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Interactions among the entomopathogenic fungus, Beauveria bassiana (Ascomycota: Hypocreales), the parasitoid, Aphidius matricariae (Hymenoptera: Braconidae), and its host, Myzus persicae (Homoptera: Aphididae)
Biological Control 50 (2009) 324–328
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Interactions among the entomopathogenic fungus, Beauveria bassiana (Ascomycota: Hypocreales), the parasitoid, Aphidius matricariae (Hymenoptera: Braconidae), and its host, Myzus persicae (Homoptera: Aphididae) M. Rashki a,*, A. Kharazi-pakdel a, H. Allahyari a, J.J.M. van Alphen b a b
Department of Plant Protection, Campus of Agriculture & Natural Resources, Tehran University, Karaj 31587-11167, Iran Institute of Biology, Animal Ecology Section, Leiden, The Netherlands
a r t i c l e
i n f o
Article history: Received 29 January 2009 Accepted 24 April 2009 Available online 5 May 2009 Keywords: Beauveria bassiana Myzus persicae Aphidius matricariae Life table Interaction
a b s t r a c t The effect of the entomopathogenic fungus Beauveria bassiana on the biological characteristics and life table of Aphidius matricariae, a parasitoid of the green peach aphid, Myzus persicae, was studied under laboratory conditions. Aphids were first infected with twice the LC95 of B. bassiana for third-instar M. persicae (2 108 conidia/ml). Subsequently, at different intervals they were exposed to 1-day-old mated parasitoid females for 24 h. The number of mummies produced per female and the percentage emergence of the F1 generation differed significantly as a function of the time interval between application of the fungus and exposure to the parasitoid. The interference of B. bassiana on parasitoid development was also studied by first exposing the aphid hosts to the parasitoid for 24 h and subsequently applying B. bassiana. The number of mummies produced by a female A. matricariae varied from 11.8 to 24.8 and was significantly different when the aphids were first exposed to the parasitoids and then treated with B. bassiana 24, 48, 72, and 96 h after exposure. There were no significantly different effects of B. bassiana on net reproductive rate (R0), mean generation time (T), intrinsic rate (rm) and the finite rate of increase (k) of A. matricariae as a result of development in hosts exposed to low or high conidial concentrations (1 102, 2 108 conidia/ml). The parasitoids developed in infected hosts had lower rm, k, T and DT (doubling time) values compared with those that developed in uninfected hosts but no differences were observed in R0 values. With proper timing, A. matricariae and B. bassiana can be used in combination in the successful biological control of M. persicae. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Intraguild interactions are not limited to closely related species and can exist between species from different kingdoms (Hochberg and Lawton, 1990). Better understanding of the factors that diminish antagonistic interaction of entomopathogens and other natural enemies could improve their integrated employment against pest insects (Lacey and Mesquita, 2002). Information on population growth parameters is necessary to perform pest control management (Maia et al., 2000). Knowledge of possible lethal or sublethal concentration effects of entomopathogens on predators and parasitoids is crucial. Likewise, information is needed on the effects of entomopathogens on fecundity, longevity and survivorship of parasitoids and predators (Fatiha et al., 2008). Aphidius matricariae Haliday is an important parasitoid of the green peach aphid, Myzus persicae (Sulzer), and 40 species of aphids belonging to 20 genera have been recognized as its hosts (Giri et al., 1982). In the laboratory, it has the highest rate of parasitism on M. persicae on canola (Desneux et al., 2006). Beauveria bassiana * Corresponding author. Fax: +98 261 2238529. E-mail address: [email protected] (M. Rashki). 1049-9644/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2009.04.016
(Balsamo) Vuilemin is one of the major fungal entomopathogens. In one study, it infected nearly 95% of migratory alate aphids, especially M. persicae (Chen et al., 2008). Laboratory studies have shown that whilst some strains of B. bassiana are highly virulent to the aphid and its coccinellid predator Coleomegilla maculata lengi Timberlake, three strains were found to be highly virulent to the aphid but caused low mortality to the predator indicating a potential for their use to control the green peach aphid (Todorova et al., 2000). There is no information on the compatible utilization of B. bassiana and A. matricariae for integrated pest management of M. persicae. Accordingly, the objective of our study was to determine the type of interaction between the entomopathogenic fungus and parasitoid wasp and its host, the green peach aphid, and to evaluate the impact of B. bassiana on life history and life table parameters of A. matricariae. 2. Materials and methods 2.1. Host plant and insect cultures Eggplants, Solanum melongena L. var. black beauty, were grown individually in plastic pots (15 cm diameter, 12 cm height). M. per-
M. Rashki et al. / Biological Control 50 (2009) 324–328
sicae and its mummies were collected from the field of eggplants in the Karaj region of Tehran. The emerged parasitoids were identified as A. matricariae. M. persicae was reared on eggplants in a controlled environment room (21 ± 1 °C, 60 ± 5% RH, 16L: 8D). A. matricariae was reared on M. persicae inside a plexiglas box (50 60 60 cm) in a controlled environment room (25 ± 1 °C, 70 ± 5% RH, 16L: 8D). One-day-old A. matricariae females were used for all experiments. 2.2. Fungus Beauveria bassiana strain EUT116 from the culture collection at the Biocontrol and Biocenology Laboratory of Tehran University was used. After passage of the fungus through M. persicae, it was cultured on Sabouraud dextrose agar with yeast extract (SDAY) for 2 weeks at 25 °C. The cultures were dried, and the harvested conidia were maintained in glass tubes using the procedure described by Hansen and Steenberg (2007) and storing them at 5 °C over silica gel. The viability of the conidia was determined by inoculating plates of SDAY with a conidial suspension which was then incubated for 18 h at 25 °C. The number of germinated conidia per 100 conidia in four areas of each plate was counted and four plates were used to assess viability. Germination was considered positive when the length of germ tube was as long as the diameter of the conidia. The percentage germination of the conidia was determined to be 100%. During the experiments, fungal suspensions of 1 102 or 2 108 B. bassiana conidia/ml were sprayed once (1 ml per spray application) onto aphids using a fine mist held above the aphids with 90° angle (Groszek, Kwazar, Jaktorow, Poland, http:// www.galactic-sprayers.com/KWAZAR/presentation.html). These concentrations were selected because they are equal to the lethal concentration that killed 10% (LC10) (sublethal) and two times of lethal concentration that killed 95% (2 LC95) of the sprayed aphids, respectively, for third-instar M. persicae.
