Biocontrol Science and Technology, 2006; 16(3): 221 /232
Effect of combination treatment with entomopathogenic fungi Beauveria bassiana and Nomuraea rileyi (Hypocreales) on Spodoptera litura (Lepidoptera: Noctuidaeae)
C. UMA MAHESWARA RAO, K. UMA DEVI, & P. AKBAR ALI KHAN Department of Botany, Andhra University, Visakhapatnam, India (Received 5 January 2005; returned 23 February 2005; accepted 12 July 2005)
Abstract In biocontrol of insect pests, efficacy of treatment with multiple pathogens has not been frequently investigated but may have potential for effective management. The possible advantage of a combination treatment with two entomopathogenic fungi / Beauveria bassiana and Nomuraea rileyi / was assessed in laboratory bioassays on second instar Spodoptera litura . From among the fungal isolates of an epizootic population, two isolates of each fungus differing in virulence to S. litura were chosen, one highly virulent and the other with low virulence. The bioassays were carried out at either a continuous temperature of 259/18C or at a temperature cycle of 329/28C 8 h/219/28C 16 h to mimic the field temperatures during the epizootic. Treatments with the two fungi were done both simultaneously and sequentially. In combination treatments at 259/18C, in all isolate combinations, a majority of the larvae showed N. rileyi induced mycosis; the percentage mortality and speed of kill of insects in these treatments was similar to the N. rileyi isolate used in the combination treatments. At the temperature cycle of 329/28C 8 h/219/28C 16 h, in all combination treatments, all the dead insects exhibited B. bassiana mycosis; the mortality pattern was similar to the B. bassiana isolate used in the combination treatments. The adverse effect of high temperature on virulence of N. rileyi was however, not evident in in vitro growth assays. Combination treatment with both fungi did not have a synergistic effect on insect mortality.
Keywords: Bioassays, combination treatment, entomopathogenic fungi, epizootics, Beauveria bassiana, Nomuraea rileyi, Spodoptera litura, temperature effect, India
Introduction In inundative applications of microbial control agents, combination treatment with two entomopathogens offers an attractive biorational strategy. If the two entomopathogens complement each other, or act synergistically, a beneficial effect can be obtained. One of the limitations of fungi as microbial control agents is that, each species and strains within a species are usually efficacious in a narrow window of climatic conditions. Farmers must manage pest complexes under various weather conditions and therefore, prefer using broad spectrum pesticides. Theoretically, this
Correspondence: K. Uma Devi, Department of Botany, Andhra University, Visakhapatnam, 530 003, AP, India. Tel: 91 891 2525582/2844563. Fax: 91 891 2755547. E-mail:
[email protected] ISSN 0958-3157 print/ISSN 1360-0478 online # 2006 Taylor & Francis DOI: 10.1080/09583150500335632
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issue could partly be addressed through the development of an appropriate co-formulation of two or more fungi or fungal strains with different host ranges and ecological tolerances (Wang et al. 2002). However, very few studies have reported the combined application of two or more entomopathogenic fungi with other pathogens with the aim of increasing efficacy (Lecuona & Alves 1988; Glare 1994; Timper & Brodie 1995; Inglis et al. 1997b, 1999, 2001). Wang et al. (2002) observe that there is a need for co-formulation studies to examine if fungi will act synergistically or in a complementary fashion. Fungal ‘cocktails’ have been tested in a few combinations. Synergistic effects have been reported in treatments involving a combination treatment with Beauveria bassiana and Bacillus thuringiensis on Colorado potato beetle (Lacey et al. 1999) and European corn borer (Lewis et al. 1996) and in combination treatments with multiple entomopathogens (Ferron 1978; Fuxa 1979). Inglis et al. (1997b, 1999) tested the efficacy of simultaneous treatment of the grasshopper, Melanoplus sanguinipes with B. bassiana and Metarhizium anisopliae to determine if efficacy over different temperatures could be increased. Co-inoculation of Galleria mellonella with two strains of B. bassiana was done to test if it would widen the effective window of water activities (Wang et al. 2002). Two co-formulated strains of M. anisopliae were assayed against the mustard beetle, Phaedon cochleariae (Leal-Bertioli et al. 2000). In both these experiments with two strains of the same species, one of the strains dominated. Some studies have attempted to understand the factors involved in dominance of any one entomopathogen when both of them exist in the field (Soper & Ignatowicz 1986), or, when they are applied in combination in vitro (Inglis et al. 1997b). In treatments involving two insect pathogens, temperature (Inglis et al. 1997b, 1999), virulence of the pathogens (Wang et al. 