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Boletín de la Sociedad Argentina de Botánica

versión On-line ISSN 1851-2372

Bol. Soc. Argent. Bot. v.43 n.3-4 Córdoba ago./dic. 2008


Host range findings on Beauveria bassiana and Metarhizium anisopliae (Ascomycota: Hypocreales) in Argentina


A. V. Toledo1*, A. M. M. De Remes Lenicov2 y C. C. López Lastra3

1*Centro de Investigaciones de Fitopatología (CIDEFI). Facultad de Ciencias Agrarias y Forestales, UNLP. Calle 60 y 119 s/n, (1900) La Plata, Buenos Aires, Argentina. E-mail:
2 División Entomología, Facultad de Ciencias Naturales y Museo, UNLP, Paseo del Bosque s/n, (1900) La Plata, Buenos Aires, Argentina.
3Centro de Estudios Parasitológicos y de Vectores (CEPAVE), UNLP-CONICET. Calle 2 Nro. 584, (1900) La Plata, Buenos Aires, Argentina.


Summary: The natural insect host range of the entomopathogenic fungi (EPF) Beauveria bassiana (Bb) and Metarhizium anisopliae (Ma) was investigated in Argentina during the winter of 2003 through spring of 2004. Fungi- infected insect samples (153) were collected from cornfields and the surrounding uncultivated areas in different localities of Buenos Aires (7), Tucumán (2), and Corrientes (3) provinces. The rates of Bb-infected host range varied among the Coleoptera (37%), Hemiptera (27%) and Dermaptera (1.3%). While the rates of Ma-infected host range varied between the Coleoptera (0.7%) and Hemiptera (34%). The greater host range resulted with B. bassiana found from eight species of Coleoptera (four families), one species of Dermaptera and four species of Hemiptera (three families), than the host range of M. anisopliae found infecting one species of Coleoptera and three species of Hemiptera (two families). We obtained 75 pure fungal isolates (48 Bb-isolates and 27 to Ma-isolates), and 56 of them (33 Bb-isolates and 23 Ma-isolates) were morphologically characterized.

Key words: Beauveria bassiana; Biological control; Coleoptera; Dermaptera; Hemiptera; Entomopathogenic fungi; Metarhizium anisopliae.

Resumen: Espectro de hospedadores hallado en Beauveria bassiana y Metarhizium anisopliae (Ascomycota: Hypocreales) en Argentina. El espectro natural de hospedadores de los hongos entomopatógenos (HEP) Beauveria bassiana (Bb) y Metarhizium anisopliae (Ma) fue investigado en Argentina desde el invierno de 2003 hasta la primavera de 2004. Las muestras de insectos con infecciones fúngicas (153) fueron recolectadas a partir de campos de maíz y las áreas no cultivadas circundantes a los mismos, en diferentes localidades de las provincias de Buenos Aires (7), Tucumán (2) y Corrientes (3). El espectro de hospedadores infectados con Bb varió entre los Coleoptera (37%), Hemiptera (27%) y Dermaptera (1,3%). Mientras que el espectro de hospedadores infectados con Ma varió entre los Coleoptera (0,7%) y los Hemiptera (34%). El mayor espectro lo presentó Bb, encontrado en ocho especies de Coleoptera (cuatro familias), una especie de Dermaptera y cuatro especies de Hemiptera (tres familias), mientras que Ma fue encontrado infectando una especie de Coleoptera y tres especies de Hemiptera (dos familias). Nosotros obtuvimos 75 aislamientos fúngicos puros (48 de Bb y 27 de Ma), de los cuales 56 (33 de Bb y 23 Ma) fueron caracterizados morfológicamente.

Palabras clave: Beauveria bassiana; Control biológico; Coleoptera; Dermaptera; Hemiptera; Hongos entomopatógenos; Metarhizium anisopliae.



