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Entomophthora fresenii, parasitic on aphids in India
439
NOTES
do not recover if transferred to clean water and fresh seed. A more direct action is suspected. Isolation, puriilcation, and identification of the toxic metabolites are under way, and a more complete toxicologic study of the potency and mode of action of these materials is now in progress.
Entomophthora
fresenii,
RAIMON
The Connecticut Agricultural Experiment Station New Haven, Connecticut 06504 Received February 5,1968
Parasitic
Entomophthora fresenii ( Nowakowski) Gustafsson is reported as a parasite of aphids in Africa, Europe, and North America. M. Gustafsson (LantbMc. Hogs/c. An&., 31, 103-212, 1965), while investigating fungus pathogens of the bean aphid, Aphis fabae Scopoli in Sweden recorded it on A. fabae, Aphis pomi DeGeer, Aphis craccae Linnaeus, Cavariella konoi Takahashi, Chaitophorus tremulae Koch, and Hyperomyzus lampsanae ( Bomer ) . Other host aphids include Myzus braggi Gillette in Louisiana (T. H. Jones, U. S. Dept. Agri., Bull. 703, 19 pp., 1918). In addition to aphids, it has also been reported on Pseudococcus citri Risso in Indonesia (H. J. De Fluiter, Bergcultures, 13,760-765,1939) and on Periphyllus lyropticus Kessler in New Hampshire (V. K. Charles, Plant Diseases Reporter, 23, 340, 1939, and on coccus viridis (green) in India (G. H. Venkataramaiah, Curr. Sci., 36, 387, 1967). During the course of the present investigations on the natural enemies of aphids, E. fresenii was found to be parasitic on Aphis gossypii Glover and Aphis craccivora Koch in Bangalore, and on Aphis spiraecola Patch in Nilambur, Mundakayam (Kerala) , and Shillong (Assam). This is the first record of the fungus on aphids in India. The fungus was found during wet spells in the months of January, August, and October-November, in Bangalore. Infection was noticed during July-August in
L. BEARD
on Aphids
in India
Shillong, and in September in Mundakayam. The cadaver (Fig. 1) infected by the fungus appears gray or violet-gray with a pulverulent appearance when seen through a hand lens. The infected insect remains loosely attached to the leaf by the stylets inserted into the plant tissue. Cladosporium sp. was often associated with this fungus in later stages. The primary conidia produced on unbranched conidiophores issuing from the host body (Fig. 2) are globose to subglobose with a small papilla and appear faintly fuliginous. The secondary conidia, which are almond-shaped with smoky tint, are formed on narrow capillaries (Fig. 3). The resting spores (zygospores) are usually observed during mid-November in Bangalore. The aphids which carry resting spores differ distinctly from those carrying the usual conidia, as shown in Fig. 1. The cadaver of an infected aphid with freshly formed zygospores internally, appears black and very soft, with the stylets inserted into the plant tissue. The internal tissues of the insect are broken down to a dark fluid of viscid consistency harboring a number of dark resting spores. The body wall ruptures when the spores come out, the entire mass becoming stuck to the substratum (in situ) of the cadaver and then hardening. The cadaver with a mass of resting spores appears as a glistening black mass. A high-
FIGS. I-5. Cadaver of Aphb craccicoru infected by Entornophtho~a fresenii. 2. Conidia of E. fresenii on the leg of Aphis gossypii; most of them have germinated giving rise to capillary conidiophores. A few almond-shaped secondary conidia are also seen. 3. Conidia of E. fresenii in the process of formation spores
on the conidiophores. 4. Enlarged view (zygospores) on a cadaver of A. craccioora
of a zygospore in Bangalore.
ly enlarged view of infected A. crucciuora with resting spores is shown in Fig. 5. The spores are ovoid and black. Figure 4 shows an enlarged view of a single resting spore. The resting spores did not germinate in
of
E.
fresenii.
5. A
mass
of
resting
the hemolymph of A. craccivora held in a moist chamber at 28°C for a fortnight, but the dark epispore disappeared. Attempts were made to obtain the fungus in culture using egg yolk or egg yolk with crushed
441
NOTES
aphids as media, but without any success. The conidia that were lodged on the media produced only secondary and tertiary conidia without undergoing further development. These attempts to culture the fungus were also handicapped by the rapid growth of the contaminant Cladosporium sp. which was very frequently found associated with the cadavers resulting from infection of the aphid by E. fresenii.
G.
RAMASESHIAII
Commonwealth institute of Biological Control Indian Station, Bangalore, India Received February 6,1968 Thanks are due to Dr. J. Weiser for confirming the identification of the fungus, and to Dr. V. P. Rao for his interest and encouragement. The research was financed, in part, by the U. S. Department of Agriculture (PL-480).
