Journal of Invertebrate Pathology 102 (2009) 6–13
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Efficacy of indigenous entomopathogenic nematodes (Rhabditida: Heterorhabditidae, Steinernematidae), from Rio Grande do Sul Brazil, against Anastrepha fraterculus (Wied.) (Diptera: Tephritidae) in peach orchards Carla R.C. Barbosa-Negrisoli a, Mauro S. Garcia a, Claudia Dolinski b,*, Aldomario S. Negrisoli Jr. a, Daniel Bernardi a, Dori Edson Nava c a b c
Laboratório de Biologia de Insetos e Controle Biológico, Universidade Federal de Pelotas, C.P. 354, 96010-900 Pelotas, RS, Brazil Laboratório de Fitopatologia e Entomologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos de Goytacazes/RJ, Brazil Laboratório de Entomologia, EMBRAPA/CPACT, Pelotas, Brazil
a r t i c l e
i n f o
Article history: Received 9 February 2009 Accepted 12 May 2009 Available online 19 May 2009 Keywords: Biological control Brazilian native strains Heterorhabditis bacteriophora Steinernema riobrave Fruit flies Rosaceae
a b s t r a c t Laboratory, greenhouse, and field experiments were performed with the objective of selecting efficient indigenous strains of entomopathogenic nematodes (EPNs) from Rio Grande do Sul (RS) state, Brazil, for controlling the South American fruit fly, Anastrepha fraterculus (Wied.). Laboratory experiments were conducted in 24 well-plates filled with sterile sand and one insect per well. In greenhouse experiments, plastic trays filled with soil collected from the field were used, while in field experiments, holes were made in soil under the edge of peach tree canopies. Among 19 EPN strains tested, Heterorhabditis bacteriophora Poinar RS88 and Steinernema riobrave Cabanillas, Poinar, & Raulston RS59 resulted in higher A. fraterculus larval (pre-pupal) and pupal mortality, with LD90 of 1630, 457 and 2851, 423 infective juveniles (IJs)/cm2, respectively. Greenhouse experiments showed no differences in pupal mortality at 250 and 500 IJs/cm2 of either nematode. In the field, H. bacteriophora RS88 and S. riobravae RS59 sprayed individually over natural and artificially infested fruit (250 IJs/cm2) resulted in A. fraterculus larval mortality of 51.3%, 28.1% and 20%, 24.3%, respectively. There was no significant difference in A. fraterculus pupal mortality sprayed with an aqueous suspension of either nematode; however, when using infected insect cadavers, H. bacteriophora RS88 was more efficient than S. riobrave RS59. Our results showed that H. bacteriophora RS88 was more virulent to insect larvae, with an efficient host search inside the infested fruit and control of pupae in the soil after being applied by aqueous suspension or infected cadavers. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Rio Grande do Sul (RS) state, Brazil, has great climatic variation, partially from topographical characteristics that facilitate agricultural diversification with possibilities for temperate or tropical fruit cultivation, depending on the micro-region. With such characteristics this Brazilian state is important for national productivity of grapes (53.96%), peaches (50.58%) and apples (42.34%), among others (Agrianual, 2008). The southern region of the state is an important producer of peaches, especially for industry (Kovaleski et al., 2000a). Many problems, such as exotic and native plant pests and diseases, are present in these fruit orchards. Among the native pests, the South American fruit fly Anastrepha fraterculus (Wied.) (Diptera: Tephritidae), is of great importance and is considered the key-pest in apple and peach crops (Kovaleski et al., 2000a). * Corresponding author. Fax: +55 22 27346863. E-mail address:
