended ’ The electron microscopy was accomplished the Service Institute for Applied hfechanics Technical Physics in Agriculture, Wageningen. Netherlands.
at and The
uranyl acetate. double-walled
10,000 X. nucleus. Coloration:
in a 3% agar medium just before it hardened. Pieces of this agar were prefixed in a solution of glutaraldehyde (6% ) in Siirensen phosphate buffer (4-5 hours), washed with Sijrensen buffer pH 6, 8 and postfixed in osmium tetroxide ( 1% ) in Siirensen buffer for 42 hours. Spores were washed again in the same buffer, dehydrated in ethanol, and embedded in Vestopal W via styrene (48 hours 60°C) following the method used by S. M. Kurtx i J. Ultrastr. Res., 5, 468, 1961) . Clearly differentiated nuclei could 1,~ distinguished in only a few of the electron micrographs (e.g., Figs. 1 and 2), and only one nucleus per spore was observed. Similar results were obtained with another strain of the fungus, a forma major, isolated from Oryctes nasicornis and observed by aid of an interference microscope. Since the spores of these two strains of Metarrhixium anisopliae have only one nucleus
NOTES
278
per spore, monospore cultures of them should have a minimum of variability. Monospore cultures were prepared as follows: Small circles (diam i 2 mm) were engraved on the bottom lid of petri dishes with the aid of a Leitz marker objective
(Type MAKKI, photograph 3). Sterile, filtered, water agar was poured into warm, sterile petri dishes to give a very thin film of agar. The center of the circle was marked with an ink dot on the underside of the petri dish. Small droplets containing, on the average, one Metarrhizium-spore were delivered on the agar above the ink dots by means of a sterile Pasteur pipette. The dots were then removed and the number of spores in each circle mounted under phase contrast with a magnification of 16x (objective), 8X (ocular). The field corresponded with the field of the engraved circles. Circles with only one spore were marked again with an ink dot to aid in their transfer to Sabouraud maltose agar with a sterile loop. K. H. VEEN Agricultural University Department of Entomology Wageningen, The Netherlands
FIG. n. Device dish with engraved
for monospore circles. Photo
Multiplication
cultures; petri by G. Eimers.
Received March 9, 1967
of
Tipula
and
Cells
of
Antheraea
The continuous line of Antheraea eucdypti ovarian cells which was established by T. D. C. Grace (Nature, 195, 788-789, 1962) is susceptible to infection by Sericesthb iridescent virus (A. J. D. Bellett and E. H. Mercer, Virology, 24, 645-653, 1964). This note reports the successful infection of this cell line with Tip&a iridescent virus (TIV), and with Chile iridescent virus (CIV) described by M. Fukaya and S. Nasu (Appl. Entomol. Zool., 1, 69-72, 1966). The suspensionsof the two viruses were prepared as follows: Larvae of the greater Gallerk mellonella, which had wax moth,
Chile
Iridescent
Viruses
in
eucalypti
been infected with either TIV or CIV, were surface-sterilized with 1% hyamine. Their abdominal legs were clipped and the oozing hemolymph, containing fragments of fat-body tissue, was collected in capillary tubes. The hemolymph was added to a modification of the Grace’s culture medium described previously (T. Hukuhara, J. Sericult. Sci. Japan, 35, 349-354, 1966). The cell cultures were incubated overnight with the virus suspension (1.5-2.0 ml) at 20-21°C. After this period the cultures were supplied with fresh culture medium. For light- and electron-microscope examinations the TIV-infected cells were fixed