2002; Leal-Bertioli et al. 2000) and time interval between the inoculation of the two pathogens (Malakar et al. 1999a) were found to affect the infection process and the subsequent expression of either or both of the pathogens. Epizootics of Nomuraea rileyi are a regular annual phenomenon in the cotton and peanut fields in Guntur district of Andhra Pradesh state of south India (Uma Devi et al. 2003). Epizootics during mid-1980s were reported to be due to B. bassiana (Uma Devi et al. 2003). Since 2000, sporadic occurrences of B. bassiana in N. rileyi epizootics have been observed (Uma Devi et al. 2003). Mixed infection of a single Spodoptera litura larva with both these fungi has also been observed (Uma Devi et al. 2003). Co-occurrence of two fungi in epizootics is rare though not impossible (Humber, pers. comm.; Aoki 1974; Papierok et al. 1986). Here, we report the results of bioassays with a combination treatment of B. bassiana and N. rileyi. Our objectives were to: (a) explore the possibility of co-formulating them for increased efficacy and (b) develop hypotheses to help explain the sporadic occurrence of B. bassiana in epizootics predominantly induced by N. rileyi . Materials and methods Insects A laboratory stock (egg masses on paper strips) of S. litura in the fifth generation of breeding from the field collected insects, was obtained from the project directorate of biological control, Bangalore, India. The egg masses were placed on castor leaf bouquets with petioles dipped in water in conical flasks. The larvae that hatched out were transferred to fresh castor leaves (washed and wiped) / three to four larvae per
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leaf. The stalk of the leaf was wrapped with a cotton plug dipped in water to keep the leaf fresh (Goettel & Inglis 1997). Two to three castor leaves were placed in one plastic bowl (15 /10 cm). The larvae were transferred to fresh castor leaves every day. Until treatment, the bowls with larvae were placed in an environmental chamber set to 259/ 18C with a 16/8-h (light/dark) cycle and 90% ambient humidity. Fungal isolates A sample of 50 isolates (46 of N. rileyi and four B. bassiana ) from an epizootic population on S. litura in peanut fields of south India (Guntur district) collected in 2000, were characterized for their virulence in laboratory bioassays (unpublished results). From this sample, two isolates of each species / one with high and the other with low virulence against S. litura were selected. They are labeled as HBb and LBb for highly virulent and isolate with low virulence B. bassiana and similarly, HNr and LNr for N. rileyi . The cultures were obtained from conidia stored as glycerol stocks at /208C on Sabouraud medium (dextrose for B. bassiana and maltose for N. rileyi ) (Goettel & Inglis 1997). For bioassays, conidia were harvested from 10- to 14-day-old cultures. Aqueous conidial suspensions of a concentration of 108 conidia/mL were made and the viability of the conidia was checked a day prior to the bioassays as described in Goettel and Inglis (1997). Conidia which showed more than 90% germination were used in the bioassays and growth assays. Insect treatments The combination treatments were done in two ways: (a) simultaneous treatment of larvae with both fungi; and (b) sequential treatment, one fungal pathogen followed by the other fungal pathogen at 6-h time intervals up to 24 h. The treatments were done in all possible combinations: (i) HNr/HBb; (ii) HNr/LBb; (iii) HBb/LNr; and (iv) LNr/LBb. For sequential treatment, two sets of treatments were set up differing in the pathogen applied first. Treatments with single fungal isolates: HBb, LBb, HNr and LNr were done to serve as positive controls. Insects treated with water with 0.02% Tween 80 (Sigma Aldrich) served as negative controls. For each treatment batch, 50 second instar larvae were chosen at random. The fungal inoculum (20 mL/insect of aqueous suspension of 108 conidia/mL) was dispensed with a micropipette on the entire surface of each larva. In combination treatments, the 20 mL of conidial suspension consisted of 10 mL (108 conidia/mL) of each of the two fungal species. To avoid crowding of larvae (which affects their response), the 50 larvae of each treatment batch were sorted into five bowls (15 / 10 cm) with 10 larvae per bowl. Fresh castor leaves cleaned and wiped and their stalks wrapped in cotton plugs dipped in water were placed in the bowls (five to six per bowl) to serve as food for the larvae. The treated larvae were monitored daily for mortality. The bowls were cleaned of litter every day and fresh castor leaves were placed. Mortality was recorded until all insects either died or pupated. Pupae and larval cadavers were transferred individually into Petri plates lined with moist filter paper to facilitate mycosis and placed at the temperature at which the treated insects were kept. The fungal pathogen expressed on each of them was identified and recorded.