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to observe the emergence of adult parasitoids. During the experiment, the petri dishes were kept inverted and incubated at 25 °C, 85–90% RH under a 16-h photoperiod. 2.3.2. Aphid first treated with fungus and then exposed to the parasitoid The objective of this experiment was to determine the development and longevity of A. matricariae when the aphid hosts were exposed to B. bassiana first and then to the parasitoid. Five treatments were examined over time on separate dates including of exposure to parasitoid simultaneously, 24, 48, or 72 h after treating with the fungus or no fungus (control). Forty third-instar aphids were used in each of fifteen replicates. They were sprayed with 2 108 of B. bassiana in 0.02% Tween 80. The aphids were then parasitized by a 1-day-old female A. matricariae at different time intervals (0, 24, 48, or 72 h). Also, as a control, 40 parasitized aphids were exposed to aphids sprayed with 0.02% Tween 80. Fifteen control replicates were set up. Each group of 40 aphids was maintained on a leaf set up in 2% water-agar in a petri dish (580 mm diameter). The inverted petri dishes were kept at 85– 90% RH, 25 °C, and 16-h photoperiod. As described in the previous experiment, dead aphids were counted daily, surface sterilized and were placed on wet filter paper to confirm infection by B. bassiana. The number of mummies produced was recorded. In addition, fifteen replicates with 40 aphids were only sprayed with fungus and an equal replicates were sprayed with 0.02% Tween 80 without exposure to parasitoid. The time of pre-imaginal parasitoid development, percentage of emergence, sex ratio, longevity of the females of F1 generation and the lengths of the metathoracic tibia and the anterior costal vein of emerged females were determined. To investigate longevity of female parasitoids, they were individually maintained in a petri dish (580 mm diameter) at 85–90% RH, 25 °C, and 16-h photoperiod and were fed honey (60%). Lengths of the mounted metathoracic tibia and the anterior costal vein of emerged females placed in Faure’s liquid (Izdebska, 2006) were determined by ImageJ software (ImageJ 1.41v, Wayne Rasband, National Institutes of Health, USA, http://rsb.info.nih.gov/ij/).
2.3. Influence of B. bassiana on development of A. matricariae 2.4. Life table parameters 2.3.1. Aphid first exposed to the parasitoid and then treated with the fungus The objective of this experiment was to assess the development of aphids exposed to the parasitoid first and then to B. bassiana. Five treatments were tested over time on separate dates, each with four groups of 40 third-instar aphids. The treatments consisted of spraying conidia at 24, 48, 72, or 96 h after host exposure to the parasitoid. Each group was placed in a separate petri dish (580 mm diameter) containing a S. melongena leaf that fit in the petri dish and fixed with the abaxial side up in 2% water-agar with a hole (2 cm diameter) in the lid covered with a fine mesh screen for ventilation (Baverstock et al., 2006). Each group of aphids was exposed to a 1-day-old female A. matricariae for 24 h. After 24 h, the parasitoids were removed from the dish and the remaining potentially parasitized aphids were sprayed with 2 108 conidia/ ml of B. bassiana in 0.02% Tween 80 after 24, 48, 72, or 96 h. The control was treated with 0.02% Tween 80. In addition, four replicates with 40 aphids were only sprayed with fungus or with 0.02% Tween 80. During the 24 h after the fungal conidia were applied to the aphids, the lid of petri dishes were replaced with new ones without hole and sealed with parafilm to maintain saturated humidity in order to facilitate conidial germination. During the following 7 days, dead aphids were counted daily, surface sterilized by putting them in 96% ethanol for 1 min, followed by 2.5% bleach (5% NaOCl) plus 0.02% Tween 80 for 3 min and two rinses of sterile distilled water plus Tween 80 for 1 min (Shipp et al., 2003) and were placed on wet filter paper to confirm infection by B. bassiana. Mummies were counted and separated individually
To measure life table parameters of A. matricariae that had developed in hosts infected with B. bassiana, third-instar M. persicae were first infected either with a sublethal concentration or twice the LC95 of B. bassiana conidia (1 102 and 2 108 conidia/ml, respectively) and Tween 80 only as control. After 24 h, the aphids were exposed to one mated female A. matricariae for another 24 h before the parasitoid was removed from the dish. The emerged parasitoids of F1 generation were tested for evaluating the effect of the fungus on the progeny of A. matricariae. Sixteen groups of 40 third-instar aphids were used for each treatment and control. Each group was maintained in a petri dish described in previous experiments at 25 °C, 70 ± 5% RH (16L: 8D). The parasitoid wasps were introduced to new aphid groups every 24 h until they died. The aphids were incubated at 25 °C, 70 ± 5% RH (16L: 8D) until adults emerged. The number of female parasitoids was recorded daily. 2.5. Data analyses The intrinsic rate of increase (rm) and other life table parameters were calculated using the following equations (Carey, 1993):
r m ¼ ln R0 =T; X lx mx ;
R0 ¼
k ¼ expðr m Þ; T ¼ lnðR0 Þ=rm ; r w ¼ ðerm Þ7 ;
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where lx is the proportion of survivorship at the age time x, and mx is the number of females produced per female per day. The net reproductive rate R0 is the mean number of female produced by a female during one generation. T, k, rw and DT parameters are mean generation time, finite rate of increase, increase rate in 1 week and doubling time, respectively. PROC GLM was used for analyses of variance (ANOVA) of all life table parameters. All data were subjected to analyses of variance (ANOVA) and the averages compared with Duncan’s test at the 0.05 level. Percentage data were arcsine square-root transformed. The Statistical Analysis System (SAS, 1989) was used for computations. 3. Results The aphids sprayed with fungus only (2 108 conidia/ml) and 0.02% Tween 80 only without exposure to parasitoid had 100 and 10% average mortality during 7 days, respectively. After maintenance on wet filter paper, the fungus appeared on 98.4% of aphids sprayed with fungus. 3.1. Influence of B. bassiana on development of A. matricariae 3.1.1. Aphid first exposed to the parasitoid and then treated with the fungus The number of mummies produced by female A. matricariae varied from 11.8 ± 0.3 to 24.8 ± 0.5 and was significantly different among different intervals and between treatments and control (F = 147.68; df = 4, 15; P = 0.0001) (Table 1). The lowest percentage of emergence of the F1 generation of A. matricariae was found for those which developed in aphids treated with B. bassiana 24 h after exposure to the female parasitoid. (F = 30.33; df = 4, 15; P = 0.0001). 3.1.2. Aphid first treated with fungus and then exposed to the parasitoid The number of mummies varied significantly among treatments. The lowest numbers of the mummies were found when