[email protected] (C. Dolinski). 0022-2011/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2009.05.005
Fruit damage may be external or internal. External damage is produced by the female insect’s oviposition into the fruit during the period early ripening (Kovaleski et al., 2000a). Internal damage occurs from larvae feeding inside the fruit (Kovaleski et al., 2000b). Control has been achieved by spraying chemicals (Salles, 1995). Despite effective control, many problems arise from the constant and excessive use of these chemicals, resulting in human contamination, as well as water, air, soil, and general environmental pollution. Consequently, international market demands, and changes in Brazilian society’s environmental awareness require healthier products for consumption, such as chemical free or low residue levels in fruit (Fachinello et al., 2004). In this context, biological control of A. fraterculus has been used as an alternative, due to mortality caused by natural enemies at different life stages. A reduction in the fruit fly population occurs due to parasitoids, predators and pathogens (Wharton, 1993). Among these, entomopathogenic nematodes (EPNs) show potential for controlling Diptera (Toledo et al., 2006a) and may help to control of A. fraterculus (Lindegren et al., 1990), since various tests
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have been performed with different species of the same genus: A. ludens Loew (Lezama-Gutierrez et al., 1996; Hernández, 2003), A. obliqua (Macquart) (Toledo et al., 2005a), A. serpentina Wied. (Toledo et al., 2006b), and A. suspensa (Loew) (Beaver and Calkins, 1984). Thus, the objective here was to select EPN strains native to RS state, and to verify their influence on the South American fruitfly in laboratory, greenhouse and field tests. This is the first study on susceptibility, virulence and efficiency of EPNs indigenous to RS state on pre-pupae and pupae of A. fraterculus, suggesting the use of these entomopathogens as biological control agent for this pest in peach orchards in Brazil. 2. Materials and methods Pre-pupae and pupae of A. fraterculus were obtained from EMBRAPA/CPACT in Brazil, and were grown on artificial diet according to Salles (1992). Selection of strains and determination of lethal concentration were performed in the laboratory, and the strain efficiency was tested in a greenhouse at the Universidade Federal de Pelotas, Pelotas, RS, Brazil. Were considered pre-pupae those larvae that left the fruit presenting a ‘‘jumping habit”. All pupae used in the experiments were one to 2-days-old pupae with tegument in processo of sclerotization. Entomopathogenic nematodes used were: Steinernema rarum (Doucet) (RS47, RS55, RS70, RS89, RS90, RS102, RS106), S. glaseri (Steiner) RS38, S. feltiae (Filipjev) RS76, S. riobrave (Cabanillas, Poinar, & Raulston) RS59, Steinernema sp. (RS69, RS92), and Heterorhabditis bacteriophora Poinar (RS33, RS56, RS57, RS58, RS72, RS88, RS107) isolated from soils of RS state (Table 1). The isolation method was according to Bedding and Akhurst (1975). Table 1 Collecting point of species and isolates used in described tests. Nematode species
Isolate
Origen
Heterorhabditis bacteriophora, Poinar, 1976
RS33
H. bacteriophora
RS88
H. bacteriophora
RS107
H. bacteriophora
RS72
H. bacteriophora
RS57
H. bacteriophora
RS56
H. bacteriophora
RS58
Steinernema feltiae (Filipjev, 1934) Wouts, Mracek, Gerdin, & Bedding, 1982 Steinernema glaseri (Steiner, 1929) Wouts, Mracek, Gerdin, & Bedding, 1982 Steinernema rarum (Doucet, 1986) Mamiya, 1998
RS76
S. rarum
RS55
S. rarum
RS89
S. rarum
RS106
S. rarum
RS70
S. rarum
RS102
S. rarum
RS47
Steinernema riobrave (Cabanillas, Poinar, & Raulston, 1994) Steinernema sp.
RS59
Steinernema sp.
