BIOLOGICAL AND MICROBIAL CONTROL Efficacy of the Biofumigant Fungus Muscodor albus (Ascomycota: Xylariales) for Control of Codling Moth (Lepidoptera: Tortricidae) in Simulated Storage Conditions 1 L. A. LACEY, D. R. HORTON, D. C. JONES, H. L. HEADRICK, AND L. G. NEVEN USDAÐARS, Yakima Agricultural Research Laboratory, 5230 Konnowac Pass Road, Wapato, WA 98951 J. Econ. Entomol. 102(1): 43Ð49 (2009) ABSTRACT Codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), a serious pest of pome fruit, is a threat to exportation of apples (Malus spp.) because of the possibility of shipping infested fruit. The need for alternatives to fumigants such as methyl bromide for quarantine security of exported fruit has encouraged the development of effective fumigants with reduced side effects. The endophytic fungus Muscodor albus Worapong, Strobel and Hess (Ascomycota: Xylariales) produces volatile compounds that are biocidal for several pest organisms, including plant pathogens and insect pests. The objectives of our research were to determine the effects of M. albus volatile organic compounds (VOCs) on codling moth adults, neonate larvae, larvae in infested apples, and diapausing cocooned larvae in simulated storage conditions. Fumigation of adult codling moth with VOCs produced by M. albus for 3 d and incubating in fresh air for 24 h at 25ЊC resulted in 81% corrected mortality. Four- and 5-d exposures resulted in higher mortality (84 and 100%, respectively), but control mortality was also high due to the short life span of the moths. Exposure of neonate larvae to VOCs for3donapples and incubating for 7 d resulted in 86% corrected mortality. Treated larvae were predominantly Þrst instars, whereas 85% of control larvae developed to second and third instars. Exposure of apples that had been infested for 5 d, fumigated with M. albus VOCs for 3 d, and incubated as described above resulted in 71% corrected larval mortality. Exposure of diapausing cocooned codling moth larvae to VOCs for 7 or 14 d resulted in 31 and 100% mortality, respectively, with negligible control mortality. Our data on treatment of several stages of codling moth with M. albus VOCs indicate that the fungus could provide an alternative to broad spectrum chemical fumigants for codling moth control in storage and contribute to the systems approach to achieve quarantine security of exported apples. KEY WORDS microbial control, apple exportation, postharvest, quarantine treatments Codling moth, Cydia pomonella L. (Lepidoptera: Tor- Insecticide resistance in codling moth is one of the tricidae), is the key insect pest of apple (Malus spp.) factors responsible for survival of larvae within and in most regions of the world where apple and other emerging from fruit (Reyes et al. 2007). Exportation of pome fruits are grown (Barnes 1991). The following apples and other fruit from the United States has been life cycle of codling moth is summarized from Barnes threatened by the presence and possibility of infested (1991) and Beers et al. (1993). Diapausing larvae over- fruit and diapausing larvae in cartons and shipping winter in cocoons in cryptic habitats (under loose containers (NWHC 2008). Fumigation with broad- bark, leaf liter at the base of trees, in nearby woodpiles spectrum chemicals has been the principal method for and fruit bins). In the spring, they pupate and emerge protection of fruit exported to certain countries (Fol- as adults soon afterward. Eggs are deposited on foliage lett and Neven 2006). Although efÞcacious, some of or fruit, and neonate larvae bore into fruit and remain these fumigants, such as methyl bromide, have serious there until fully grown Þfth instars exit and search for environmental consequences (Yagi et al. 1995, cocooning sites. In summer generations, larvae pupate Thomas 1996, UNEP 2008). The need for alternatives soon after constructing cocoons and emerge as second to these chemicals has encouraged the search for and generation adults. A small percentage of Þrst gener- development of effective fumigants with reduced side ation larvae may enter diapause, but diapause usually effects. begins in subsequent generations in late summer or The fungus Muscodor albus Worapong, Strobel and early fall. There are one to four generations per year Hess (Ascomycota: Xylariales) is an endophyte of depending on climatic conditions. cinnamon, Cinnamomum zeylanicum Breyne (Laura- ceae), with biofumigant activity (Strobel et al. 2001, 1 Corresponding author, e-mail: [email protected]. Worapong et al. 2001). The biocidal properties of the 44 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 102, no. 1 fungus are due to the production of a mixture of soon after closing the chambers. The light, tempera- volatile organic compounds (VOCs) (alcohols, esters, ture, and %RH in the walk-in incubator used for