parasitism occurred 72 h after exposure to B. bassiana and the highest number of mummies was recorded when exposure to the fungus and parasitoid occurred at the same time. In all cases, the number of mummies produced in the treatment groups was significantly lower than the number of mummies produced in the control group (F = 343.99; df = 4, 70; P = 0.0001) (Table 2). The percentage emergence of the F1 generation of A. matricariae was significantly lower in the fungus treated aphids than in uninfected aphids and differed significantly among treatments with lower emergence recorded as the interval between fungal infection and parasitism increased (F = 42.49; df = 4, 70; P = 0.0001) (Table 2). The sex ratio did not differ among treatments (F = 0.58; df = 4, 59; P = 0.6802). There was no significant difference in the duration from oviposition to mummification ranging from 7.0 ± 0.1 to 7.3 ± 0.1 days among treatments (F = 1.70; df = 4, 62; P = 0.1625) (Table 3). The presence of the fungus caused a longer duration between parasitoid mummy formation and female emergence, with the longest duration when the aphids were exposed to the parasitoids 24 h after fungal infection (F = 47.54; df = 4, 57; P = 0.0001). However, fungal infection of the aphids only increased the time between mummification and male emergence of the parasitoids when the hosts where exposed to parasitoid attack 72 h after fungal infection (F = 5.40; df = 4, 48; P = 0.0011) (Table 3). Therefore, compared with the development of A. matricariae in uninfected hosts, total developmental time (=the time from oviposition to adult emergence) was significantly longer for female parasitoids that had developed in host attacked by A. matricariae 24 h after fungal infection (F = 22.40; df = 4, 57; P = 0.0001), and for males when parasitoid attack had occurred 72 h after fungal infection (F = 4.30; df = 4, 48; P = 0.0047). No significant variation between treatments was found in longevity of females or in their morphometric measurements (P > 0.05). Longevity of females ranged from 6.3 ± 0.2 to 6.7 ± 0.2 days (F = 0.78; df = 4, 319; P = 0.5394) among treatments. Length of anterior costal vein and metathoracic tibia was 0.5 ± 0.0
Table 1 The mean (±SE) number of Aphidius matricariae (Am) mummies and percentage of adult parasitoids that emerged per replicate from Myzus persicae hosts that had been exposed to the parasitoid and then sprayed with Beauveria bassiana (Bb) (2 108 conidia/ml) 24, 48, 72, and 96 h later. Treatments
No. aphids per replicate exposed to A. matricariae
No. aphids treated with fungus after exposure to A. matricariae
No. mummies produced by A. matricariae
% Emergence of F1 generation of A. matricariaea
Only Am Am + Bb (24 h) Am + Bb (48 h) Am + Bb (72 h) Am + Bb (96 h)
40 40
— 37.3 ± 1.0
30.0 ± 0.4a 11.8 ± 0.3d
92.6 ± 3.3a 31.3 ± 4.1c
40
36.3 ± 1.7
21.8 ± 0.5c
34.6 ± 4.7c
40
34.8 ± 2.1
24.0 ± 0.5b
61.8 ± 3.5b
40
35.3 ± 3.2
24.0 ± 0.9b
79.2 ± 7.7a
Means followed by the same letter in the same column are not significantly different (Duncan’s test, P < 0.05). a Values are a percentage of the number of mummies produced.
Table 2 The mean (±SE) number of Aphidius matricariae (Am) mummies produced, percentage adult emergence of F1 generation and percentage of female in the F1 generation per replicate from Myzus persicae hosts that had been sprayed with Beauveria bassiana (Bb) and then parasitized 0, 24, 48, and 72 h later. Treatments
No. of mummies produced
% Emergence of F1 generationa
Percentage of females in F1 generationb
Only Am Bb + Am (0 h) Bb + Am (24 h) Bb + Am (48 h) Bb + Am (72 h)
20.7 ± 0.6a 12.9 ± 0.4b 5.7 ± 0.4c 4.7 ± 0.3c 3.0 ± 0.3d
84.0 ± 2.1a 72.3 ± 2.3b 62.2 ± 3.2c 61.6 ± 2.4c 38.7 ± 2.8d
60.6 ± 3.0a 66.4 ± 4.7a 67.8 ± 5.4a 60.6 ± 4.2a 63.9 ± 4.5a
Means followed by the same letter in the same column are not significantly different (Duncan’s test, P < 0.05). a Values are a percentage of the number of mummies produced. b Values are a percentage of the number of emerged adults.
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M. Rashki et al. / Biological Control 50 (2009) 324–328 Table 3 Development time (Mean ± SE) of Aphidius matricariae (Am) when aphids were first exposed to Beauveria bassiana (Bb) and then parasitized 0, 24, 48, and 72 h later. Treatments
Oviposition until mummification (day)
Only Am Bb + Am (0 h) Bb + Am (24 h) Bb + Am (48 h) Bb + Am (72 h)
7.1 ± 0.0a 7.3 ± 0.1a 7.3 ± 0.2a 7.1 ± 0.1a 7.2 ± 0.1a
Mummification until emergence (day)
Oviposition until emergence (day)
$
#
$
#
4.2 ± 0.1c 5.0 ± 0.1b 7.4 ± 0.3a 4.7 ± 0.2b 5.0 ± 0.0b
4.4 ± 0.2b 4.7 ± 0.2b 4.8 ± 0.2b 4.9 ± 0.2b 5.8 ± 0.2a
11.2 ± 0.1b 12.1 ± 0.1b 15.2 ± 0.7a 11.3 ± 0.3b 12.0 ± 0.0b
11.4 ± 0.2b 11.6 ± 0.2b 11.8 ± 0.3b 11.6 ± 0.3b 12.8 ± 0.2a
Means followed by the same letter in the same column are not significantly different (Duncan’s test, P < 0.05).
Table 4 Life table parameters (Mean ± SE) of Aphidius matricariae that had developed in hosts treated with different concentrations of Beauveria bassiana. Life table parameters
Tween-80
1 102 (conidia/ml)
2 108 (conidia/ml)
rm R0 K T DT rw
0.3 ± 0.0a 40.9 ± 3.6a 1.3 ± 0.0a 13.1 ± 3.3b 2.4 ± 0.0b 9.3 ± 0.0a
0.3 ± 0.0b 45.3 ± 4.7a 1.3 ± 0.0b 15.0 ± 3.8a 2.7 ± 0.1a 9.0 ± 0.0b
0.3 ± 0.0b 33.9 ± 6.5a 1.294 ± 0.0b 14.1 ± 3.2a 2.7 ± 0.1a 9.0 ± 0.1b
Means followed by different letter in the same row are significantly different (Duncan’s test, P < 0.05). rm, intrinsic rate of increase, R0, net reproductive rate, K, finite rate of increase, T, mean generation time, DT, doubling time, rw, increase rate in 1 week.
(F = 0.25; df = 4, 319; P = 0.9088) and 0.4 ± 0.0 mm (F = 0.60; df = 4, 319; P = 0.6621), respectively, at different time intervals.
Fig. 1. Survival rate (lx) of Aphidius matricariae influenced by two concentrations of Beauveria bassiana compared to control.