Beauveria (Ascomycota: Hypocreales) is one of the earliest entomopathogenic fungi (EPF) ever discovered (Rehner, 2005); it is considered as one of the principal organisms on fungal insect pathology research (Steinhaus, 1963). Among its most relevant characteristics are the cosmopolitan distribution, easy recognition and isolation, frequent occurrence in nature and the wide broad host range including more than 700 insect species (Goettel et al., 1990; Rehner, 2005). On the other hand, Metarhizium spp. (Ascomycota: Hypocreales) are known to infect more than 200 insect species, many of which are major agricultural pests such as spittlebugs, sugar cane borer, termites, scarab grubs, and grasshoppers (St. Leger, 1993). Since Metarhizium EPF species have the potential to be of economic importance, several commercial companies have registered several strains for insect pest management (Rehner, 2005). Currently, the natural host range of B. bassiana (Bals.) Vuill. and M. anisopliae (Metsch.) Sorokin in Argentina, include several species of insects such as Diatraea sacharalis (Fabricius) (Lepidoptera: Pyralidae), Spilosoma virginica (Fabricius) (Lepidoptera: Lymantriidae), Doru lineare (Eschscholtz) (Dermaptera: Forficulidae), Musca domestica L. (Diptera: Muscidae), Nezara viridula L. (Hemiptera: Pentatomidae), Phyrdenus muriceus Germar (Coleoptera: Curculionidae), Diabrotica speciosa (Germar) (Coleoptera: Chrysomelidae), Cycloneda sanguinea L. (Coleoptera: Coccinellidae), Diloboderus abderus Sturm (Coleoptera: Scarabeidae), Aleuas lineatus Stal, Dichroplus elongatus Giglio Toss, Rhammatocerus pictus (Bruner) (Orthoptera: Acrididae) and Triatoma infestans Klug (Hemiptera: Reduviidae) (Fresa, 1979; López Lastra, 1988; Mariani et al., 1996; Lecuona & Díaz, 2001; Luna Rodríguez & Lecuona, 2002; Marti et al., 2005; Siri et al., 2005).
The objectives of this paper were to search for new insect host of B. bassiana and M. anisopliae and to provide morphological characterization of some of the native isolates from Argentina.

Materials and Methods

Sampling and identification of insects and fungi

The sample localities were not only the cornfields but also the surrounding uncultivated field with Eryngium sp. L. (Apiaceae) plants growing as predominant weed. Samples were obtained from localities with different agroecological characteristics of Argentina during the winter of 2003 through the spring of 2004. These localities were from the provinces of Buenos Aires (Abasto, Berazategui, Colonia Urquiza, La Plata, Los Hornos, San Antonio de Areco and San Vicente), Tucumán province (El Manantial and Horco Molle), and Corrientes province (Bella Vista, Esteros del Iberá and Mercedes). Dead insects covered by cottony mycelia were collected, placed in sterilized plastic containers and then processed in the laboratory. Healthy insects were also collected, fixed in 70% ethanol, and taken to the laboratory for their taxonomic identification.