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To examine the effect of temperature on each fungal species, experiments of simultaneous treatment with the two fungal pathogens were conducted at two temperature regimes: one at a simulated temperature at the time of the epizootic (March): 329/28C 8 h/219/28C 16 h and the other, at a constant temperature of 259/ 18C in an environmental chamber set to the test temperature with a 16/8-h (light/ dark) cycle and 90% ambient humidity. The sequential treatment experiment was done only at 259/18C. The treatments were set up as a completely randomized block design with two replicates per treatment (Goettel & Inglis 1997). The experiment was repeated once. Growth assay In vitro growth assay of the isolates of the two fungi were done at the two temperature regimes at which the insect bioassays were conducted to determine if infection potential of the fungal isolates can be assessed from their in vitro growth rate at that temperature. The growth assays were done in liquid medium as described by Goettel and Inglis (1997). Assays were set up in 250-mL conical flasks with 100 mL of liquid Sabouraud medium. Each flask was inoculated with 1 mL of an aqueous conidial suspension of (108 conidia/mL) and incubated at the test temperatures (a continuous temperature of 259/18C or an oscillating temperature cycle of 329/28C 8 h/219/28C 16 h) at 120 rpm for 10 days. On the 11th day, the fungal culture was filtered and dried to constant weight in an oven at 808C. The dry weight of the culture was noted. The experiments were set up in duplicate and repeated twice (three experiments in total). Data analysis There was no mortality in the controls (untreated) in any of the experiments. Percentage mycosis was estimated as proportion of dead insects that showed mycosis. The mortality and mycosis values were arcsine percent square root transformed and the means were back-transformed (Gomez & Gomez 1984). Mortality data was subjected to ANOVA with test temperatures and treatment types (combination versus single treatments with their respective fungal isolates) as the main effects. In ANOVA analysis, the data of the replicates within an experiment and the data from experiments repeated in time were not pooled. The differences between the replicates were not significant in all the treatments. To assess the speed and pattern of mortality of the insects, the data was tested for fit to Weibull distribution pattern for computing median lethal time. In some of the treatments, insect mortality was below or near 50%. Hence, the data did not fit Weibull distribution. Therefore the time to mortality response in each combination treatment was plotted as a graph along with the two respective positive controls (single treatments with respective fungal isolates). Dry mass values of the isolates in growth assays at the two test temperatures were analyzed for any significant effect of temperature on the growth of the fungal isolates through ANOVA. The data of the replicates within an experiment and the experiments repeated in time were not pooled for ANOVA analysis. The statistical software STATISTICA ver. 5.0 (StatSoft Inc. 1995) was used for data analysis.
