Crop Protection 28 (2009) 333–337 Contents lists available at ScienceDirect Crop Protection journal homepage: www.elsevier.com/locate/cropro Pathogenicity of Isaria sp. (Hypocreales: Clavicipitaceae) against the sweet potato whitefly B biotype, Bemisia tabaci (Hemiptera: Aleyrodidae)q H. Enrique Cabanillas*, Walker A. Jones 1 USDA-ARS, Kika de la Garza Subtropical Agricultural Research Center, Beneficial Insects Research Unit, 2413 E. Hwy. 83, Weslaco, TX 78596, USA article info abstract Article history: The pathogenicity of a naturally occurring entomopathogenic fungus, Isaria sp., found during natural Received 13 May 2008 epizootics on whiteflies in the Lower Rio Grande Valley of Texas, against the sweet potato whitefly, Received in revised form Bemisia tabaci (Gennadius) biotype B, was tested under laboratory conditions (27 C, 70% RH and 26 November 2008 a photoperiod of 14:10 h light:dark). Exposure of second-, third- and fourth-instar nymphs to 20, 200, Accepted 28 November 2008 and 1000 spores/mm2, on sweet potato leaves resulted in insect mortality. Median lethal concentrations for second-instar nymphs (72–118 spores/mm2) were similar to those for third-instar nymphs Keywords: (101–170 spores/mm2), which were significantly more susceptible than fourth-instar nymphs Biological control 2 Entomopathogenic fungi (166–295 spores/mm ). The mean time to death was less for second instars (3 days) than for third instars 2 Whitefly (4 days) when exposed to 1000 spores/mm . Mycosis in adult whiteflies became evident after delayed infections of sweet potato whitefly caused by this fungus. These results indicate that Isaria sp. is path- ogenic to B. tabaci nymphs, and to adults through delayed infections caused by this fungus. Its low lethal concentrations and high virulence against B. tabaci make this fungus a promising biocontrol agent to control whitefly. Published by Elsevier Ltd. 1. Introduction 1985; Horowitz and Ishaaya, 1996). Because of the negative impact of pesticides on natural enemies of the whitefly, and toxic effects on The sweet potato whitefly, Bemisia tabaci (Gennadius) biotype B, non-target organisms, including humans and livestock, sound has become the world’s most damaging whitefly pest species; it is alternative means of control as part of integrated pest management a major pest of economically important crops worldwide, especially are needed (Kunimi, 2007). in warm climates (Cock, 1993; Bellows et al., 1994; De Barro et al., Entomopathogenic fungi (EPF) have been recognized as impor- 2005). For example, it is considered as an economically important tant biocontrol agents of Aleyrodidae pests (Fransen, 1990; Lacey pest of cotton and other vegetable crops in California and Arizona. et al., 1996; Wraight et al., 1997). Unlike other biocontrol agents, In addition, it vectors economically important plant viruses (De EPF infect by penetrating the external insect cuticle and need not be Barro et al., 2005). The recent discovery of the Q biotype of B. tabaci, ingested to initiate disease. They continue to offer the only a highly insecticide-resistant biotype in the United States and biological control option involving entomopathogens against plant- abroad, presents greater challenges to control this insect pest than sucking insects (Lacey and Goettel, 1995). Isaria sp. is an unde- the introduction of the B biotype in the 1980s (Brown et al., 1995; scribed entomopathogenic fungus, which was found causing Horowitz et al., 2003a,b; Chu et al., 2006). natural epizootics in the sweet potato whitefly in the Lower Rio The heavy use of chemical pesticides to control the sweet potato Grande Valley of Texas in September 2001 [Agricultural Research, whitefly is not always effective, as this insect has the ability to May–June 2007 issue, vol. 55, No. 5, p. 11. Published by the Agri- develop resistance to a wide range of pesticides (Prabhaker et al., cultural Research Service (ARS), U.S. Department of Agriculture – USDA]. Since then, it has periodically killed whiteflies at the ARS insect-rearing facilities in Weslaco, Texas. This newly discovered q Mention of trade names or commercial products in this publication is solely for pathogen seems to cause insect mortality; however, Koch’s postu- the purpose of providing specific information and does not imply recommendation lates (Lacey and Brooks, 1997) should be used to verify the or endorsement by the U.S. Department of Agriculture. hypothesis that the isolated fungus is the cause of insect death. If * Corresponding author. Tel.: 1 956 969 4873; fax: 1 956 969 4888. þ þ there is a pathogenic relationship, it is important to determine the E-mail address: [email protected] (H.E. Cabanillas). 