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 mycoparasite of fungi and nematodes and as saprobe in soils, but this fungus has not been reported previously to be entomopathogenic. Many species 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, Seifert, and Gams [formerly known as Gliocladium roseum Bainier; teleomorph: Bionectria ochroleuca (Schweinitz)] (Schroers et al., 1999) is known from temperate and tropical regions (Schroers, 2001) and is common in an extraordinary range of habitats in tropical, temperate, sub-arctic, and desert regions of the world. It has been reported from cultivated grassland and woodland, forest, heathland, freshwater, and coastal soils, particularly those of neutral to alkaline pH (Sutton et al., 1997). This fungus has been frequently associated with cysts of Heterodera spp., Globodera spp. and other nematodes in soil and with sclerotia of Sclerotina sclerotorium (Lib.) de Bary, Phymatotrichum omnivorum Duggar, Rhizoctonia solani Kühn, Botrytis spp., and Verticillium spp. and other soil fungi and plants materials (Sutton et al., 1997). As a mycoparasite C. rosea was *
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tested successfully as a biological control agent against diVerent fungal plant pathogens including S. sclerotorium (Lib.) de Bary (Ervio et al., 1994), Verticillium dahliae Kleb. (Keinath et al., 1991), and several species of Botrytis (James and Sutton, 1996). In Argentina the presence of Gliocladium roseum in soil was reported (Cabello and Arambarri, 2002) but this fungus has not been reported previously to be entomopathogenic. Previous reports of entomopathogenic fungi aVecting hemipteran insects in South America mostly involve cercopid pests of sugar cane in Brazil (Marques et al., 1981). The few records of entomopathogenic fungi from hemipterans in Argentina represent hosts in the families Coccidae, Aphididae, Cixiidae, and Aleyrodidae (Marchionatto, 1935; Toledo et al., 2004; Yasem de Romero, 1984, 1985). Cicadellid leafhoppers represent a large group of insects important to agriculture, with approximately 22,000 species described worldwide (McKamey, 2002). The Cicadellinae is the largest subfamily of this group and has a cosmopolitan distribution; many species of this group can
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A.V. Toledo et al. / Journal of Invertebrate Pathology 92 (2006) 7–10
be abundant and cause considerable damage to crops as vectors of plant pathogens (Nielson, 1968). Sonesimia grossa Signoret (Cicadellidae: Cicadellinae, Cicadellini) occurs in Southern Brazil, Paraguay, Bolivia and in the provinces of Misiones and Tucumán in Argentina (Remes Lenicov et al., 1999). This species has been found to vector the bacterium Xylella fastidiosa wells in Brazilian citrus crops (Yamamoto et al., 2002). The genus Oncometopia Stål (Cicadellidae: Cicadellinae) contains the largest number of species of any genus in the tribe Proconiini, and some are economically important (Young, 1968). For example, Oncometopia orbona (Fabricius) is an important vector of phony peach disease in the southern United States (Young, 1968). Oncometopia facialis is implicated in X. fastidiosa transmission in citrus and coVee in Brazil, as the cause of citrus variegated chlorosis (CVC) (Lopes, 1999; Yamamoto et al., 2002). The distribution of Oncometopia is from Northern United States to Brazil, Argentina, and Bolivia (Young, 1968); in Argentina these insects are found in Tucumán and Misiones provinces (Remes Lenicov et al., 1999). The objective of our study was to search for fungal pathogens of the Cicadellidae, to characterize two isolates of
C. rosea obtained from insects, and to evaluate the pathogenicity of C. rosea by means of preliminary laboratory bioassays against two species of hemipteran pests. Clonostachys rosea was isolated from adult females of Oncometopia tucumana Schroder collected in March 2003 on Lantana camara L. (Fam. Verbenaceae) plants at Horco Molle, Tucumán province (26°46⬘50.1⬙S and 65°19⬘38.3⬙W, 703 m elevation), and from adult females of S. grossa collected in September 2003 on Eryngium sp. (Tourn) L. (Fam. Apiaceae) plants from Colón, Entre Ríos province (31°51⬘15.1⬙S and 58°19⬘23.5⬙W, 25 m elevation). These sites are located in the Northwestern and Northeastern regions of Argentina, respectively. Infected insects covered by white cottony mycelia (Figs. 1A and B) and strongly Wxed to the plant substrate were collected in sterilized plastic containers and returned to the laboratory for isolation, identiWcation, and fungal characterization. Fungal cultures were obtained from monosporic isolates in the manner described by Lecuona (1996) and incubated for three days at 25 °C on Sabouraud dextrose agar (SDA) + 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