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Results Insect bioassays Effect of temperature on the virulence of Nomuraea rileyi and Beauveria bassiana. Mortality and speed of kill of the highly virulent isolate of N. rileyi, decreased significantly (F /45.1; P /0.001; df for treatments /1; df for error /3) with change of temperature regime from a continuous 259/18C to a fluctuating cycle of 329/28C 8 h/219/28C 16 h). With the low virulence N. rileyi isolate, there was a slight decrease in mortality (F /13.4; P /0.07; df for treatments /1; df for error /3) but speed of kill was unaffected (Figures 1/3). At both temperature regimes, all the insects that died of infection showed profuse mycosis (Figure 1). In contrast, in both B. bassiana isolates, virulence increased when the temperature regime was changed from a continuous 259/18C to a fluctuating cycle of 329/28C 8 h/219/28C 16 h. There was an increase in speed of kill and a significant increase in
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Figure 1. Percent mortality and mycosis in bioassays of second instar larvae of Spodoptera litura with Beauveria bassiana , Nomuraea rileyi and a combination treatment (Cb) simultaneously with both fungi (a) at 259/18C; (b) at a temperature cycle of 329/28C 8 h/219/28C 16 h. HNr and LNr are high and low virulence N. rileyi; HBb and LBb are high and low virulence B. bassiana. At 259/18C (A), in HBb/LNr combination, combined treatment had a negative effect compared to the more virulent isolate in the combination (F /11.3; P /0.00; df (treat) /1; df (error) /3). At a temperature cycle of 329/28C 8 h/219/28C 16 h (B), in HBb/HNr and LBb/LNr combinations, combination treatments had a negative effect compared to the more virulent isolate in the combination (F /11.3; P /0.00; df (treat) /2 df (error) /3; F /7.3; P /0.00; df (treat) /2; df (error) /7). The difference in combination treatment was slightly synergistic in LBb/HNr combination (F /7.8; P /0.00; df (treat) /1; df (error) /3).
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Figure 2. Cumulative mortality of second instar larvae of Spodoptera litura bioassayed with Beauveria bassiana and Nomuraea rileyi alone and in combination (Cb) with both fungi at 259/18C. In all combination treatments, pattern and magnitude of mortality is similar to N. rileyi . HNr and LNr are high and low virulence N. rileyi ; HBb and LBb are high and low virulence B. bassiana.
mortality (F /45.1; P B/0.003; df for treatments /1; df for error /3) (Figures 1/3) but the proportion of mycotic insects decreased significantly (F /13; P /0.04; df for treatments /1; df for error /3) at fluctuating temperature cycle of 329/28C 8 h/219/ 28C 16 h (Figure 1). Thus, a fluctuating temperature cycle had a negative effect on mortality and speed of kill with N. rileyi , but a positive effect on B. bassiana isolates used in the study. Both high and low virulence isolates of N. rileyi have a high mycotic potential (expressed on all dead insects) and it is unaffected by temperature. The effect of temperature on mycotic potential of the two B. bassiana isolates was not consistent. The mycotic potential was less in the low virulence isolate than in the highly virulent one and it reduced with increase in temperature. Effect of combination treatment with Nomuraea rileyi and Beauveria bassiana. Mortality. In bioassays conducted at 259/18C, both in the simultaneous and sequential combination treatments, no synergistic effect is evident. Proportion and pattern of mortality was similar to N. rileyi even when the B. bassiana isolate was more virulent than the partner N. rileyi isolate (e g., HBb and LNr combination) (Figure 1a, 2, 4, Figure 5)
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Figure 3. Cumulative mortality of second instar larvae of Spodoptera litura bioassayed with Beauveria bassiana and Nomuraea rileyi alone and in combination (Cb) with both fungi at a temperature cycle of 329/ 28C 8 h/219/28C 16 h. The mortality pattern in all combination treatments except LBb/HNr (B), is similar to B. bassiana . HNr and LNr are high and low virulence N. rileyi ; HBb and LBb are high and low virulence B. bassiana.