Isolation and characterization of fungi

Infected insects were mounted on sterile glass microscope slides, and with the aid of an sterilized insect pin (number 000) a small portion of mycelium was transferred from the surface of the cadaver onto malt extract agar (MEA) with penicillin G 40,000 units/ml (Merck, Germany) and streptomycin 80,000 units/ml (Parafarm®, Argentina). Cultures were incubated at 25° C in darkness. After 7 days they were transferred to MEA medium in glass tubes and stored at 4° C. Some isolates were deposited at the Mycological Collection of the Centro de Estudios Parasitológicos y de Vectores (CEPAVE, La Plata, Buenos Aires, Argentina), while others were deposited at the Instituto de Botánica Carlos Spegazzini (LPSC, La Plata, Buenos Aires, Argentina) and at the USDAARS Collection of Entomopathogenic Fungal Cultures (ARSEF, Ithaca, New York). Macroscopic and microscopic observations of B. bassiana and M. anisopliae isolates were made from cultures on MEA and potato dextrose agar (PDA), respectively. Monosporic isolates of each fungal species were obtained as reported by Lecuona (1996) and incubated in Petri dishes at 25º C in darkness. After 72 hours of incubation, three colony-forming units (CFU) from each plate were removed and transferred to three sterile Petri dishes containing MEA and PDA and incubated for 14 days in darkness at 23 and 25º C, respectively. Growth rates, aspect, and pigmentation of the colony were recorded. The radial growth of the colony was measured from two orthogonal diameters at 14 days after incubation. Micromorphological characteristics were recorded from 14 d-old colonies by using and mounting young colony structures in lactophenol cotton blue (0.01% w/v). Twenty-five conidia, of each isolate, were individually measured under the microscope and their mean ± standard error was calculated. Morphological descriptions were made according to Brady (1979a, b) and De Hoog (1972) monographs. To estimate conidial germination, 100ml of MEA medium were added to sterile glass microscope slides, which were placed inside Petri dishes containing sterile moistened filter paper. Three slides per isolate, each containing culture media was inoculated with 100μl of conidial suspension at a concentration of 1x104 conidia/ μl. Spore germination was estimated by examining microscopically at least 200 conidia per isolate, three times from each disk after 24 hours of incubation at 25° C in darkness.

Results and Discussion

Sampling and identification of fungi and host insects

A total of 153 infected insects were collected from different hosts (56 Coleoptera, 42 Hemiptera and 2 Dermaptera infected with B. bassiana, and 1 Coleoptera and 52 Hemiptera with M. anisopliae) from July 13, 2003 to November 15, 2004. Samplings were done between July to November due to previous results indicated that fungi were more prevalent in that period of time. All insect cadavers were covered with mycelia, and some of them had sporulated. The rates of Bb-infected host range varied among the Coleoptera (37%), Hemiptera (27%) and Dermaptera (1.3%). The rates of Ma-infected host range varied between the Coleoptera (0.7%) and Hemiptera (34%). The greater host range resulted with B. bassiana which was found infecting eight species of Coleoptera (four families: Chrysomelidae, Coccinellidae, Curculionidae, and Lampyridae), one species of Dermaptera (Forficulidae) and four species of Hemiptera (three families: Cicadellidae, Membracidae, and Pentatomidae), than the host range of M. anisopliae which was found infecting one species of Coleoptera (Cantharidae) and three species of Hemiptera (Cercopidae and Pentatomidae). Data regarding fungal species, species and number of insect hosts, origin locality, and collection date are given in Table 1.

Table 1. Data of Beauveria bassiana and Metharrhizium anisopliae found on different insect host species in Argentina: Note: Asterisk (*) shows new records from Argentina.

Isolation and characterization of fungi

Seventy-five fungal isolates (48 isolates corresponding to B. bassiana and 27 to M. anisopliae), out of 153 insects, were isolated in pure cultures. Additionally, 33 isolates of B. bassiana and 23 of M. anisopliae were morphologically characterized. Data of fungal species, host, and locality of origin, and CEPAVE, LPSC and ARSEF collection accession numbers of each isolate are given in Table 2.

Table 2. List of B. bassiana and M. anisopliae isolates found on different insect hosts, origin localities, repository agencies and their accession numbers.

Beauveria bassiana colony diameters ranged from 1.3 x 1.4 to 3.5 x 3.6 cm (Mean ± SE: 2.2 ± 0.09 x 2.2± 0.08 cm) after 14 days of incubation on MEA at 23º C in darkness (Table 3). Some morphological differences, as to mycelia growth, coloration, and the presence or absence of exudate drops were observed (Fig. 1a). Most of the isolates showed regular colony borders, white cottony and powdery mycelium without exudate drops, and colorless or pale yellow colony reverse. In contrast, the isolates B. bassiana CEP 073 and CEP 074 showed irregular colony borders; CEP 150 and CEP 175 showed colorless exudate drops; while CEP 075, CEP 091 and CEP 175 showed synnemata formation. This structure was rarely observed in B. bassiana when De Hoog (1972) and Brady (1979a) describe this species. In the field survival and transmission of B. bassiana and other pathogenic fungi could be enhanced by the production of synnemata, such as were observed by Meyer et al. (2007) in natural infections caused by Hirsutella citriformis Speare (Ascomycota: Hypocreales).