streptomycin 80,000 U/ml (Parafarm, Argentina), on darkness. Measurements of fungal structures (conidia, conidiogenous cells, and mycelia) from cultures were made to enable speciWc identiWcation. Fungal material could not be measured directly from host insects since only immature, nonsporulating mycelium was evident on host cadavers. Microscopic and macroscopic descriptions were made from potato-dextrose agar (PDA) and oatmeal agar (OA) according to the standards used by Schroers et al. (1999) and Schroers (2001). Mycelia were mounted in lactophenol cotton blue (0.01% w/v) and observed though phase contrast with an Olympus CH3 microscope. Fungal preparations were photographed using a Nikon Optiphot microscope equipped with diVerential interference contrast (DIC) and Wtted with a Canon Power Shot A80 camera. Infected insect hosts were photographed using an Olympus SZ–PT stereo microscope Wtted with a PM-C35B camera. Clonostachys rosea isolates were deposited in the Mycological Collections of the Instituto de Botánica Spegazzini (LPS; La Plata, Argentina), at the Centro de Estudios Parasitológicos y de Vectores (CEP; La Plata, Argentina), at the USDA-ARS Collection of Entomopathogenic Fungal Cultures (ARSEF; Ithaca, New York), and at the Centralbureau voor Schimmelcultures (CBS; Utrecht, Netherlands). The accession numbers of C. rosea from O. tucumana are LPS 780, CEP 050, ARSEF 7200, and CBS 115882, and the accession numbers of C. rosea isolated from S. grossa are LPS 785, CEP 066, and CBS 115883 at the four collections, respectively. Living adults of O. tucumana and Tapajosa rubromarginata Signoret (Hemiptera: Cicadellidae, Proconiini) were captured with nets and manual aspirators from Citrus limon (L.) Burman (Fam. Rutaceae) orchard sites and L. camara L. plants at Horco Molle, Tucumán, Argentina, during March 2004. Insects were reared and conditioned at the laboratory facilities of PROIMI, Tucumán, at 26 § 1 °C and quarantined for 10 days before using them for pathogenicity tests. After 10 days, 60 healthy adult insects of each species were selected for the assays. Forty individuals of each species were sprayed in groups of 10 with 600 l of a conidial suspension (in 0.01% Tween 20) of 6 £ 105 conidia/ml from a culture of C. rosea (originally isolated from O. tucumana). Conidia were harvested from 10-day-old cultures grown on PDA at 25 °C in darkness. Twenty insects (in two groups of 10) were used as control for each species, sprayed with 600 l of 0.01% Tween 20 (using a glass nozzle of 35 cc capacity). Insects were maintained in groups of 10 in 30£ 11 cm polyethylene terephthalate (PET) plastic jars, and fed with buds of C. limon and L. camara plants. Four replicates of each treatment were performed. Treated and control insects were maintained at 26 § 1 °C, 99% RH and a photoperiod of 12:12 h (light:dark). Cumulative mortality was recorded daily for 14 days. Dead insects were removed and deposited in high-humidity chambers (sterile petri dishes with Wlter paper dampened with sterile
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distilled water). Cadavers were mounted in lactophenol + cotton blue (0.01% w/v) and infection with C. rosea was checked. Our observations of morphological features of C. rosea agree with those previously reported by Schroers et al. (1999) and Schroers (2001). Colony diameters at 7 days post-incubation (19–27 mm) are closer to those described by Schroers et al. (1999). Primary conidiophores phialides measured 20–43 £ 3 m (Fig. 1C), secondary ones 10– 17 £ 2–2.9 m (Fig. 1D) and conidia 4–11.6 £ 2–3.9 m (Fig. 1E). These are more similar to measurements reported by Schroers (2001). Both Argentinean isolates presented phialides shorter and narrower than those described by Schroers et al. (1999). Mortality for O. tucumana reached 82.5% by 14 days after inoculation, but fungal infection was conWrmed for only 12.5% of the dead insects. We did observe phialides and conidia growing out of dead hosts. For T. rubromarginata mortality was 45.5% after 14 days but fungal growth was observed only in 11.8% of dead insects. Some appreciable mortality was registered in control insects (15–20%), but this might have been due to the fact that the insects were Weld-collected and of mixed ages. In conclusion, the present study provides the Wrst report of C. rosea as an entomopathogenic fungus of O. tucumana and S. grossa (Hemiptera: Cicadellidae) extending our knowledge of the occurrence and distribution of entomopathogenic fungi of Cicadellidae. Acknowledgments We thank Dr. Walter Gams for the identiWcation of C. rosea and Martijn ten Hoopen for additional information resources. References Cabello, M., Arambarri, A., 2002. Diversity in soil fungi undisturbed and disturbed Celtis tala and Scutia buxifolia forest in the eastern Buenos Aires province (Argentina). Microbiol. Res. 157 (2), 115–125. Ervio, L.R, Halkilahti, A.M., Pohjakallio, O., 1994. The survival in soil of Sclerotinia species and their ability to form mycelia. Adv. Front. Plant Sci. 8, 121–133. James, T.D.W., Sutton, J.C., 1996. Biological control of Botrytis leaf blight of onion by Gliocladium roseum applied as sprays and with fabric applications. Eur. J. Plant Pathol. 102, 265–275. Keinath, A.P, Fravel, D.R., Papavizas, G.C., 1991. Potential of Gliocladium roseum for biocontrol of Verticillium dahliae. Phytopathology 81, 644–648. Lecuona, R.E., 1996. Técnicas empleadas con hongos entomopatógenos. In: Lecuona, R.E. (Ed.), Microorganismos Patógenos Empleados en el Control Microbiano de Insectos Plaga. M. Mas, Buenos Aires, pp. 143–150. Lopes, J.R.S., 1999. Estudos com vetores de Xylella fastidiosa e implicacoes no manejo da clorose variegada dos cirtros. Laranja (Cordeirópolis) 20 (2), 329–344. McKamey, S.H., 2002. Leafhoppers of the world database: progress report. In: Hoch, H., Asche, M., Homberg, C., Kessling, P. (Eds.), 11th International Auchenorrhyncha Congress, August 5–9, 2002, Potsdam, Berlin, Germany, p. 85.
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