In the simultaneous treatments at 329/28C 8 h/219/28C 16 h, the mortality pattern was similar to the B. bassiana isolate except in HNr/LBb combination, where it was similar to N. rileyi (Figure 3). Percentage mortality was, however, lower than the B. bassiana isolate used in the combination and similar to N. rileyi in all except the HBb/LNr combination where it was similar to B. bassiana (Figure 1b and 3). In all these combinations, except HNr/LBb, the B. bassiana isolate was more virulent than N. rileyi isolate. Mycosis. In all the simultaneous combination treatments, a proportion of dead insects showed no mycosis. In these treatments at 259/18C, N. rileyi- induced mycotic cadavers were predominant with only a few B. bassiana- induced mycotic cadavers (Figure 1a).There was a considerable ( /35%) proportion of B. bassiana -induced mycotic cadavers only in the HBb/LNr combination treatment (Figure 1a). At 329/28C 8 h/219/28C 16 h, all the cadavers exhibited B. bassiana- induced mycosis (Figure 1b). The main purpose of the sequential treatments was to examine which fungus would predominate in the larva if there was a time gap between the exposure of the insect to the two fungal species. In the simultaneous exposure treatment, cadavers expressing either of the fungi were found only at 259/18C. Therefore, the sequential inoculation experiments were carried out only at this temperature.
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Figure 4. Percent values of mortality and mycosis in bioassays (at 259/18C) of second instar larvae of Spodoptera litura with Beauveria bassiana , Nomuraea rileyi and in combination (Cb) with application of one fungal species followed sequentially by the second fungal species at different time intervals. Mortality in all combination treatments is similar to N. rileyi . Mycosis in all combination treatments except HBb/LNr (C) is predominantly due to N. rileyi . The isolate mentioned first is the one applied first. HNr and LNr are high and low virulence N. rileyi ; HBb and LBb are high and low virulence B. bassiana.
In sequential treatments, the fungus that was expressed on the cadaver depended on the isolates involved and the timing of the inoculation of the two fungi. In all combination treatments except with HBb/LNr, cadavers with N. rileyi -induced mycosis were predominant with some (2/20%) cadavers in some treatments exhibiting B. bassiana (Figure 4). In vitro growth assay A significant increase in biomass was observed in low virulence B. bassiana and high virulent N. rileyi at a temperature cycle of 329/28C 8 h/ 219/28C 16 h compared to culture at a continuous temperature of 259/18C. Temperature did not have significant effect on the growth of the low virulence N. rileyi. In the high virulent B. bassiana, a significant decrease in growth was observed at the oscillating temperature cycle (Table I). Discussion Temperature influenced the infection potential of both B. bassiana and N. rileyi but in different ways. Change in temperature from a continuous 259/18C to a fluctuating temperature cycle of 329/28C 8 h/219/28C 16 h, had a negative impact on the virulence of N. rileyi isolates and a positive influence on B. bassiana isolates. The extent to which virulence was affected varied with the isolate in both fungi. The in vitro growth rate of isolates of N. rileyi and B. bassiana at these two test temperatures did not, however, match their infection potential at these temperatures. Similarly, in
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Figure 5. Cumulative mortality of second instar larvae of Spodoptera litura bioassayed with Beauveria bassiana and Nomuraea rileyi alone and in combination (Cb) (sequential inoculation of the two fungi) at 259/18C. In all sequential combination treatments at this temperature (259/18C), the pattern and magnitude of mortality is very similar to N. rileyi . A representative graph of each set of sequential treatments with 6-h time gap between inoculation of the two fungi is shown (the figures representing entire data in supplementary material). HNr and LNr are high and low virulence N. rileyi ; HBb and LBb are high and low virulence B. bassiana.