Isolation of a Nuclear-Polyhedrosis Virus from the Almond Moth, Cadra cautellu A previously unreported nuclear-polyhedrosis virus was isolated from larvae of laboratory-reared almond moths (Cadra cautella) submitted to the Insect Pathology Pioneering Research Laboratory for disease diagnosis in 1966. It had been brought into the Tifton, Georgia, laboratory with collections of almond-moth larvae made in peanut warehouses at various locations in Georgia as part of a study of the buildup of resistance to Malathion. While cultures of these strains of the almond moth were being established, the disease was spread through the rearing room-probably by contaminated equipment. The symptoms were quickly noticed, the cultures were isolated, and a sample of larvae and pupae was submitted for disease diagnosis. Several weeks later when the examination began, all specimens were dead and desiccated. The sample contained both light and dark larvae, but the light-colored larvae predominated five to one. Under low magnification (10x ), the light larvae were seen to have localized white patches along the dorsum, and all water mounts prepared from these specimens had numerous polyhedral inclusions. Some darkcolored specimens were heavily infected with Bacillus thuringiensis, serological type IX Tolworth, and others were infected with
both the bacillus and the virus. All pupae were free of disease-producing organisms. The polyhedra were isolated by surfacecleaning dried but intact infected larvae in 0.5% sodium hypochlorite and rinsing them several times in distilled water. Then the larvae were macerated in a glass tissue grinder with a small amount of sterile, distilled water, hemacytometer counts were made (average yield was 29 X 10’ polyhedra/larva) , and the suspension was passed successively through a coarse, fritted glass filter and a 400-wire mesh screen. The resultant suspension was further purified by sonification and differential centrifugation. In the process of cleaning and purification, 45% of the polyhedra were lost. Tissues attacked by the virus were the fat body, the hypodermis, and the tracheae. The inclusion bodies were irregularly shaped (Fig. 1) and varied in size. APproximate diameters of untreated polyhedra from a fresh suspension prepared from living, diseased insects ranged from 0.95 to 4.00 p. These measurements were obtained by using bright-field illumination, a magnification of 950 X, and a calibrated optical system with a Leitz 1 screw micrometer eyepiece. The size and structure of the inclusion bodies and of the isolated viral rods
NOTES
do not recover if transferred to clean water and fresh seed. A more direct action is suspected. Isolation, puriilcation, and identification of the toxic metabolites are under way, and a more complete toxicologic study of the potency and mode of action of these materials is now in progress.
Entomophthora
fresenii,
RAIMON
The Connecticut Agricultural Experiment Station New Haven, Connecticut 06504 Received February 5,1968
Parasitic
Entomophthora fresenii ( Nowakowski) Gustafsson is reported as a parasite of aphids in Africa, Europe, and North America. M. Gustafsson (LantbMc. Hogs/c. An&., 31, 103-212, 1965), while investigating fungus pathogens of the bean aphid, Aphis fabae Scopoli in Sweden recorded it on A. fabae, Aphis pomi DeGeer, Aphis craccae Linnaeus, Cavariella konoi Takahashi, Chaitophorus tremulae Koch, and Hyperomyzus lampsanae ( Bomer ) . Other host aphids include Myzus braggi Gillette in Louisiana (T. H. Jones, U. S. Dept. Agri., Bull. 703, 19 pp., 1918). In addition to aphids, it has also been reported on Pseudococcus citri Risso in Indonesia (H. J. De Fluiter, Bergcultures, 13,760-765,1939) and on Periphyllus lyropticus Kessler in New Hampshire (V. K. Charles, Plant Diseases Reporter, 23, 340, 1939, and on coccus viridis (green) in India (G. H. Venkataramaiah, Curr. Sci., 36, 387, 1967). During the course of the present investigations on the natural enemies of aphids, E. fresenii was found to be parasitic on Aphis gossypii Glover and Aphis craccivora Koch in Bangalore, and on Aphis spiraecola Patch in Nilambur, Mundakayam (Kerala) , and Shillong (Assam). This is the first record of the fungus on aphids in India. The fungus was found during wet spells in the months of January, August, and October-November, in Bangalore. Infection was noticed during July-August in
L. BEARD
on Aphids
in India
Shillong, and in September in Mundakayam. The cadaver (Fig. 1) infected by the fungus appears gray or violet-gray with a pulverulent appearance when seen through a hand lens. The infected insect remains loosely attached to the leaf by the stylets inserted into the plant tissue. Cladosporium sp. was often associated with this fungus in later stages. The primary conidia produced on unbranched conidiophores issuing from the host body (Fig. 2) are globose to subglobose with a small papilla and appear faintly fuliginous. The secondary conidia, which are almond-shaped with smoky tint, are formed on narrow capillaries (Fig. 3). The resting spores (zygospores) are usually observed during mid-November in Bangalore. The aphids which carry resting spores differ distinctly from those carrying the usual conidia, as shown in Fig. 1. The cadaver of an infected aphid with freshly formed zygospores internally, appears black and very soft, with the stylets inserted into the plant tissue. The internal tissues of the insect are broken down to a dark fluid of viscid consistency harboring a number of dark resting spores. The body wall ruptures when the spores come out, the entire mass becoming stuck to the substratum (in situ) of the cadaver and then hardening. The cadaver with a mass of resting spores appears as a glistening black mass. A high-
FIGS. I-5. Cadaver of Aphb craccicoru infected by Entornophtho~a fresenii. 2. Conidia of E. fresenii on the leg of Aphis gossypii; most of them have germinated giving rise to capillary conidiophores. A few almond-shaped secondary conidia are also seen. 3. Conidia of E. fresenii in the process of formation spores
on the conidiophores. 4. Enlarged view (zygospores) on a cadaver of A. craccioora
of a zygospore in Bangalore.
