Infection byEntomophthoraSensu Stricto (Entomophthoromycota: Entomophthorales) inDiaphorina citri(Hemiptera: Liviidae) in Veracruz, Mexico

Abstract

The Asian citrus psyllid, Diaphorina citri Ku wayama is one of the most important pests of cit rus. It vectors the pathogenic bacterium causing the disease Huanglongbing (HLB) or Greening of citrus trees (Halbert & Manjunath 2004). Ento mopathogenic fungi are important biological con trol agents of insects and often cause epizootics that reduce host populations dramatically (Mc Coy et al. 1988). Among the entomopathogenic fungi infecting D. citri in the field are Beauveria bassiana (Bals.) Vuill. (Rivero-Aragon & Grillo Ravelo 2000; Yang et al. 2006), Hirsutella citrifor mis Speare, (Rivero-Aragon & Grillo-Ravelo 2000; Subandiyah et al. 2000; Meyer et al. 2007; Ca sique-Valdes & Sanchez-Pena 2010; Casique-Val des et al. 2011), Isaria fumosorosea Wize (Samson 1974; Subandiyah et al. 2000; Casique-Valdes & Sanchez-Pena 2010), Isaria javanica (Friederichs & Bally) Samson & Hywel-Jones (=Paecilomyces javanicus (Friederichs & Bally) Brown & Smith) (Yang et al. 2006), and Lecanicillium lecanii (Zimm.) Zare & W. Gams (=Acrostalagmus aphi dum Oudem., Verticillium lecanii (Zimm.) Viegas) (Rivero-Aragon & Grillo-Ravelo 2000; Yang et al. 2006; Casique-Valdes & Sanchez-Pena 2010). Au bert (1987) also lists Cladosporium nr. oxysporum Berk. & M.A. Curtis and the sooty mold, Capno dium citri Berk, and Desm. attacking!), citri, but the pathogenicities of these 2 usually saprophytic fungi are not clear. In a survey of entomopathogenic fungi of D. citri, about 600 plants of Citrus spp. (Persian lime, sweet orange and tangerine) and orange jessamine (Murraya paniculata (L.) Jack) were examined for dead infected insects on foliage on 9-13 Oct 2012, across 90 km in Veracruz state, Mexico. The area (described below) includes the main production zone of Persian lime in Mexico (Sanchez-Torres et al. 2011). We also collected live D. citri adults in our search for fungal infections, On 12 October 2012, live adults were collected on M. paniculata, at the localities of Ixtacuaco, La Guadalupe, San Andres, San Pedro, Totomoxtle and Venustiano Carranza, in the municipali ties of Papantla, San Rafael, and Tlapacoyan in north-central Veracruz state (highway distance: aprox. 90 km). At Ixtacuaco, insects were col lected from sweet orange (Citrus x sinensis (L.) Osbeck) also. Psyllids (n = 210) were placed in brown paper bags (38.5 x 16 cm) by shaking the insects off infested branches into the bags. The opening of each bag was folded and sealed with masking tape. The puffed bags containing D. citri adults were carefully placed in large plastic bags, moistened slightly and held at 25-30 °C during transport to the laboratory in Saltillo. In this way live healthy insects can be maintained in bags for a few days. We expected to find mainly infections by H. citriformis, a slow-growing fungus that is commonly found attacking D. citri adults in the Gulf Coast region of Mexico (Casique-Valdes et al. 2011). After 3 days of keeping insects in paper bags, dead (n = 203) and live in = 7) insects were placed in petri dishes at room temperature, under dif fuse fluorescent light (10:14 h L:D) with a piece of wet cotton to maintain high humidity. Insects were observed daily to detect fungal development. One dead D. citri showing the typical signs of in fection by an Entomophthora species (Entomoph thorales: Entomophthoraceae) was observed in a paper bag on day 3 after collection; on day 6, there were 2 additional similar dead insects (all from Venustiano Carranza, Papantla) in petri dishes (total 1.4% infection) (Fig. 1A-C). These insects had swollen abdomens, that angled away from the substrate; wings spread latero-dorsally and raised above the abdomen, and conspicuous masses of glutinous hyphae and conidiophores on the abdomen and thorax, similar to the well known fungus Entomophthora muscae (Cohn) Fres., infecting flies (Diptera: Muscidae) (Mullens 1990; Krasnoff et al. 1995). All infected insects were collected at Venustiano Carranza, Papantla: N 20° 27' 18 W 97° 17' 06. No similarly killed insects were detected in the field survey. Fungi in the Entomophthorales forcibly eject their prima ry conidia (PC) (Keller 2007). Thus, slides were placed in the petri dish under infected insects in an attempt to collect discharged conidia. Two of these insects became rapidly overgrown by saprophytic fungi (mainly Cladosporium sp.; Capnodiales: Davidiellaceae), which completely ob scured the original pathogen. In petri dishes, some dead adults in typical Entomophthora-infected pos ture were also overgrown by fungal saprophytes. This rapid secondary fungal growth is frequently observed on other insects killed by Entomophtho rales (Sanchez-Pena 1993). Thus the percent infec tion by Entomophthora is possibly underestimated.