Journal of Invertebrate Pathology 92 (2006) 7–10 www.elsevier.com/locate/yjipa First record of Clonostachys rosea (Ascomycota: Hypocreales) as an entomopathogenic fungus of Oncometopia tucumana and Sonesimia grossa (Hemiptera: Cicadellidae) in Argentina A.V. Toledo a,¤, E. Virla b, R.A. Humber c, S.L. Paradell d, C.C. López Lastra a a Centro de Estudios Parasitológicos y de Vectores (CEPAVE) UNLP-CONICET, Calle 2 Nro. 584 (1900) La Plata, Buenos Aires, Argentina b Planta Piloto de Procesos Industriales Microbiológicos (PROIMI) CONICET, Av. Belgrano y Pje. Caseros (T 4001 MVB), San Miguel, Tucumán, Argentina c USDA-ARS Plant Soil and Nutrition Laboratory, Tower Road, Ithaca, NY 14853, USA d División Entomología. Facultad de Ciencias Naturales y Museo. UNLP. Paseo del Bosque s/n. (1900), La Plata, Buenos Aires, Argentina Received 4 August 2005; accepted 11 October 2005 Available online 31 March 2006 Abstract Clonostachys rosea (Link: Fries) Schroers, Samuels, Seifert, and Gams (Ascomycota: Hypocreales) has been reported as a mycopara- site of fungi and nematodes and as saprobe in soils, but this fungus has not been reported previously to be entomopathogenic. Many spe- cies of cicadellid leafhoppers cause economic damage to crops as vectors of plant pathogens. In the present work, we report the Wrst record of C. rosea as an entomopathogenic fungus of two leafhoppers pest, Oncometopia tucumana and Sonesimia grossa (Hemiptera: Cicadellidae), in Argentina and evaluate the pathogenicity of C. rosea against them. © 2006 Elsevier Inc. All rights reserved. Keywords: Argentina; Cicadellidae; Clonostachys rosea; Entomopathogenic fungi; Hemiptera; Oncometopia tucumana; Sonesimia grossa Clonostachys rosea (Link: Fries) Schroers, Samuels, Seif- tested successfully as a biological control agent against ert, and Gams [formerly known as Gliocladium roseum Bai- diVerent fungal plant pathogens including S. sclerotorium nier; teleomorph: Bionectria ochroleuca (Schweinitz)] (Lib.) de Bary (Ervio et al., 1994), Verticillium dahliae Kleb. (Schroers et al., 1999) is known from temperate and tropi- (Keinath et al., 1991), and several species of Botrytis (James cal regions (Schroers, 2001) and is common in an extraordi- and Sutton, 1996). In Argentina the presence of Gliocladium nary range of habitats in tropical, temperate, sub-arctic, roseum in soil was reported (Cabello and Arambarri, 2002) and desert regions of the world. It has been reported from but this fungus has not been reported previously to be cultivated grassland and woodland, forest, heathland, entomopathogenic. Previous reports of entomopathogenic freshwater, and coastal soils, particularly those of neutral fungi aVecting hemipteran insects in South America mostly to alkaline pH (Sutton et al., 1997). This fungus has been involve cercopid pests of sugar cane in Brazil (Marques frequently associated with cysts of Heterodera spp., Globo- et al., 1981). The few records of entomopathogenic fungi dera spp. and other nematodes in soil and with sclerotia of from hemipterans in Argentina represent hosts in the fami- Sclerotina sclerotorium (Lib.) de Bary, Phymatotrichum lies Coccidae, Aphididae, Cixiidae, and Aleyrodidae (Mar- omnivorum Duggar, Rhizoctonia solani Kühn, Botrytis spp., chionatto, 1935; Toledo et al., 2004; Yasem de Romero, and Verticillium spp. and other soil fungi and plants materi- 1984, 1985). Cicadellid leafhoppers represent a large group als (Sutton et al., 1997). As a mycoparasite C. rosea was of insects important to agriculture, with approximately 22,000 species described worldwide (McKamey, 2002). The * Corresponding author. Fax: +54 221 423 2327. Cicadellinae is the largest subfamily of this group and has a E-mail address: [email protected] (A.V. Toledo). cosmopolitan distribution; many species of this group can 0022-2011/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2005.10.005 8 A.V. Toledo et al. / Journal of Invertebrate Pathology 92 (2006) 7–10 be abundant and cause considerable damage to crops as C. rosea obtained from insects, and to evaluate the patho- vectors of plant pathogens (Nielson, 1968). Sonesimia genicity of C. rosea by means of preliminary laboratory grossa Signoret (Cicadellidae: Cicadellinae, Cicadellini) bioassays against two species of hemipteran pests. occurs in Southern Brazil, Paraguay, Bolivia and in the Clonostachys rosea was isolated from adult females of provinces of Misiones and Tucumán in Argentina (Remes Oncometopia tucumana Schroder collected in March 2003 Lenicov et al., 1999). This species has been found to vector on Lantana camara L. (Fam. Verbenaceae) plants at Horco the bacterium Xylella fastidiosa wells in Brazilian citrus Molle, Tucumán province (26°46Ј50.1ЉS and 65°19Ј38.3ЉW, crops (Yamamoto et al., 2002). The genus Oncometopia Stål 703 m elevation), and from adult females of S. grossa col- (Cicadellidae: Cicadellinae) contains the largest number of lected in September 2003 on Eryngium sp. (Tourn) L. (Fam. species of any genus in the tribe Proconiini, and some are Apiaceae) plants from Colón, Entre Ríos province economically important (Young, 1968). For example, (31°51Ј15.1ЉS and 58°19Ј23.5ЉW, 25 m elevation). These Oncometopia orbona (Fabricius) is an important vector of sites are located in the Northwestern and Northeastern phony peach disease in the southern United States (Young, regions of Argentina, respectively. 