ly enlarged view of infected A. crucciuora with resting spores is shown in Fig. 5. The spores are ovoid and black. Figure 4 shows an enlarged view of a single resting spore. The resting spores did not germinate in
of
E.
fresenii.
5. A
mass
of
resting
the hemolymph of A. craccivora held in a moist chamber at 28°C for a fortnight, but the dark epispore disappeared. Attempts were made to obtain the fungus in culture using egg yolk or egg yolk with crushed
441
NOTES
aphids as media, but without any success. The conidia that were lodged on the media produced only secondary and tertiary conidia without undergoing further development. These attempts to culture the fungus were also handicapped by the rapid growth of the contaminant Cladosporium sp. which was very frequently found associated with the cadavers resulting from infection of the aphid by E. fresenii.
G.
RAMASESHIAII
Commonwealth institute of Biological Control Indian Station, Bangalore, India Received February 6,1968 Thanks are due to Dr. J. Weiser for confirming the identification of the fungus, and to Dr. V. P. Rao for his interest and encouragement. The research was financed, in part, by the U. S. Department of Agriculture (PL-480).
Isolation of a Nuclear-Polyhedrosis Virus from the Almond Moth, Cadra cautellu A previously unreported nuclear-polyhedrosis virus was isolated from larvae of laboratory-reared almond moths (Cadra cautella) submitted to the Insect Pathology Pioneering Research Laboratory for disease diagnosis in 1966. It had been brought into the Tifton, Georgia, laboratory with collections of almond-moth larvae made in peanut warehouses at various locations in Georgia as part of a study of the buildup of resistance to Malathion. While cultures of these strains of the almond moth were being established, the disease was spread through the rearing room-probably by contaminated equipment. The symptoms were quickly noticed, the cultures were isolated, and a sample of larvae and pupae was submitted for disease diagnosis. Several weeks later when the examination began, all specimens were dead and desiccated. The sample contained both light and dark larvae, but the light-colored larvae predominated five to one. Under low magnification (10x ), the light larvae were seen to have localized white patches along the dorsum, and all water mounts prepared from these specimens had numerous polyhedral inclusions. Some darkcolored specimens were heavily infected with Bacillus thuringiensis, serological type IX Tolworth, and others were infected with
both the bacillus and the virus. All pupae were free of disease-producing organisms. The polyhedra were isolated by surfacecleaning dried but intact infected larvae in 0.5% sodium hypochlorite and rinsing them several times in distilled water. Then the larvae were macerated in a glass tissue grinder with a small amount of sterile, distilled water, hemacytometer counts were made (average yield was 29 X 10’ polyhedra/larva) , and the suspension was passed successively through a coarse, fritted glass filter and a 400-wire mesh screen. The resultant suspension was further purified by sonification and differential centrifugation. In the process of cleaning and purification, 45% of the polyhedra were lost. Tissues attacked by the virus were the fat body, the hypodermis, and the tracheae. The inclusion bodies were irregularly shaped (Fig. 1) and varied in size. APproximate diameters of untreated polyhedra from a fresh suspension prepared from living, diseased insects ranged from 0.95 to 4.00 p. These measurements were obtained by using bright-field illumination, a magnification of 950 X, and a calibrated optical system with a Leitz 1 screw micrometer eyepiece. The size and structure of the inclusion bodies and of the isolated viral rods