3.2. Life table parameters Calculated values of the intrinsic rate of increase (rm) were significantly different (F = 6.73; df = 2, 48; P = 0.003) between parasitoid females from treated and untreated aphids, but no difference was observed in the net reproductive rate (R0) between the two concentrations (1 102 and 2 108 conidia/ml) and the control (Table 4). The intrinsic rate of increase was higher for parasitoids that developed in untreated aphids compared with those that developed in aphids exposed to B. bassiana. The mean generation time was shorter in the controls than in treated wasps. The other parameters including the finite rate of increase (k), the mean generation time (T), the doubling time (DT) were not significantly different between the two concentrations of B. bassiana used. The proportion of wasps surviving at time x (lx) and number of females produced per female per day (mx) were almost similar among the two treatments and between the treatments and control (Figs. 1 and 2).
Fig. 2. Number of females produced per female per day (mx) of Aphidius matricariae influenced by two concentrations of Beauveria bassiana compared to control.
4. Discussion The numbers of mummies from aphids first parasitized then treated with fungus, depended on the time interval between exposure to A. matricariae and application of B. bassiana. Similar results have been obtained by Powell et al. (1986) who demonstrated the successful development of Aphidius rhopalosiphi de Stefani Perez in the aphid host Metopolophium dirhodum (Walker) also depends on the time interval between exposure to the parasitoid and infection with the fungus Pandora (Erynia) neoaphidis (Brefeld) Batko. However, such a difference was not found for Aphelinus asychis (Walker) after exposure to Paecilomyces fumosoroseus Brown and Smith at various intervals (Mesquita and Lacey, 2001). The severe decrease in successful parasitoid mummification when developing in hosts that were infected with B. bassiana 24 h after being parasitized suggests that the presence of an early instar parasitoid larva provides a
competitive advantage to the fungus, while older parasitoid larvae have a high probability of winning the competition for the host aphid, possibly due to a low susceptibility of older larvae to fungus infection (Fransen and van Lenteren, 1994). Competition for food between larvae of A. matricariae and B. bassiana reduces the percentage of parasitoid emergence when the interval between oviposition and fungal infection of the host is less than 96 h. In most cases, there is no evidence for the invasion of the parasitoid larval tissue by the fungus when aphids have first been exposed to the parasitoid and then inoculated with fungus (Powell et al., 1986; Askary and Brodeur, 1999). The lower number of mummies obtained from infected aphids subsequently exposed to parasitoids is likely to be caused by lower survival of parasitoid larvae and/or by host selection; that is, parasitoids
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may have rejected the infected aphids for oviposition (Mesquita and Lacey, 2001). Brobyn et al. (1988) demonstrated that aphids treated with fungus 72 h before exposure to parasitoids were less often attacked and parasitized than healthy aphids. The decrease in the percentage emergence of the F1 generation as a function of the time interval between fungus treatment and parasitism shows that the probability that the fungus outcompetes the parasitoid increases with the time interval. A possible mechanism is that the fungus reduces the host quality for the parasitoid larva, causing mortality of the parasitoid larva. It also results in the pupal stage being prolonged, especially for the development of female parasitoids. In our study, female parasitoids seem to be more sensitive than males in competition with B. bassiana. This is possibly due to the higher growth rate of females (Sequeira and Mackauer, 1992). However, the longevity and morphometrics of F1 A. matricariae were not adversely affected as a consequence of developing within hosts infected with B. bassiana. Females of A. matricariae did not change sex allocation in response to infected aphids in different stages of fungal development. There is little information on the fecundity of parasitoids that emerged from infected host insects (Mesquita and Lacey, 2001), especially about sublethal and chronic effects of entomopathogenic fungi. Our results demonstrated that B. bassiana strain EUT116 did not influence the life-time number of offspring of females (R0) but it reduced the rm value, although similar rm values were obtained with the low and high concentrations. The rm values decreased because the entomopathogenic fungus resulted in an increase of the mean generation time of the parasitoids (T). Fatiha et al. (2008) showed that direct application of Verticillium (=Lecanicillium) lecanii (Zimm.) Viegas decreased the R0 value of Serangium japonicum (Coleoptera: Coccinellidae), a predator of whiteflies, and that different concentrations of the fungus resulted in similar values of the intrinsic rate of increase (rm). Also they indicated that the mean generation time (T) was not significantly different between fungal treatment and control. It is important to consider complementary activity of biocontrol agents (Lacey et al., 1997). Mesquita et al. (1997) reported better control of Diuraphis noxia (Mordvilko) when A. asychis and P. fumosoroseus were combined under field conditions. However, A. colemani Viereck is very susceptible to B. bassiana (commercial formulation, strain JW-1) in the laboratory but shows lower rates of infection in the greenhouse (Ludwig and Oetting, 2001). Scopes (1970) observed that application of A. matricariae was not a suitable method for controlling M. persicae when the aphids were infected with the entomopathogenic fungus Cephalosporium aphidicola Petch (Ascomycota: Hypocreales) as the parasitoids did not complete its development in infected hosts. Our research indicates that B. bassiana strain EUT116 could have an adverse effect on the development of the endoparasitoid, A. matricariae if the parasitoids are released after B. bassiana has been applied. However, our results also show that with good timing, A. matricariae and B. bassiana could potentially be used in combination for the successful biological control of M. persicae. Therefore, application of these two biological agents requires effective time management to avoid antagonistic interactions. Acknowledgments Our sincere thanks are extended to Dr. A. Shirvani, Dr. G. Mohammadinezhad from Shahid Bahonar University of Kerman, Dr. A. Zamani from Razi University of Kermanshah and M. Askari Seyahooei from Institute of Biology, Leiden University for their
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Ludwig, S.W., Oetting, R.D., 2001. Susceptibility of natural enemies to infection by Beauveria bassiana and impact of insecticides on Ipheseius degenerans (Acari: Phytoseiidae). Journal of Agricultural and Urban Entomology 18, 169–178. Maia, A.H.N., Luiz, A.J.B., Campanhola, C., 2000. Statistical inference on associated fertility life table parameters using jackknife technique: computational aspects. Journal of Economic Entomology 93, 511–518. Mesquita, A.L.M., Lacey, L.A., 2001. Interactions among the entomopathogenic fungus, Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes), the parasitoid, Aphelinus asychis (Hymenoptera: Aphelinidae), and their aphid host. Biological Control 22, 51–59. Mesquita, A.L.M., Lacey, L.A., Leclant, F., 1997. Individual and combined effects of the fungus, Paecilomyces fumosoroseus and parasitoid, Aphelinus asychis Walker (Hym., Aphelinidae) on confined populations of Russian wheat aphid, Diuraphis noxia (Mordvilko) (Hom., Aphidiidae) under field conditions. Journal of Applied Entomology 121, 155–163. Powell, W., Wilding, N., Brobyn, P.J., Clark, S.J., 1986. Interference between parasitoids (Hym.: Aphidiidae) and fungi (Entomophthorales) attacking cereal aphids. Entomophaga 31, 293–302. SAS, 1989. SAS/STAT Users Guide, version 6, Vols. 1 and 2. SAS Institute Inc. Cary, NC. Scopes, N.E.A., 1970. Control of Myzus persicae on year-round chrysanthemums by introducing aphids parasitized by Aphidius matricariae into boxes of rooted cuttings. Annals of Applied Biology 66, 323–327. Sequeira, R., Mackauer, M., 1992. Covariance of adult size and development time in the parasitoid wasp Aphidius ervi in relation to the size of its host, Acyrthosiphon pisum. Evolutionary Ecology 6, 34–44. Shipp, J.L., Zhang, Y., Hunt, D.W.A., Ferguson, G., 2003. Influence of humidity and greenhouse microclimate on the efficacy of Beauveria bassiana (Balsamo) for control of greenhouse arthropod pests. Environmental Entomology 32, 1154– 1163. Todorova, S.I., Coderre, D., Cote, J.C., 2000. Pathogenicity of Beauveria bassiana isolates toward Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), Myzus persicae (Homoptera: Aphididae) and their predator Coleomegilla maculata lengi (Coleoptera: Coccinellidae). Phytoprotection 81, 15–22.