1 Current address: European Biological Control Laboratory, USDA-ARS, Campus severity of the virulence of the infection. This fungus which is being International de Baillarguet, 34980 Montferrier sur Lez, France. described elsewhere (Cabanillas et al. Unpub.), is closely related to 0261-2194/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.cropro.2008.11.015 334 H.E. Cabanillas, W.A. Jones / Crop Protection 28 (2009) 333–337 some of the entomopathogenic Paecilomyces species which has the time of application to collect spore deposit samples. Six been transferred to the genus Isaria (Hodge et al., 2005; Luangsa- microscopic fields (two each at 0.125, 0.5, and 3.25 mm2 corre- ard et al., 2005), it appears to be a promising candidate for control sponding to high, medium, and low spore concentrations) were of B. tabaci. Virulence, infectivity, and pathogenicity are considered examined on each block of agar (at 400Â magnifications). Conidia important properties of entomopathogens (Thomas and Elkinton, were counted, and counts were averaged and expressed as dosages 2004; Casadevall and Pirofski, 1999, 2001). The objectives of this applied per mm2. In our assays the estimated doses of 20 (low study were to determine the pathogenicity and virulence of this concentration), 200 (medium concentration) and 1000 spores/mm2 fungus against different nymphal stages of the sweet potato (high concentration) were produced by spray- whitefly on sweet potato, based on lethal concentrations to kill the ing spore suspensions containing approximately 1 Â 106,1Â 107, insect pest and the lethal time from infection until death of the 1 Â 108 spores/ml, respectively. insect. Spore viability was determined at the time of treatment by spraying 1-ml aliquots of spore suspension of the lowest concen- 2. Materials and methods tration through the spray tower onto a 10-cm diameter Petri dish with SDAY and incubating the plate at 25 C for 18 h. Spore 2.1. Pathogen and insect cultures germination was halted by adding three separate drops of lacto- phenol–cotton blue and adding a coverslip. The proportion of Isaria sp. used in these experiments was isolated from naturally viable conidia was determined by examining 100 spores in each of infected sweet potato whitefly nymphs and adults on eggplant these three different fields of view at 400Â magnification with (Solanum melongena L.) leaves in greenhouses at the USDA-ARS, a phase contrast microscope, and determining the proportion of Kika de la Garza Subtropical Agricultural Research Center (KSARC) spores that possessed a distinct germ tube, as defined by germ tube at Weslaco, Texas in September 2001. It was cultured on Sabouraud lengths that were two times the diameter of the spore (Goettel and dextrose agar with yeast extract (SDAY) and incubated at 25 C Inglis, 1997). (12:12 h light:dark). Conidia were harvested from three-week-old The treatments consisted of low, medium and high spore cultures by rinsing them with sterile distilled water containing concentrations and the control with water and Silwet. For assess- 0.02% Silwet L-77 (Loveland Industries, Greeley, CO). A culture of ment of natural mortality, untreated nymphs (untreated control) Isaria sp. was deposited in the ARS Collection of Entomopathogenic and nymphs treated with 0.02% Silwet L-77 (application control) Fungal Cultures (Ithaca, NY) as ARSEF 7028. were also included in all experiments. Each treatment was applied The sweet potato whitefly, biotype B, used for these bioassays separately onto six excised leaves each with 50 marked second-, originated from our colony maintained at the KSARC, USDA-ARS in third-, and fourth-instar nymphs. After evaporation of water, Weslaco, Texas on sweet potato leaves (Ipomoea batata L.) under rooted leaves were covered with plastic bags, and incubated at laboratory conditions at 25 Æ 1 C, 65 Æ 5% RH, and a photoperiod of 27 C and a relative humidity of 95–100% for 24 h. Thereafter, leaves 14:10 h (L:D) for four years. were kept in an environmental chamber at 27 C(Æ1 C), 60–80% RH and a photoperiod of 14:10 h light:dark. Insect mortality for the 2.2. Pathogenicity and virulence studies pathogenicity and virulence studies was evaluated 7 days after treatment and daily for 7 days after treatment, respectively. Death The pathogenicity of this fungus was determined as the esti- due to mycosis was verified by detaching treated dead nymphs mated spore concentration required to kill 50% of the test insects from the leaf surface, surface-disinfecting with 0.03% chlorine 2 (LC50, expressed as conidia/mm ), and its virulence was estimated bleach solution, rinsing twice in sterile distilled water, and plating by the median time to death of infected insects (LT50)(Thomas and on SDAY. Plates were incubated at 25 C, and cadavers were scored Elkinton, 2004). The fungus was passed through B. tabaci and the B. for fungal growth after 7 days. The entire bioassay was repeated tabaci reisolates were used for bioassays on virulence. Bioassays three times under the same conditions. were performed on rooted sweet potato leaves in test tubes with hydroponic nutrient solution. Leaves were infested by placing 50– 3. Statistical analysis 60 adults (both male and female) into clip-screen cages with a sweet potato leaf in a test tube for 12 h until they laid 50–100 The insect mortality data, including the three replicate experi- eggs.