Biological Control 17, 29–34 (2000) Article ID bcon.1999.0767, available online at http://www.idealibrary.com on The Time–Dose–Mortality Modeling and Virulence Indices for Two Entomophthoralean Species, Pandora delphacis and P. neoaphidis, against the Green Peach Aphid, Myzus persicae Jun-Huan Xu and Ming-Guang Feng1 Department of Biological Sciences, Huajiachi Campus, Zhejiang University, Hangzhou 310029, People’s Republic of China Received August 11, 1998; accepted June 30, 1999 mophthorales (Feng et al., 1995), but none has been Bioassays of two entomophthoralean fungi, Pandora well investigated. More effort is needed on microbial delphacis and P. neoaphidis, were conducted on the agents for aphid control (Milner, 1997) because of aphid green peach aphid, Myzus persicae. For inoculation, resistance to chemical insecticides (Devonshire, 1989) batches of I3-day-old nymphs on detached cabbage and increasing limitations of their use on vegetables leaves were exposed to conidial showers of varying grown in the outskirts of central cities in China (Feng time lengths from sporulating fungal mats produced in and Li, 1995). liquid culture. Eight dosages of each pathogen were The entomophthoralean fungus Pandora neoaphidis used to inoculate 88–345 nymphs each. The nymphs (Remaudiere & Hennebert) Humber is a well-known inoculated were maintained at 18–20°C and L:D (12:12) pathogen frequently causing aphid epizootics (De- at high humidity and were examined daily for mortal- dryver, 1983; Pickering et al., 1989; Feng et al., 1991, ity. The resulting data were analyzed using a time–dose– 1992), whereas P. delphacis (Hori) Humber usually mortality modeling technique, yielding the param- eters for time and dose effects of the two fungal infects planthoppers (Melissa and James, 1987) but species. P. delphacis killed the aphid more rapidly rarely aphids, though it has been found infecting the than P. neoaphidis, though both had similar slopes for cotton aphid, Aphis gossypii Glover, in controlled bioas- say (Shimazu, 1977). The two Pandora species have dose effect. The values of LD50 estimated on days 3–7 after exposure decreased from 18.1 to 0.9 conidia/mm2 similar morphological characteristics and are distin- for P. delphacis and from 17.0 to 0.04 conidia/mm2 for P. guished primarily on the basis of their hosts, i.e., neoaphidis. The estimates of LT50 for the two patho- aphids or planthoppers and leafhoppers (Humber, 1989). gens were similar at dosages of more than 5 conidia/ In recent years, we obtained isolates of the two fungal mm2 but differed at lower dosages. Based on the time– species from cadavers of the English grain aphid, dose–mortality relationships fitted and the virulence Sitobion avenae (F.), and the brown planthopper, indices estimated, P. delphacis is a promising micro- Nilaparvata lugens Stål, in the late-season rice crop in bial agent for aphid control and is competitive with P. Hangzhou, Zhejiang Province. Despite similarities in neoaphidis. ௠ 2000 Academic Press morphology, the isolates from the planthopper species Key Words: Pandora delphacis; Pandora neoaphidis; grow much more rapidly on plates or in liquid medium Myzus persicae; virulence. than those from the aphid species (Xu and Feng, 1998). In the present study we attempt to determine the time–dose–mortality relationships between the two INTRODUCTION pathogens and M. persicae, estimate their virulence indices, and evaluate their potential for use in aphid The green peach aphid, Myzus persicae (Sulzer), control. infests up to 40 families of plants, including various important crops in the field as well as in the greenhouse (Blackman and Eastop, 1984). It commonly devastates MATERIALS AND METHODS cruciferous vegetables in China (Liu, 1991). Microbial agents naturally occurring on aphids in China include Preparation of Inocula a few species of entomopathogenic fungi, mostly Ento- The two isolates, P. delphacis F95129 and P. neoaphi- dis F97006, were obtained from two cadavers of N. 1 To whom correspondence should be addressed. Fax: 86-571-697- lugens and S. avenae collected on rice plants in Hang- 1278. E-mail: [email protected]. zhou, Zhejiang in October 1995 and 1997, respectively. 29 1049-9644/00 $35.00 Copyright ௠ 2000 by Academic Press All rights of reproduction in any form reserved. 