Infection by Entomophthora Sensu Stricto (Entomophthoromycota: Entomophthorales) in Diaphorina citri (Hemiptera: Liviidae) in Veracruz, Mexico Author(s): L. Guizar-Guzman and S. R. Sanchez-Peña Source: Florida Entomologist, 96(2):624-627. 2013. Published By: Florida Entomological Society DOI: http://dx.doi.org/10.1653/024.096.0230 URL: http://www.bioone.org/doi/full/10.1653/024.096.0230 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. 624 Florida Entomologist 96(2) June 2013 INFECTION BY ENTOMOPHTHORA SENSU STRICTO (ENTOMOPHTHOROMYCOTA: ENTOMOPHTHORALES) IN DIAPHORINA CITRI (HEMIPTERA: LIVIIDAE) IN VERACRUZ, MEXICO L. GUIZAR-GUZMAN AND S. R. SANCHEZ-PEÑA* Departamento de Parasitología, Universidad Autonoma Agraria Antonio Narro, Saltillo, Coahuila, 25315, Mexico *Corresponding author; E-mail: [email protected] The Asian citrus psyllid, Diaphorina citri Ku- insects off infested branches into the bags. The wayama is one of the most important pests of cit- opening of each bag was folded and sealed with rus. It vectors the pathogenic bacterium causing masking tape. The puffed bags containing D. citri the disease Huanglongbing (HLB) or Greening of adults were carefully placed in large plastic bags, citrus trees (Halbert & Manjunath 2004). Ento- moistened slightly and held at 25-30 °C during mopathogenic fungi are important biological con- transport to the laboratory in Saltillo. In this way trol agents of insects and often cause epizootics live healthy insects can be maintained in bags for that reduce host populations dramatically (Mc- a few days. We expected to find mainly infections Coy et al. 1988). Among the entomopathogenic by H. citriformis, a slow-growing fungus that is fungi infecting D. citri in the field are Beauveria commonly found attacking D. citri adults in the bassiana (Bals.) Vuill. (Rivero-Aragon & Grillo- Gulf Coast region of Mexico (Casique-Valdes et Ravelo 2000; Yang et al. 2006), Hirsutella citrifor- al. 2011). mis Speare, (Rivero-Aragon & Grillo-Ravelo 2000; After 3 days of keeping insects in paper bags, Subandiyah et al. 2000; Meyer et al. 2007; Ca- dead (n = 203) and live (n = 7) insects were placed sique-Valdes & Sanchez-Peña 2010; Casique-Val- in petri dishes at room temperature, under dif- des et al. 2011), Isaria fumosorosea Wize (Samson fuse fluorescent light (10:14 h L:D) with a piece 1974; Subandiyah et al. 2000; Casique-Valdes & of wet cotton to maintain high humidity. Insects Sanchez-Peña 2010), Isaria javanica (Friederichs were observed daily to detect fungal development. & Bally) Samson & Hywel-Jones (=Paecilomyces One dead D. citri showing the typical signs of in- javanicus (Friederichs & Bally) Brown & Smith) fection by an Entomophthora species (Entomoph- (Yang et al. 2006), and Lecanicillium lecanii thorales: Entomophthoraceae) was observed in (Zimm.) Zare & W. Gams (=Acrostalagmus aphi- a paper bag on day 3 after collection; on day 6, dum Oudem., Verticillium lecanii (Zimm.) Viegas) there were 2 additional similar dead insects (all (Rivero-Aragon & Grillo-Ravelo 2000; Yang et al. from Venustiano Carranza, Papantla) in petri 2006; Casique-Valdes & Sanchez-Peña 2010). Au- dishes (total 1.4% infection) (Fig. 1A-C). These bert (1987) also lists Cladosporium nr. oxysporum insects had swollen abdomens, that angled away Berk. & M.A. Curtis and the sooty mold, Capno- from the substrate; wings spread latero-dorsally dium citri Berk. and Desm. attacking D. citri, but and raised above the abdomen, and conspicuous the pathogenicities of these 2 usually saprophytic masses of glutinous hyphae and conidiophores fungi are not clear. on the abdomen and thorax, similar to the well- In a survey of entomopathogenic fungi of D. known fungus Entomophthora muscae (Cohn) citri, about 600 plants of Citrus spp. (Persian Fres., infecting flies (Diptera: Muscidae) (Mullens lime, sweet orange and tangerine) and orange 1990; Krasnoff et al. 1995). All infected insects jessamine (Murraya paniculata (L.) Jack) were were collected at Venustiano Carranza, Papantla: examined for dead infected insects on foliage on N 20° 27’ 18” W 97° 17’ 06”. No similarly killed 9-13 Oct 2012, across 90 km in Veracruz state, insects were detected in the field survey. Fungi in Mexico. The area (described below) includes the the Entomophthorales forcibly eject their prima- main production zone of Persian lime in Mexico ry conidia (PC) (Keller 2007). Thus, slides were (Sánchez-Torres et al. 2011). We also collected live placed in the petri dish under infected insects in D. citri adults in our search for fungal infections, an attempt to collect discharged conidia. On 12 October 2012, live adults were collected on Two of these insects became rapidly overgrown M. paniculata, at the localities of Ixtacuaco, La by saprophytic fungi (mainly Cladosporium sp.; Guadalupe, San Andres, San Pedro, Totomoxtle Capnodiales: Davidiellaceae), which completely ob- and Venustiano Carranza, in the municipali- scured the original pathogen. In petri dishes, some ties of Papantla, San Rafael, and Tlapacoyan in dead adults in typical Entomophthora-infected pos- north-central Veracruz state (highway distance: ture were also overgrown by fungal saprophytes. aprox. 