1968). Oncometopia facialis is implicated in X. fastidiosa Infected insects covered by white cottony mycelia (Figs. transmission in citrus and coVee in Brazil, as the cause of 1A and B) and strongly Wxed to the plant substrate were citrus variegated chlorosis (CVC) (Lopes, 1999; Yamamoto collected in sterilized plastic containers and returned to the et al., 2002). The distribution of Oncometopia is from laboratory for isolation, identiWcation, and fungal charac- Northern United States to Brazil, Argentina, and Bolivia terization. (Young, 1968); in Argentina these insects are found in Tuc- Fungal cultures were obtained from monosporic isolates in umán and Misiones provinces (Remes Lenicov et al., 1999). the manner described by Lecuona (1996) and incubated for The objective of our study was to search for fungal patho- three days at 25°C on Sabouraud dextrose agar (SDA)+ gens of the Cicadellidae, to characterize two isolates of antibiotic:penicillin G 40,000 U/ml (Merck, Germany) and Fig. 1. Clonostachys rosea. (A and B) On Sonesimia grossa. (C) Primary conidiophores, phialides, and conidia. (D) Secondary conidiophores, phialides, and conidia. (E) Conidia. Scale bars: (A and B) 3 mm; (C) 15 m; (D) 11 m; (E) 10 m. A.V. Toledo et al. / Journal of Invertebrate Pathology 92 (2006) 7–10 9 streptomycin 80,000 U/ml (Parafarm, Argentina), on dark- distilled water). Cadavers were mounted in lactophenol + ness. Measurements of fungal structures (conidia, conidi- cotton blue (0.01% w/v) and infection with C. rosea was ogenous cells, and mycelia) from cultures were made to checked. enable speciWc identiWcation. Fungal material could not Our observations of morphological features of C. rosea be measured directly from host insects since only imma- agree with those previously reported by Schroers et al. ture, nonsporulating mycelium was evident on host (1999) and Schroers (2001). Colony diameters at 7 days cadavers. post-incubation (19–27 mm) are closer to those described Microscopic and macroscopic descriptions were made by Schroers et al. (1999). Primary conidiophores phialides from potato-dextrose agar (PDA) and oatmeal agar (OA) measured 20–43 £ 3 m (Fig. 1C), secondary ones 10– according to the standards used by Schroers et al. (1999) 17 £ 2–2.9 m (Fig. 1D) and conidia 4–11.6 £ 2–3.9 m and Schroers (2001). Mycelia were mounted in lactophenol (Fig. 1E). These are more similar to measurements reported cotton blue (0.01% w/v) and observed though phase con- by Schroers (2001). Both Argentinean isolates presented trast with an Olympus CH3 microscope. Fungal prepara- phialides shorter and narrower than those described by tions were photographed using a Nikon Optiphot Schroers et al. (1999). microscope equipped with diVerential interference contrast Mortality for O. tucumana reached 82.5% by 14 days (DIC) and Wtted with a Canon Power Shot A80 camera. after inoculation, but fungal infection was conWrmed for Infected insect hosts were photographed using an Olympus only 12.5% of the dead insects. We did observe phialides SZ–PT stereo microscope Wtted with a PM-C35B camera. and conidia growing out of dead hosts. For T. rubromargi- Clonostachys rosea isolates were deposited in the Myco- nata mortality was 45.5% after 14 days but fungal growth logical Collections of the Instituto de Botánica Spegazzini was observed only in 11.8% of dead insects. Some apprecia- (LPS; La Plata, Argentina), at the Centro de Estudios Para- ble mortality was registered in control insects (15–20%), but sitológicos y de Vectores (CEP; La Plata, Argentina), at the this might have been due to the fact that the insects were USDA-ARS Collection of Entomopathogenic Fungal Cul- Weld-collected and of mixed ages. tures (ARSEF; Ithaca, New York), and at the Centralbu- In conclusion, the present study provides the Wrst report reau voor Schimmelcultures (CBS; Utrecht, Netherlands). of C. rosea as an entomopathogenic fungus of O. tucumana The accession numbers of C. rosea from O. tucumana are and S. grossa (Hemiptera: Cicadellidae) extending our LPS 780, CEP 050, ARSEF 7200, and CBS 115882, and the knowledge of the occurrence and distribution