30 XU AND FENG Both were maintained on SEMA (80% Sabouraud dex- min with P. neoaphidis because P. delphacis sporulated trose agar, 11.5% fresh milk, 8.5% egg yolk) plates at more abundantly than P. neoaphidis. Colonies consist- 15°C and a photoperiod of L:D (12:12) by subculture ing of 186 and 250 nymphs on detached leaves without every month. Preparation of P. delphacis inocula started exposure to the conidial shower were included as from the growth of the isolate on SEMA at 25°C for 7 controls for bioassays 1 and 2, respectively. days. The mycelia of the resulting fungal colony were After exposure, the aphid colonies on detached leaves moved into a 100-ml flask containing 30 ml Sabouraud in the petri dishes were maintained in a growth cham- dextrose broth [SDB: 1% peptone, 1% yeast extract, and ber at 18–20°C and L:D (12:12). Live and dead aphids 4% glucose plus 20 µg PSN antibiotic mixture (penicil- were recorded daily for 7 days. Dead aphids were lin 25%, streptomycin 25%, and neomycin 50%; Gibco- removed from the colonies and examined individually BRL, Grand Island, NY) per milliliter]. Shaken in the for verification of infection after overnight incubation dark at 28 Ϯ 1°C for 24 h (150 rpm), the liquid culture in a moist chamber. There was no need for leaf change was transferred into 80 ml liquid medium in a 200-ml during the observation period because the nutrient flask for further growth for another 24 h under the solution around the petioles kept the leaves fresh for same regime. Finally, 10 ml liquid culture was poured approximately 2 weeks. onto a 90-mm petri dish containing 1.5% agar only. Excessive water was removed using filter paper. Incu- Data Analysis bated at 25°C and L:D (12:12) for 24 h, the fungal mats on the plates sporulated uniformly and were ready for A time–dose–mortality modeling technique (Robert- use. The procedures for preparation of P. neoaphidis son and Preisler, 1992; Nowierski et al., 1996; Feng et inocula were the same as above but took much longer: al., 1996, 1998) was used to analyze the resulting 15 days on SEMA at 20°C, 2 days in 30 ml SDB, and 5 time–dose–mortality data of P.delphacis and P.neoaphi- days in 80 ml SDB. dis for M. persicae. Considering a bioassay that in- cludes I dosages and J times of observation, the cumu- lative mortality probability, pij, caused by the dose di Aphid Colonies for Bioassay (i ϭ 1,2,···,I) at the time tj ( j ϭ 1,2,···,J) can be A laboratory M. persicae population initiated from expressed as individuals collected in the field was maintained on ϭ Ϫ Ϫ ␶ ϩ␤ cabbage, Brassica oleracea L. (cv. Jingfeng 1), at 20– pij 1 exp [ exp ( j log10(di ))], (1) 24°C and L:D (12:12). For bioassay, vigorous apterae were selected from the population to produce offspring where ␤ is the slope to describe the dose effect and ␶j is on detached cabbage leaves (15 aphids/leaf) in 150-mm the parameter(s) for the time effect of di during the petri dishes. For longer maintenance, each petiole was period from start to the jth observation (t1, t2,···,tjϪ1, wrapped with cotton saturated with Hoagland–Snyder tj, tjϩ1,···,tJ). To guarantee that the observed mortal- nutrient solution (Adams and van Emden, 1972). Three ity probability is independent of time, the true mortal- days later, the apterae were removed and the nymphal ity induced with di at the interval [tjϪ1, tj] must be colonies left on the leaves were ready for use in the considered. This true mortality, qij, is called the condi- bioassay. tional mortality probability (Robertson and Preisler, 1992) and is given as follows Exposure of Aphids ϭ Ϫ Ϫ ␥ ϩ␤ qij 1 exp [ exp ( j log10(di ))], (2) Bioassays of the two fungal pathogens against the aphid species were conducted in March 1998 (bioassay where ␤ is equal to that in Equation 1 and ␥j describes 1) and repeated in May 1999 (bioassay 2). Following the conditional time effect of di at the interval [tjϪ1, tj]. Feng and Johnson (1991) and Feng et al. (1998), The independence between time intervals of observa- detached leaves with nymphs were gently moved to tion allowed the fitting of data to Eq. [2] by approaching 150-mm petri dishes lined with moistened filter paper. the binomial response variable to the maximum likeli- A sporulating fungal mat in a dish was inverted onto hood equation (Feng et al., 1996, 1998; Nowierski et al., each dish containing one or two leaves with aphids. The 1996), yielding ␥j and ␤. Then, the values of ␶j were sporulating fungal mat was rotated 90° each quarter of calculated using the formula (Robertson and Preisler, the exposure time. One 20-mm glass coverslip was 1992) below: placed at the center of each dish to receive conidia discharged from the plate. After exposure the conidial j dosage was determined based on the counts of the ␶ ϭ ␥k j ln ͚ e . (3) conidia from five random fields of the coverslip (0.785 1kϭ1 2 mm2/field) under microscope. Eight dosages were in- cluded in each bioassay, resulting from the exposures of The procedures, including modeling, estimation of 1–120 min, 0.3–30 min with P. delphacis, and 1–180 time- and dose-effect parameters for both conditional Pandora SPP. AGAINST GREEN PEACH APHID 31 and cumulative models, test for goodness of fit, and killed by P.