90 km). At Ixtacuaco, insects were col- This rapid secondary fungal growth is frequently lected from sweet orange (Citrus × sinensis (L.) observed on other insects killed by Entomophtho- Osbeck) also. Psyllids (n = 210) were placed in rales (Sanchez-Peña 1993). Thus the percent infec- brown paper bags (38.5 × 16 cm) by shaking the tion by Entomophthora is possibly underestimated. Scientific Notes 625 Fig. 1. Entomophthora sp. on Diaphorina citri. Figs. 1A-1C. Dead D. citri adults with typical position of Ento- mophthora-infected insects (see text); the same insect is in A and B. Figs. 1D-1F. Conidia of Entomophthora sp. Fig. 1D. Empty or dehydrated primary conidium (22.7 x 16.6 μm) with typical shape of Entomophthora species. Fig. 1E. Empty primary conidium (*) and production of secondary conidium (14.1 × 11.6 μm) with small germ tube. Fig. 1F. Empty campanulate primary conidium (*) and production of secondary conidium. The halos in D-E (arrows) formed upon conidial attachment to hard surfaces. Bar = 1mm (Figs. 1A-1C); Bar =11 μm (Fig. 1D), Bar = 7 μm (Figs. 1E-1F). Unfortunately no PC landed on the slides We also observed the mucous cytoplasm halo placed under infected insects for microscopical ex- around the discharged PC, typical of Entomoph- amination; they landed on the bottom of the petri thora (Fig. 1D-F) (Eilenberg et al. 1986). Most PC dish. Pictures of unstained conidia shown were were empty (had germinated) when detected, and taken from the dish bottom under the compound no nuclei were observed; they also lysed a few hours microscope. Most PC observed had germinated, later. Thus it is not possible to assign this fungus to producing long germ tubes and/or secondary co- a species with certainty since number and size of nidia (SC) (Humber 1981; Keller et al. 2007). The conidial nuclei are essential criteria for the identi- pictures herein are either of PC emptied of cyto- fication of Entomophthora species (Humber 1981; plasmic contents, or perhaps slightly dehydrated, Keller 2007). Entomophthorales have been occa- and measured with the software Axioma Vision sionally reported to attack psyllids in temperate 4.5 (n = 4). Average measurements are provided. regions of Europe (Prischepa et al. 2011; Jankevica Both PC and SC observed are typical of the genus 2004) and the tropics of South Asia (see review in Entomophthora Cohn in the strict sense (Humber Villacarlos & Wilding 1994). In the New World 1981). PC are campanulate, with a flat base, sur- tropics, Alves et al. (2009) reported Zoophthora rounded with a halo of mucilage upon discharge radicans on psyllids (Gyropsylla spegazziniana) (Fig. 1 D-F), with prominent apical point and in Brazil; however, the globose spores and coarse broad basal papilla, 16.6 × 22.7 μm; SC were glo- hyphae shown do not correspond to the capillary bose, with broad basal papilla, 14.1 × 11.6 μm, conidia characteristic of Zoophthora and perhaps borne apically on a short conidiophore arising describe a mixed infection with the fungus, Bat- from PC (Fig. 2). This is similar to the spore size koa, or other fungus. Felix-Alvarez et al. (2003) (PC: 11.5-20.5 × 9-18.5 μm; SC: 11.5-16 × 9-11.5 mentioned infections by Entomophthorales on D. μm) of Entomophthora philippinensis Villacarlos citri in Matanzas, Cuba, but provide no further de- & Wilding infecting the leucaena psyllid, Hetero- scription of the fungi involved. psylla cubana Crawford, in the Philippines (Vil- Most Entomophthora species are restricted lacarlos & Wilding 1994). to a narrow range of closely related host species 626 Florida Entomologist 96(2) June 2013 (Geden et al. 1993). It is possible that in the New colectados de limonaria, Murraya paniculata, en World this fungus host-jumped to the exotic D. el pueblo de Venustiano Carranza, municipio de citri from another (psyllid?) host. This was pos- Papantla. Diaphorina citri es un nuevo registro sibly the case for E. philippinensis on H. cubana, de hospedero para este género de hongo entomo- which is exotic in the Philippines (Villacarlos & patógeno. Wilding 1994).