Biological Control xxx (2014) xxx–xxx Contents lists available at ScienceDirect Biological Control journal homepage: www.elsevier.com/locate/ybcon Entomopathogenic fungi as biological control agents for the vector of the laurel wilt disease, the redbay ambrosia beetle, Xyleborus glabratus (Coleoptera: Curculionidae) ⇑ D. Carrillo a, , C.A. Dunlap b, P.B. Avery c, J. Navarrete a, R.E. Duncan a, M.A. Jackson b, R.W. Behle b, R.D. Cave c, J. Crane a, A.P. Rooney b, J.E. Peña a a University of Florida, Tropical Research and Education Center, Homestead, FL 33031, United States b U.S. Department of Agriculture, Agricultural Research Service, Crop Bioprotection Research Unit, Peoria, IL 61604, United States c University of Florida, Indian River Res. and Education Center, Fort Pierce, FL 34945, United States highlights graphical abstract Four fungal strains can infect and kill Xyleborus glabratus, vector of laurel wilt. Spores adhere to beetles walking on fungus-treated wood. Initial tunneling activity is not inhibited but beetles die inside the tunnels. Beauveria bassiana kills Xyleborus glabratus faster than other tested fungi. Fungal pathogens are potential biological control agents of Xyleborus glabratus. article info abstract Article history: The redbay ambrosia beetle (RAB), Xyleborus glabratus, is a wood-boring insect that vectors the fungal Received 16 December 2013 pathogen, Raffaelea lauricola, which causes laurel wilt, a lethal disease of avocado. The objective of this Accepted 28 October 2014 study was to determine the susceptibility of RAB to infection and subsequent death by exposure to three Available online xxxx commercial strains of entomopathogenic fungi [two strains of Isaria fumosorosea (Ifr 3581 and PFR), and strain GHA of Beauveria bassiana]. RAB females were dipped in fungal spore solutions and their median Keywords: survivorship times (MST) determined. Contact with any of the biopesticides resulted in death of all Avocado RAB females. MSTs of RAB females ranged from 3 days (B. bassiana) to 5 days (I. fumosorosea PFR). B. bas- Lauraceae siana killed RAB females faster, followed by Ifr 3581 and PFR. RAB females dipped in B. bassiana suspen- Xyleborus glabratus Invasive species sions had the highest number of viable spores attached to their bodies, followed by Ifr 3581. Beetles Beauveria bassiana dipped in PFR suspension had significantly less viable spores attached to their bodies. No significant dif- Isaria fumosorosea ferences were observed in the mortality of beetles exposed to entomopathogenic fungi by dipping in a fungal suspension or walking on treated avocado bolts. Beetles bored into the logs and constructed gal- leries, but they were found dead inside the galleries a few days after exposure to the entomopathogens. Entomopathogenic fungal infection in dead beetles was confirmed through molecular techniques. This is the first study to demonstrate that entomopathogenic fungi are potential biological control agents against RAB. Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Fax: +1 305 2467003. E-mail address: dancar@ufl.edu (D. Carrillo). http://dx.doi.org/10.1016/j.biocontrol.2014.10.009 1049-9644/Ó 2014 Elsevier Inc. All rights reserved. Please cite this article in press as: Carrillo, D., et al. Entomopathogenic fungi as biological control agents for the vector of the laurel wilt disease, the redbay ambrosia beetle, Xyleborus glabratus (Coleoptera: Curculionidae). Biological Control (2014), http://dx.doi.org/10.1016/j.biocontrol.2014.10.009 2 D. Carrillo et al. / Biological Control xxx (2014) xxx–xxx 1. Introduction entomopathogenic inoculation of adult females could result in reduced beetle populations in the field (Castrillo et al., 2011). Avocado is Florida’s second most important fruit crop after cit- The objective of this study was to provide baseline data on the rus, with an economic impact of $54 million annually (Evans et al., potential use of three formulations of different entomopathogenic 2010). The redbay ambrosia beetle (RAB), Xyleborus glabratus Eich- fungi to cause RAB mortality. The specific objectives were to: (1) hoff (Coleoptera: Curculionidae: Scolytinae), represents a major determine the median survivorship times of RABs exposed to the threat to the avocado industry. This beetle vectors the fungal path- entomopathogenic fungi; (2) determine the number of viable ogen, Raffaelea lauricola T.C. Harr., Fraedrich & Aghayeva spores attached to beetles exposed to fungal suspension of the (Harrington et al., 2008), which causes laurel wilt (LW), a lethal pathogens, and (3) compare infection rates between beetles dipped disease of trees in the family Lauraceae, including avocado, Persea in fungal solutions and beetles interacting with avocado wood americana Mill. (Crane et al., 2008; Mayfield et al., 2008). RAB was treated with fungal solutions. first detected during 2002 in southeast Georgia (Rabaglia et al., 2006), and the rapid spread of the vector has resulted in severe 2. Materials and methods mortality of several environmentally important native ornamental and forest tree species such as redbay [Persea borbonia (L.) Spreng.] 