hold- ketones, acids, and lipids) (Strobel et al. 2001). These ing adult moths and infested apples after exposure to VOCs are lethal for a variety of plant pathogens, rot- M. albus was 16:8 (L:D) h, 25 Ϯ 0.5ЊC, and 45.8%RH, causing organisms, plant parasitic nematodes, and in- respectively. After each exposure period, oxygen (O2) sects (Strobel et al. 2001, Mercier and Jime´nez 2004, and carbon dioxide (CO2) were measured with an Mercier and Smilanick 2005, Strobel 2006, Lacey et al. O2/CO2 meter (PaciÞc CA System Techni-Systems, 2008, Riga et al. 2008). An artiÞcial mixture of VOCs Chelan WA). A 180-cm clear reinforced PVC tube was that mimics those naturally produced by M. albus is connected to the exhaust outlet of the chambers and comparable to that produced by the fungus for control to the intake of the meter. The contents of the cham- of plant and human pathogenic fungi and bacteria ber were drawn through the meter for 5 min until (Strobel et al. 2001, Strobel 2006, Grimme et al. 2007). readings had stabilized. Mean ambient air was 20.6% However, the individual VOCs have variable activity O2 and 0.3% CO2. against different organisms (Strobel et al. 2001, Ramin Adult Mortality. Moths used for experiments had et al. 2005). The speciÞc compounds that are respon- emerged within 24 h before testing. For each repli- sible for insecticidal activity are not yet known. There- cated test, Ϸ20 moths (50:50 mixture of males and fore, our tests were conducted with the naturally pro- females) were placed in each of thirty 0.47-liter cups. duced VOCs of M. albus. A 6.4-cm diameter hole cut in the lid and covered with Control of codling moth adults and larvae with VOCs polyester mesh (Econet-T) provided ventilation. Five in cartons and shipping containers would reduce the containers with moths were placed in each of three threat of accidental exportation of codling moth. The chambers used for controls and three for M. albus objectives of this study were to determine the effects of treatments. Each cup was provisioned with 15% honey M. albus VOCs on codling moth adults, neonate larvae on water in 2 ml of Micro-Eppendorf tubes with the lids apples, early instars within apples, and diapausing co- removed and a 3.8-cm piece of cotton inserted into the cooned larvae in simulated storage conditions. tube. The tubes were taped to the side of each cup. Just before the chambers were sealed, a cup with 30 g of hydrated rye grain was placed in each of the three Materials and Methods controls. A cup containing 30 g of hydrated M. albus Source of Adult and Larval Codling Moth. Codling mycelium on rye grain was placed in the remaining moth adults, eggs, and diapause-destined cocooned lar- three chambers. Moths were left in the chambers for vae were obtained from a colony maintained at the 3, 4, or 5 d. After the desired exposure period, mortality Yakima Agricultural Research Laboratory by using the was assessed at 0, 24, and 48 h postexposure. Moths rearing system and artiÞcial medium described by Toba were kept in a walk-in incubator at 25ЊC during the and Howell (1991) and Hansen and Anderson (2006). postexposure intervals. Substantial growth of M. albus Origin and Activation of M. albus. Samples of a on the rye grain was observed after the exposure desiccated M. albus mycelium on rye (Secale spp.) periods. Five replicated tests were conducted for each grain were obtained from AgraQuest (Davis, CA) and exposure period on Þve separate dates. stored at 4ЊC until used. Before testing, aliquots of 30 g Neonate Larval Mortality, Apple Damage, and In- of the formulation were placed in 0.47-liter plastic star Distribution. Neonate codling moth larvae were cups and activated by adding 30 ml of sterile deionized exposed to M. albus VOCs on apples in 0.47 plastic cups water. The cups were then sealed with a lid and placed in the chambers described above. Ten neonate codling in an incubator at 25ЊC for 24 h before use. Cups with moth Ͻ4 h old were placed on an unwaxed Fuji apple 30 g of autoclaved rye grain and 30 ml of sterile deion- (Ϸ7.4 cm in diameter and 187.6 g per apple) in each ized water were prepared in the same manner and of 10 ventilated cups. Five cups were placed in each used for control chambers. chamber along with a sixth container with either 30 g Bioassay Procedure. After incubation of M. albus of hydrated rye grain or 30 g of M. albus mycelium on and control cups as described above, exposures of rye grain that had been prepared as described above. codling moth were made in 28.3-liter hermetically The chambers were then hermetically sealed for 72 h. sealed Þberglass chambers. For each assay, a container After exposure, the cups were removed from the with hydrated M. albus was uncovered and immedi- chambers and incubated for 7 d at 25.8 Ϯ 1.4ЊC after ately placed in treatment chambers.