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Biological Control journal homepage: www.elsevier.com/locate/ybcon
Interactions among the entomopathogenic fungus, Beauveria bassiana (Ascomycota: Hypocreales), the parasitoid, Aphidius matricariae (Hymenoptera: Braconidae), and its host, Myzus persicae (Homoptera: Aphididae) M. Rashki a,*, A. Kharazi-pakdel a, H. Allahyari a, J.J.M. van Alphen b a b
Department of Plant Protection, Campus of Agriculture & Natural Resources, Tehran University, Karaj 31587-11167, Iran Institute of Biology, Animal Ecology Section, Leiden, The Netherlands
a r t i c l e
i n f o
Article history: Received 29 January 2009 Accepted 24 April 2009 Available online 5 May 2009 Keywords: Beauveria bassiana Myzus persicae Aphidius matricariae Life table Interaction
a b s t r a c t The effect of the entomopathogenic fungus Beauveria bassiana on the biological characteristics and life table of Aphidius matricariae, a parasitoid of the green peach aphid, Myzus persicae, was studied under laboratory conditions. Aphids were first infected with twice the LC95 of B. bassiana for third-instar M. persicae (2 108 conidia/ml). Subsequently, at different intervals they were exposed to 1-day-old mated parasitoid females for 24 h. The number of mummies produced per female and the percentage emergence of the F1 generation differed significantly as a function of the time interval between application of the fungus and exposure to the parasitoid. The interference of B. bassiana on parasitoid development was also studied by first exposing the aphid hosts to the parasitoid for 24 h and subsequently applying B. bassiana. The number of mummies produced by a female A. matricariae varied from 11.8 to 24.8 and was significantly different when the aphids were first exposed to the parasitoids and then treated with B. bassiana 24, 48, 72, and 96 h after exposure. There were no significantly different effects of B. bassiana on net reproductive rate (R0), mean generation time (T), intrinsic rate (rm) and the finite rate of increase (k) of A. matricariae as a result of development in hosts exposed to low or high conidial concentrations (1 102, 2 108 conidia/ml). The parasitoids developed in infected hosts had lower rm, k, T and DT (doubling time) values compared with those that developed in uninfected hosts but no differences were observed in R0 values. With proper timing, A. matricariae and B. bassiana can be used in combination in the successful biological control of M. persicae. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Intraguild interactions are not limited to closely related species and can exist between species from different kingdoms (Hochberg and Lawton, 1990). Better understanding of the factors that diminish antagonistic interaction of entomopathogens and other natural enemies could improve their integrated employment against pest insects (Lacey and Mesquita, 2002). Information on population growth parameters is necessary to perform pest control management (Maia et al., 2000). Knowledge of possible lethal or sublethal concentration effects of entomopathogens on predators and parasitoids is crucial. Likewise, information is needed on the effects of entomopathogens on fecundity, longevity and survivorship of parasitoids and predators (Fatiha et al., 2008). Aphidius matricariae Haliday is an important parasitoid of the green peach aphid, Myzus persicae (Sulzer), and 40 species of aphids belonging to 20 genera have been recognized as its hosts (Giri et al., 1982). In the laboratory, it has the highest rate of parasitism on M. persicae on canola (Desneux et al., 2006). Beauveria bassiana * Corresponding author. Fax: +98 261 2238529. E-mail address: [email protected] (M. Rashki). 1049-9644/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2009.04.016
(Balsamo) Vuilemin is one of the major fungal entomopathogens. In one study, it infected nearly 95% of migratory alate aphids, especially M. persicae (Chen et al., 2008). Laboratory studies have shown that whilst some strains of B. bassiana are highly virulent to the aphid and its coccinellid predator Coleomegilla maculata lengi Timberlake, three strains were found to be highly virulent to the aphid but caused low mortality to the predator indicating a potential for their use to control the green peach aphid (Todorova et al., 2000). There is no information on the compatible utilization of B. bassiana and A. matricariae for integrated pest management of M. persicae. Accordingly, the objective of our study was to determine the type of interaction between the entomopathogenic fungus and parasitoid wasp and its host, the green peach aphid, and to evaluate the impact of B. bassiana on life history and life table parameters of A. matricariae. 2. Materials and methods 2.1. Host plant and insect cultures Eggplants, Solanum melongena L. var. black beauty, were grown individually in plastic pots (15 cm diameter, 12 cm height). M. per-
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sicae and its mummies were collected from the field of eggplants in the Karaj region of Tehran. The emerged parasitoids were identified as A. matricariae. M. persicae was reared on eggplants in a controlled environment room (21 ± 1 °C, 60 ± 5% RH, 16L: 8D). A. matricariae was reared on M. persicae inside a plexiglas box (50 60 60 cm) in a controlled environment room (25 ± 1 °C, 70 ± 5% RH, 16L: 8D). One-day-old A. matricariae females were used for all experiments. 2.2. Fungus Beauveria bassiana strain EUT116 from the culture collection at the Biocontrol and Biocenology Laboratory of Tehran University was used. After passage of the fungus through M. persicae, it was cultured on Sabouraud dextrose agar with yeast extract (SDAY) for 2 weeks at 25 °C. The cultures were dried, and the harvested conidia were maintained in glass tubes using the procedure described by Hansen and Steenberg (2007) and storing them at 5 °C over silica gel. The viability of the conidia was determined by inoculating plates of SDAY with a conidial suspension which was then incubated for 18 h at 25 °C. The number of germinated conidia per 100 conidia in four areas of each plate was counted and four plates were used to assess viability. Germination was considered positive when the length of germ tube was as long as the diameter of the conidia. The percentage germination of the conidia was determined to be 100%. During the experiments, fungal suspensions of 1 102 or 2 108 B. bassiana conidia/ml were sprayed once (1 ml per spray application) onto aphids using a fine mist held above the aphids with 90° angle (Groszek, Kwazar, Jaktorow, Poland, http:// www.galactic-sprayers.com/KWAZAR/presentation.html). These concentrations were selected because they are equal to the lethal concentration that killed 10% (LC10) (sublethal) and two times of lethal concentration that killed 95% (2 LC95) of the sprayed aphids, respectively, for third-instar M. persicae.