Table 3. Radial growth, conidia size and conidial germination of B. bassiana and M. anisopliae isolates from different host insects and localities.

Fig. 1: Morphological colony differences, 14 days-old on malt extract agar or potato dextrose agar. a. Beauveria bassiana isolates CEP 069, CEP 075, CEP 081, and CEP 091. b - c. Metarhizium anisopliae isolate CEP 094. d. M. anisopliae isolate CEP 178. e. M. anisopliae isolate CEP 090. f. M. anisopliae isolate CEP 088, bar = 0.7 cm

Conidia were hyaline, smooth, and globose to broadly ellipsoidal, measuring 2.0 - 2.8 x 1.9 - 2.4 μm (average: 2.4 ± 0.04 x 2.1 ± 0.02 μm) (Table 3). The morphology and conidial measurements of the isolates fit the descriptions of Brady (1979a). Most of B. bassiana isolates showed 90% or higher spore germination. The lower spore viabilities were found in isolates: CEP 153 (61.9%), CEP 154 (68.9%), CEP 152 (72.0%), CEP 151 (80.5%), and CEP 115 (82.4%) (Table 3).
Metarhizium anisopliae colony diameters ranged from 2.2 x 2.2 to 4.2 x 4.1 cm (Mean ± SE: 3.2 ± 0.12 x 3.1 ± 0.12 cm) after 14 days of incubation on PDA at 25º C in darkness (Table 3). Some morphological differences related to mycelia growth, coloration, and the presence or absence of exudate drops were noted (Fig. 1 b-f). Colony borders of the majority of isolates were regular, with white mycelia bearing clumps of conidiophores which became colored with the development of conidia, varying from yellow green or olivaceous green to dark herbage green, with the presence of colorless exudate drops; the reverse was pale yellow. In contrast, M. anisopliae isolates CEP 094, CEP 096, CEP 097, CEP 095, CEP 120, CEP 121 and CEP 122 showed irregular colony borders; CEP 093, CEP 090, CEP 159, CEP 160, CEP 178 and CEP 179 lacked exudate drops; while CEP 078, CEP 084 and CEP 087 showed orange to brownish colony reverse. In all the cases the sporulation started after the sixth or seventh day of incubation. M. anisopliae conidia were cylindrical, tapering and truncate at both ends, olivaceous to green and aseptate, measuring 6.5 - 9.2 x 2.4 - 2.9 μm (average: 7.1 ± 0.12 x 2.8 ± 0.03 μm). However, some conidia were longer (usually 5 - 8 x 1.5 - 3.5 μm) than those measurements reported by Brady (1979b) (Table 3). The conidial germination in the majority of isolates was 100%, although only two isolates presented of 85.3% (CEP 078) and 90.8% (CEP 079) (Table 3).
Our results show new insect host range findings for these indigenous fungal isolates, which exhibit some morphological differences among B. bassiana and M. anisopliae isolates in Argentina.
Further studies, including molecular examinations,
will determine their genetic variability among isolates from different host and origin and their potential as biocontrol agents against insect pests.


We wish to thank Dr. Lanteri, Dr. Paradell, Dr. Cabrera, Dr. Melo, Professor Cicchino (Natural Science Museum, La Plata, Buenos Aires, Argentina) for the insect's identification, Dr. Richard Humber and Karen Hansen for the fungal cultures preservation in the USDA-ARS Collection of Entomopathogenic Fungal Cultures (ARSEF), Dr. Vilma Rosato for the fungal material preservation in the Institute of Botany Carlos Spegazzini, and Dr. Cabanillas (USDA, ARS, Weslaco, Texas) for his manuscript critical review. This study was partially supported by the National Research Council of Argentina (CONICET).


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Recibido el 15 de Febrero del 2008,
aceptado el 15 de Octubre del 2008.

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