Beauveria brongniartii an isolate that showed increased mycelial growth at 458C (than at optimal temperature) was not found infectious at 458C (Xavier & Khachatourians 1996). The different impact of temperature on the infection potential of the two fungi was also reflected in combination treatments. At a temperature cycle of 329/28C 8 h/219/ 28C 16 h, all the cadavers in combination treatments with both fungi expressed B. bassiana- induced mycosis. In the combination treatments at 259/18C, cadavers expressing either N. rileyi or B. bassiana were observed, the former being predominant with a few cadavers of the latter type in most treatments. Thus, temperature had a major influence on which of the two fungi sporulated from the cadaver in combination treatments. Similarly, temperature influenced competitive colonization by one of the fungi in dual treatments with B. bassiana and M. anisopliae (Inglis et al. 1997b). But, unlike the results in this experiment with B. bassiana and N. rileyi, in combination treatments with B. bassiana and M. anisopliae at temperature cycles with extreme oscillating temperatures, i.e., 15/358C and 10 /408C, M. anisopliae had a competitive advantage (Inglis et al. 1999). At temperature cycles with a smaller range in temperature oscillations (20 /308C), no significant advantage for one species was observed (Inglis et al. 1997b). Temperature was found to influence the expression of one of the entomopathogens (virus/fungus) in a combination treatment of gypsy moth with nuclear polyhedrosis virus and Entomophaga maimaiga (Malakar et al. 1999a).
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Table I. In vitro growth assay of Nomuraea rileyi and Beauveria bassiana (Hyphomycetes) at two temperature regimes. Dry weight (in mg) atb a
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103.29/4.8
123.89/6.3
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144.59/12.3
131.59/15.4
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76.39/4.8
HNr
53.59/6.4
68.59/0.96
% Difference over controlsc (ANOVA values for temp effect) 10.59 (F /765.5; P /0.001; df (treat) /1; df (error) /5)* /9 (F /115.1; P /0.001; df (treat) /1; df (error) /5)* 3.7(F /0.4; P /0.6; df (treat) /1; df (error) /5) 28.1 (F /47.6; P /0.006; df (treat) /1; df (error) /5)*
a
LBb, low virulence Beauveria bassiana ; HBb, highly virulent B. bassiana ; LNr, low virulence Nomuraea rileyi ; HNr, high virulent N. rileyi. bThe values represent mean9/SE of six replicates (in time and in space). c Negative value when test value is lower than control. *Highly significant.
In all the combination treatments with B. bassiana and N. rileyi, only one of the fungus sporulated on the larval cadaver but never both. Similar observations of one member in a combination treatment being dominant have been reported in almost all experiments involving dual treatments (Inglis et al. 1997b, 1999; Soper & Ignatowicz 1986; Leal-Bertioli et al. 2000; Wang et al. 2002). In the experiments of B. bassiana and N. rileyi described here, the mortality pattern of the insects (day of onset of mortality and nature of the time mortality response line) in combination treatments, resembled the mortality pattern of the fungus which sporulated from the cadaver. Similarly, in treatments of gypsy moth larvae treated simultaneously with an entomophthoralean fungus Entomophaga maimaiga and nuclear polyhedrosis virus, dead larvae that showed fungal sporulation showed a mortality pattern similar to the fungus and those with occlusion bodies of the virus, showed a mortality pattern similar to the virus (Malakar et al. 1999b). Treatment of S. litura larvae with a combination of B. bassiana and N. rileyi did not yield a beneficial result in laboratory bioassays. Inglis et al. (1997b, 1999) also reported that there was only a marginal advantage (depending on temperature) in combination treatment of grasshoppers with M. flavoviridae and B. bassiana . Wang et al. (2002) and Leal-Bertioli et al. (2000) observed that in combination treatments, the dose of the effective isolate is diluted due to co-formulation and hence, synergistic effect is not possible; instead, the time to death of the insect may be extended. Manipulation of the concentration of the components in the combination concoctions resulted in synergistic effect in Heliothis zea (Fuxa 1979). In treatments with N. rileyi alone, all dead larvae exhibited mycosis but in all combination treatments, some proportion of the dead insects did not show mycosis. This could be an outcome of an antagonism between the two fungi. B. bassiana and N. rileyi were found mutually compatible in in vitro tests conducted at a continuous temperature of 259/18C (Mohan 2002). Mutual relationship between two fungal species is reported to be influenced by temperature (Inglis et al. 1997b). Moreover, in vitro relationships do not always reflect Interactions in vivo (Soper & Ignatowicz 1986; Inglis et al. 1999).
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