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to observe the emergence of adult parasitoids. During the experiment, the petri dishes were kept inverted and incubated at 25 °C, 85–90% RH under a 16-h photoperiod. 2.3.2. Aphid first treated with fungus and then exposed to the parasitoid The objective of this experiment was to determine the development and longevity of A. matricariae when the aphid hosts were exposed to B. bassiana first and then to the parasitoid. Five treatments were examined over time on separate dates including of exposure to parasitoid simultaneously, 24, 48, or 72 h after treating with the fungus or no fungus (control). Forty third-instar aphids were used in each of fifteen replicates. They were sprayed with 2 108 of B. bassiana in 0.02% Tween 80. The aphids were then parasitized by a 1-day-old female A. matricariae at different time intervals (0, 24, 48, or 72 h). Also, as a control, 40 parasitized aphids were exposed to aphids sprayed with 0.02% Tween 80. Fifteen control replicates were set up. Each group of 40 aphids was maintained on a leaf set up in 2% water-agar in a petri dish (580 mm diameter). The inverted petri dishes were kept at 85– 90% RH, 25 °C, and 16-h photoperiod. As described in the previous experiment, dead aphids were counted daily, surface sterilized and were placed on wet filter paper to confirm infection by B. bassiana. The number of mummies produced was recorded. In addition, fifteen replicates with 40 aphids were only sprayed with fungus and an equal replicates were sprayed with 0.02% Tween 80 without exposure to parasitoid. The time of pre-imaginal parasitoid development, percentage of emergence, sex ratio, longevity of the females of F1 generation and the lengths of the metathoracic tibia and the anterior costal vein of emerged females were determined. To investigate longevity of female parasitoids, they were individually maintained in a petri dish (580 mm diameter) at 85–90% RH, 25 °C, and 16-h photoperiod and were fed honey (60%). Lengths of the mounted metathoracic tibia and the anterior costal vein of emerged females placed in Faure’s liquid (Izdebska, 2006) were determined by ImageJ software (ImageJ 1.41v, Wayne Rasband, National Institutes of Health, USA, http://rsb.info.nih.gov/ij/).
2.3. Influence of B. bassiana on development of A. matricariae 2.4. Life table parameters 2.3.1. Aphid first exposed to the parasitoid and then treated with the fungus The objective of this experiment was to assess the development of aphids exposed to the parasitoid first and then to B. bassiana. Five treatments were tested over time on separate dates, each with four groups of 40 third-instar aphids. The treatments consisted of spraying conidia at 24, 48, 72, or 96 h after host exposure to the parasitoid. Each group was placed in a separate petri dish (580 mm diameter) containing a S. melongena leaf that fit in the petri dish and fixed with the abaxial side up in 2% water-agar with a hole (2 cm diameter) in the lid covered with a fine mesh screen for ventilation (Baverstock et al., 2006). Each group of aphids was exposed to a 1-day-old female A. matricariae for 24 h. After 24 h, the parasitoids were removed from the dish and the remaining potentially parasitized aphids were sprayed with 2 108 conidia/ ml of B. bassiana in 0.02% Tween 80 after 24, 48, 72, or 96 h. The control was treated with 0.02% Tween 80. In addition, four replicates with 40 aphids were only sprayed with fungus or with 0.02% Tween 80. During the 24 h after the fungal conidia were applied to the aphids, the lid of petri dishes were replaced with new ones without hole and sealed with parafilm to maintain saturated humidity in order to facilitate conidial germination. During the following 7 days, dead aphids were counted daily, surface sterilized by putting them in 96% ethanol for 1 min, followed by 2.5% bleach (5% NaOCl) plus 0.02% Tween 80 for 3 min and two rinses of sterile distilled water plus Tween 80 for 1 min (Shipp et al., 2003) and were placed on wet filter paper to confirm infection by B. bassiana. Mummies were counted and separated individually
To measure life table parameters of A. matricariae that had developed in hosts infected with B. bassiana, third-instar M. persicae were first infected either with a sublethal concentration or twice the LC95 of B. bassiana conidia (1 102 and 2 108 conidia/ml, respectively) and Tween 80 only as control. After 24 h, the aphids were exposed to one mated female A. matricariae for another 24 h before the parasitoid was removed from the dish. The emerged parasitoids of F1 generation were tested for evaluating the effect of the fungus on the progeny of A. matricariae. Sixteen groups of 40 third-instar aphids were used for each treatment and control. Each group was maintained in a petri dish described in previous experiments at 25 °C, 70 ± 5% RH (16L: 8D). The parasitoid wasps were introduced to new aphid groups every 24 h until they died. The aphids were incubated at 25 °C, 70 ± 5% RH (16L: 8D) until adults emerged. The number of female parasitoids was recorded daily. 2.5. Data analyses The intrinsic rate of increase (rm) and other life table parameters were calculated using the following equations (Carey, 1993):
r m ¼ ln R0 =T; X lx mx ;
R0 ¼
k ¼ expðr m Þ; T ¼ lnðR0 Þ=rm ; r w ¼ ðerm Þ7 ;