2.1. Beetle collection and rearing and swamp bay [Persea palustris (Raf.) Sarg.] in South Carolina, Georgia, and Florida (Fraedrich et al., 2008; Hanula et al., 2008). Wilting trees from a natural area in Miami-Dade County In 2007, LW eliminated a homeowner’s backyard avocado tree in (25°43037.9600N–80°28036.1600W) with abundant swamp bay trees northeastern Florida (Mayfield et al., 2008). A rapid progression infested with RAB were cut and measured, and wood >10 cm £ of this disease was observed during 2010 in an abandoned com- was stored inside emergence chambers (0.17 m3, Brute container mercial avocado grove located in Highlands County (central Flor- 2643-60, RubbermaidÒ, with a mason jar placed in a hole in one ida) (J.E. Peña/D. Carrillo, personal observation). of its sides to collect emerging beetles), transported to, and held Over 95% of avocados in Florida are grown in Miami-Dade at the Containment Facility of the University of Florida, Tropical County in the southeastern coast of the peninsula. During January Research and Education Center (TREC), Homestead, Florida. Wood 2010, the first RAB was detected in a natural area in Miami-Dade was kept at 14:10 h L:D, 80% RH, and 25 °C. RAB females that County about 19 km north of the commercial avocado production emerged from the wood were collected daily and placed inside area (Ploetz et al., 2011). By February 2011, the first native Petri dishes (5 cm £) provided with a moistened filter paper. Bee- swamp bay trees were confirmed positive for laurel wilt, and tles were kept inside a refrigerator at 12 °C before conducting the by February 2012 the disease was detected in a commercial avo- bioassays. All RAB adult females used in the bioassays were used cado grove (FDACS, 2012). To date, over 3000 avocado trees in 52 within 2 days of collection (Carrillo et al., 2014). groves have been destroyed due to this insect–disease complex (J. Crane, personal observation), and research in other parts of 2.2. Fungi the southeastern US and north Florida has demonstrated that anywhere this insect–disease complex is left unchallenged for Two strains of I. fumosorosea (ARSEF 3581 = Ifr 3581, a TX strain 18–24 months, 95% of the host (susceptible) trees are killed. isolated from Bemisia tabaci (Gennadius), and PFR 97Ò 20% WDG, an However, at this initial stage of the RAB’s invasion of the com- Apopka, FL commercial strain isolated from a mealybug, Phenacoc- mercial avocado growing area, vector densities are low (Carrillo cus solani Ferris (Certis USA, Columbia, MD)), and one commercial et al., 2012), which indicates that there is an urgent need to strain of B. bassiana GHA (Balsamo) Vuillemin (BotaniGardÒ quickly mitigate the spread of this problem to reduce its impact ES, Laverlam International, Butte, MT) were tested. The two on the entire Florida avocado industry. Current management I. fumosorosea strains were blastospore powder formulations in strategies for RAB have focused on evaluating chemical and vacuum-sealed bags containing 109 colony forming units (CFU)/g, cultural control practices. Several contact insecticides have been and B. bassiana was an emusifiable suspension. All products were tested and, so far, their effectiveness is limited (Peña et al., commercial formulations and were refrigerated at 4 °C until use. 2011; Carrillo et al., 2013). Growers are cutting, chipping, and All conidial or blastospore counts were made using a plastic burning infested/infected trees with the hope of reducing beetle disposable C-Chip Neubauer Improved hemocytometer (Incyto populations and the spread of the pathogen. One technique not yet DHC-No. 1, Chungnam, Korea) and viewed under a Leica tested is the use of entomopathogenic fungi, which has produced DM500 Brightfield (Leica Microsystems, Wetzlar, Germany) at good results with other Scolytinae. Several fungal entomopatho- 400Â. Suspensions were diluted to 2.4 Â 106 blastospores or gens are associated with scolytine beetles (Popa et al., 2011). conidia/mL. Changing focal depth with the microscope was Among them, Beauveria bassiana (Balsamo), Metarhizium brunne- required to distinguish B. bassiana spores, which have conspicuous um (Metschnikoff) Sorokin (formerly Metarhizium anisopliae cell walls, from droplets formed after adding water to the emulsified (Metschnikoff) Sorokin), Isaria amoenerosea P. Hennings, Isaria suspension in the BotaniGard formulation. Viability for all farinosa Holm ex Gray, and Isaria fumosorosea Wize have shown strains was determined by spreading 100 lL of suspension of 106 efficacy against coleopterans and some scolytines. B. bassiana blastospores or conidia/mL on three separate Petri dishes contain- has been used against the coffee berry borer, Hypothenemus ham- ing potato dextrose agar (PDA) and incubating them at 25 °C for pei (Ferrari) (Bustillo et al., 1998; Posada and Vega, 2005). More- 16–24 h under a 14:10 h L:D photoperiod. Germination was over, the impact of entomopathogens on scolytinae broods determined by counting 200 spores/plate (100 spores/area) with a appears to cause significant reductions in survival.