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where lx is the proportion of survivorship at the age time x, and mx is the number of females produced per female per day. The net reproductive rate R0 is the mean number of female produced by a female during one generation. T, k, rw and DT parameters are mean generation time, finite rate of increase, increase rate in 1 week and doubling time, respectively. PROC GLM was used for analyses of variance (ANOVA) of all life table parameters. All data were subjected to analyses of variance (ANOVA) and the averages compared with Duncan’s test at the 0.05 level. Percentage data were arcsine square-root transformed. The Statistical Analysis System (SAS, 1989) was used for computations. 3. Results The aphids sprayed with fungus only (2 108 conidia/ml) and 0.02% Tween 80 only without exposure to parasitoid had 100 and 10% average mortality during 7 days, respectively. After maintenance on wet filter paper, the fungus appeared on 98.4% of aphids sprayed with fungus. 3.1. Influence of B. bassiana on development of A. matricariae 3.1.1. Aphid first exposed to the parasitoid and then treated with the fungus The number of mummies produced by female A. matricariae varied from 11.8 ± 0.3 to 24.8 ± 0.5 and was significantly different among different intervals and between treatments and control (F = 147.68; df = 4, 15; P = 0.0001) (Table 1). The lowest percentage of emergence of the F1 generation of A. matricariae was found for those which developed in aphids treated with B. bassiana 24 h after exposure to the female parasitoid. (F = 30.33; df = 4, 15; P = 0.0001). 3.1.2. Aphid first treated with fungus and then exposed to the parasitoid The number of mummies varied significantly among treatments. The lowest numbers of the mummies were found when
parasitism occurred 72 h after exposure to B. bassiana and the highest number of mummies was recorded when exposure to the fungus and parasitoid occurred at the same time. In all cases, the number of mummies produced in the treatment groups was significantly lower than the number of mummies produced in the control group (F = 343.99; df = 4, 70; P = 0.0001) (Table 2). The percentage emergence of the F1 generation of A. matricariae was significantly lower in the fungus treated aphids than in uninfected aphids and differed significantly among treatments with lower emergence recorded as the interval between fungal infection and parasitism increased (F = 42.49; df = 4, 70; P = 0.0001) (Table 2). The sex ratio did not differ among treatments (F = 0.58; df = 4, 59; P = 0.6802). There was no significant difference in the duration from oviposition to mummification ranging from 7.0 ± 0.1 to 7.3 ± 0.1 days among treatments (F = 1.70; df = 4, 62; P = 0.1625) (Table 3). The presence of the fungus caused a longer duration between parasitoid mummy formation and female emergence, with the longest duration when the aphids were exposed to the parasitoids 24 h after fungal infection (F = 47.54; df = 4, 57; P = 0.0001). However, fungal infection of the aphids only increased the time between mummification and male emergence of the parasitoids when the hosts where exposed to parasitoid attack 72 h after fungal infection (F = 5.40; df = 4, 48; P = 0.0011) (Table 3). Therefore, compared with the development of A. matricariae in uninfected hosts, total developmental time (=the time from oviposition to adult emergence) was significantly longer for female parasitoids that had developed in host attacked by A. matricariae 24 h after fungal infection (F = 22.40; df = 4, 57; P = 0.0001), and for males when parasitoid attack had occurred 72 h after fungal infection (F = 4.30; df = 4, 48; P = 0.0047). No significant variation between treatments was found in longevity of females or in their morphometric measurements (P > 0.05). Longevity of females ranged from 6.3 ± 0.2 to 6.7 ± 0.2 days (F = 0.78; df = 4, 319; P = 0.5394) among treatments. Length of anterior costal vein and metathoracic tibia was 0.5 ± 0.0
Table 1 The mean (±SE) number of Aphidius matricariae (Am) mummies and percentage of adult parasitoids that emerged per replicate from Myzus persicae hosts that had been exposed to the parasitoid and then sprayed with Beauveria bassiana (Bb) (2 108 conidia/ml) 24, 48, 72, and 96 h later. Treatments
No. aphids per replicate exposed to A. matricariae
No. aphids treated with fungus after exposure to A. matricariae
No. mummies produced by A. matricariae
% Emergence of F1 generation of A. matricariaea
Only Am Am + Bb (24 h) Am + Bb (48 h) Am + Bb (72 h) Am + Bb (96 h)
40 40
— 37.3 ± 1.0
30.0 ± 0.4a 11.8 ± 0.3d
92.6 ± 3.3a 31.3 ± 4.1c
40
36.3 ± 1.7
21.8 ± 0.5c
34.6 ± 4.7c
40
34.8 ± 2.1
24.0 ± 0.5b
61.8 ± 3.5b
40
35.3 ± 3.2
24.0 ± 0.9b
79.2 ± 7.7a
Means followed by the same letter in the same column are not significantly different (Duncan’s test, P < 0.05). a Values are a percentage of the number of mummies produced.
Table 2 The mean (±SE) number of Aphidius matricariae (Am) mummies produced, percentage adult emergence of F1 generation and percentage of female in the F1 generation per replicate from Myzus persicae hosts that had been sprayed with Beauveria bassiana (Bb) and then parasitized 0, 24, 48, and 72 h later. Treatments
No. of mummies produced
% Emergence of F1 generationa
Percentage of females in F1 generationb
Only Am Bb + Am (0 h) Bb + Am (24 h) Bb + Am (48 h) Bb + Am (72 h)
20.7 ± 0.6a 12.9 ± 0.4b 5.7 ± 0.4c 4.7 ± 0.3c 3.0 ± 0.3d
84.0 ± 2.1a 72.3 ± 2.3b 62.2 ± 3.2c 61.6 ± 2.4c 38.7 ± 2.8d
60.6 ± 3.0a 66.4 ± 4.7a 67.8 ± 5.4a 60.6 ± 4.2a 63.9 ± 4.5a
Means followed by the same letter in the same column are not significantly different (Duncan’s test, P < 0.05). a Values are a percentage of the number of mummies produced. b Values are a percentage of the number of emerged adults.
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M. Rashki et al. / Biological Control 50 (2009) 324–328 Table 3 Development time (Mean ± SE) of Aphidius matricariae (Am) when aphids were first exposed to Beauveria bassiana (Bb) and then parasitized 0, 24, 48, and 72 h later. Treatments
Oviposition until mummification (day)
Only Am Bb + Am (0 h) Bb + Am (24 h) Bb + Am (48 h) Bb + Am (72 h)
7.1 ± 0.0a 7.3 ± 0.1a 7.3 ± 0.2a 7.1 ± 0.1a 7.2 ± 0.1a
Mummification until emergence (day)
Oviposition until emergence (day)
$
#
$
#
4.2 ± 0.1c 5.0 ± 0.1b 7.4 ± 0.3a 4.7 ± 0.2b 5.0 ± 0.0b
4.4 ± 0.2b 4.7 ± 0.2b 4.8 ± 0.2b 4.9 ± 0.2b 5.8 ± 0.2a
11.2 ± 0.1b 12.1 ± 0.1b 15.2 ± 0.7a 11.3 ± 0.3b 12.0 ± 0.0b
11.4 ± 0.2b 11.6 ± 0.2b 11.8 ± 0.3b 11.6 ± 0.3b 12.8 ± 0.2a
Means followed by the same letter in the same column are not significantly different (Duncan’s test, P < 0.05).
Table 4 Life table parameters (Mean ± SE) of Aphidius matricariae that had developed in hosts treated with different concentrations of Beauveria bassiana. Life table parameters
Tween-80
1 102 (conidia/ml)
2 108 (conidia/ml)
rm R0 K T DT rw
0.3 ± 0.0a 40.9 ± 3.6a 1.3 ± 0.0a 13.1 ± 3.3b 2.4 ± 0.0b 9.3 ± 0.0a
0.3 ± 0.0b 45.3 ± 4.7a 1.3 ± 0.0b 15.0 ± 3.8a 2.7 ± 0.1a 9.0 ± 0.0b
0.3 ± 0.0b 33.9 ± 6.5a 1.294 ± 0.0b 14.1 ± 3.2a 2.7 ± 0.1a 9.0 ± 0.1b
Means followed by different letter in the same row are significantly different (Duncan’s test, P < 0.05). rm, intrinsic rate of increase, R0, net reproductive rate, K, finite rate of increase, T, mean generation time, DT, doubling time, rw, increase rate in 1 week.
(F = 0.25; df = 4, 319; P = 0.9088) and 0.4 ± 0.0 mm (F = 0.60; df = 4, 319; P = 0.6621), respectively, at different time intervals.
Fig. 1. Survival rate (lx) of Aphidius matricariae influenced by two concentrations of Beauveria bassiana compared to control.
3.2. Life table parameters Calculated values of the intrinsic rate of increase (rm) were significantly different (F = 6.73; df = 2, 48; P = 0.003) between parasitoid females from treated and untreated aphids, but no difference was observed in the net reproductive rate (R0) between the two concentrations (1 102 and 2 108 conidia/ml) and the control (Table 4). The intrinsic rate of increase was higher for parasitoids that developed in untreated aphids compared with those that developed in aphids exposed to B. bassiana. The mean generation time was shorter in the controls than in treated wasps. The other parameters including the finite rate of increase (k), the mean generation time (T), the doubling time (DT) were not significantly different between the two concentrations of B. bassiana used. The proportion of wasps surviving at time x (lx) and number of females produced per female per day (mx) were almost similar among the two treatments and between the treatments and control (Figs. 1 and 2).
Fig. 2. Number of females produced per female per day (mx) of Aphidius matricariae influenced by two concentrations of Beauveria bassiana compared to control.
4. Discussion The numbers of mummies from aphids first parasitized then treated with fungus, depended on the time interval between exposure to A. matricariae and application of B. bassiana. Similar results have been obtained by Powell et al. (1986) who demonstrated the successful development of Aphidius rhopalosiphi de Stefani Perez in the aphid host Metopolophium dirhodum (Walker) also depends on the time interval between exposure to the parasitoid and infection with the fungus Pandora (Erynia) neoaphidis (Brefeld) Batko. However, such a difference was not found for Aphelinus asychis (Walker) after exposure to Paecilomyces fumosoroseus Brown and Smith at various intervals (Mesquita and Lacey, 2001). The severe decrease in successful parasitoid mummification when developing in hosts that were infected with B. bassiana 24 h after being parasitized suggests that the presence of an early instar parasitoid larva provides a
competitive advantage to the fungus, while older parasitoid larvae have a high probability of winning the competition for the host aphid, possibly due to a low susceptibility of older larvae to fungus infection (Fransen and van Lenteren, 1994). Competition for food between larvae of A. matricariae and B. bassiana reduces the percentage of parasitoid emergence when the interval between oviposition and fungal infection of the host is less than 96 h. In most cases, there is no evidence for the invasion of the parasitoid larval tissue by the fungus when aphids have first been exposed to the parasitoid and then inoculated with fungus (Powell et al., 1986; Askary and Brodeur, 1999). The lower number of mummies obtained from infected aphids subsequently exposed to parasitoids is likely to be caused by lower survival of parasitoid larvae and/or by host selection; that is, parasitoids
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may have rejected the infected aphids for oviposition (Mesquita and Lacey, 2001). Brobyn et al. (1988) demonstrated that aphids treated with fungus 72 h before exposure to parasitoids were less often attacked and parasitized than healthy aphids. The decrease in the percentage emergence of the F1 generation as a function of the time interval between fungus treatment and parasitism shows that the probability that the fungus outcompetes the parasitoid increases with the time interval. A possible mechanism is that the fungus reduces the host quality for the parasitoid larva, causing mortality of the parasitoid larva. It also results in the pupal stage being prolonged, especially for the development of female parasitoids. In our study, female parasitoids seem to be more sensitive than males in competition with B. bassiana. This is possibly due to the higher growth rate of females (Sequeira and Mackauer, 1992). However, the longevity and morphometrics of F1 A. matricariae were not adversely affected as a consequence of developing within hosts infected with B. bassiana. Females of A. matricariae did not change sex allocation in response to infected aphids in different stages of fungal development. There is little information on the fecundity of parasitoids that emerged from infected host insects (Mesquita and Lacey, 2001), especially about sublethal and chronic effects of entomopathogenic fungi. Our results demonstrated that B. bassiana strain EUT116 did not influence the life-time number of offspring of females (R0) but it reduced the rm value, although similar rm values were obtained with the low and high concentrations. The rm values decreased because the entomopathogenic fungus resulted in an increase of the mean generation time of the parasitoids (T). Fatiha et al. (2008) showed that direct application of Verticillium (=Lecanicillium) lecanii (Zimm.) Viegas decreased the R0 value of Serangium japonicum (Coleoptera: Coccinellidae), a predator of whiteflies, and that different concentrations of the fungus resulted in similar values of the intrinsic rate of increase (rm). Also they indicated that the mean generation time (T) was not significantly different between fungal treatment and control. It is important to consider complementary activity of biocontrol agents (Lacey et al., 1997). Mesquita et al. (1997) reported better control of Diuraphis noxia (Mordvilko) when A. asychis and P. fumosoroseus were combined under field conditions. However, A. colemani Viereck is very susceptible to B. bassiana (commercial formulation, strain JW-1) in the laboratory but shows lower rates of infection in the greenhouse (Ludwig and Oetting, 2001). Scopes (1970) observed that application of A. matricariae was not a suitable method for controlling M. persicae when the aphids were infected with the entomopathogenic fungus Cephalosporium aphidicola Petch (Ascomycota: Hypocreales) as the parasitoids did not complete its development in infected hosts. Our research indicates that B. bassiana strain EUT116 could have an adverse effect on the development of the endoparasitoid, A. matricariae if the parasitoids are released after B. bassiana has been applied. However, our results also show that with good timing, A. matricariae and B. bassiana could potentially be used in combination for the successful biological control of M. persicae. Therefore, application of these two biological agents requires effective time management to avoid antagonistic interactions. Acknowledgments Our sincere thanks are extended to Dr. A. Shirvani, Dr. G. Mohammadinezhad from Shahid Bahonar University of Kerman, Dr. A. Zamani from Razi University of Kermanshah and M. Askari Seyahooei from Institute of Biology, Leiden University for their
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