RS92
Capão do Leão, RS State, Brazil Julio de Castilhos, RS State, Brazil Arroio Grande, RS State, Brazil Rosário do Sul, RS State, Brazil Lagoa Vermelha, RS State, Brazil Bom Jesus, RS State, Brazil Lagoa Vermelha, RS State, Brazil Cacequi, RS State, Brazil Coxilha, RS State, Brazil Canguçu, RS State, Brazil Muitos Capões RS State, Brazil Canguçu, RS State, Brazil Cidreira, RS State, Brazil Dom Pedrito, RS State, Brazil São José do Herval, RS State, Brazil Planalto, RS State, Brazil Lagoa Vermelha, RS State, Brazil Dom Pedrito, RS State, Brazil Canguçu, RS State, Brazil
RS38 RS90
RS69
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These EPNs were produced according to Kaya and Stock (1997) and held in plastic (zip-lock) bags containing sponge inside. To minimize contaminants, EPNs were kept in an environment controlled chamber at 12 °C, using distilled water with 0.1% hypochlorite solution during the whole process. 2.1. Selection of entomopathogenic nematode strains to control prepupae and pupae of A. fraterculus Eighteen isolates of EPNs as shown in Table 1 were tested in virulence bioassays on pre-pupae and pupae of A. fraterculus, and the experiment was repeated. In bioassays, pre-pupae and pupae were infected according to methodology modified from Grewal et al. (1999) in 24 well-plates (1.1 cm2/well) containing 2.5 g sterilized sand (10% w/v) per well and inoculated with 100 lL of a suspension of 0 (control) and 100 infective juveniles (IJs)/well (=91 IJs/ cm2), and then incubated in a controlled chamber at 25 ± 1 °C, RH 70 ± 10% and 12 h photoperiod. Pre-pupal mortality and fly emergence was evaluated at three and 12 days, respectively. Dead pre-pupae and pupae were dissected under stereo-microscope to verify the presence of EPNs inside. The experiment had 10 replicates, with 12 individuals for each isolate. Data on pre-pupae and pupae mortality were subjected to analysis of variance (ANOVA) and differences between treatment means were established by Tukey’s HDS test at P < 0.05 probability. 2.2. Lethal dose (LD50) and (LD90) of S. riobrave RS59 and H. bacteriophora RS88 on A. fraterculus Lethal dose (LD50 and LD90), of the two isolates that caused the highest pre-pupae and pupae mortality in the previous experiment (S. riobrave RS59 and H. bacteriophora RS88), were determined using the previously mentioned methodology. These two isolates were used in the rest of the experiments and isolate names are not mentioned hereafter when only these isolates were used. Pre-pupae and pupae were inoculated with: 0 (control), 50, 100, 150, 200, 250 IJs/well (0; 45; 91; 136; 182; 227 IJs/cm2), and stored as above. Evaluation of mortality and emergence from pre-pupae and pupae was the same as the previous bioassay. Data of A. fraterculus pre-pupae and pupae mortality were subjected to Probit analysis at P < 0.05 probability using the software Polo Plus 1.0 (Leora Software, 2008). 2.3. Efficiency of S. riobrave and H. bacteriophora on A. fraterculus pupae in greenhouse The control of A. fraterculus pupae in the soil with S. riobrave and H. bacteriophora was tested in a greenhouse experiment. The treatments had three concentrations of the selected isolates: 0 (control), 250 IJs/cm2 (half of the LD90) and 500 IJs/cm2 (about LD90). One-dayold pupae were buried 2 cm deep, distributed in two rows with a distance of 13 cm between rows and 7 cm between pupae, with a total number of 10 insects per plastic tray (42 28 6 cm). Trays were filled with 2.5 kg of clay soil (argissoil) from the orchard where field experiments were performed. Using a manual sprayer (PCP-1P GuaranyÒ) was used to apply 100 mL of the IJs solution, plus an additional 100 mL of distilled water, to the trays. Subsequently, trays were covered with a thin cloth, and closed with a rubber ribbon at the edges to prevent emerged adult insects from escaping and natural enemies from entering. The experiment had 10 replicates per treatment; each tray was considered a replicate. Emergence was evaluated after 12 days. Non-emerged pupae were transferred to glass tubes (2.5 8.5 cm), closed with wet cotton, and kept in the laboratory at 25 ± 1 °C for three additional days. After this period, non-emerged insects were considered dead, since it was impossible to verify symptoms from EPN infection due to natural pupal darkening (dark
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brown) (Quesada-Allué, 1994). Dead pupae were dissected under stereo-microscope to verify the presence of EPNs. Mean air temperature and relative humidity, 25 ± 3 °C and 60 ± 20%, were recorded every day at 9:00 and 16:00 with a digital thermo-hygrometer. To keep ideal humidity and enhance the entomopathogen’s action, water was sprayed when needed. The bioassay was repeated and completely randomized. Pupal mortality data were subjected to ANOVA, and differences between treatment means were established with Tukey’s HDS test at P < 0.05 probability. 2.4. Field experiment Field experiments were performed at EMBRAPA/CPACT in a 10year-old peach orchard (cultivar ‘‘Capdebosque”) that was free of insecticides. First, soil samples were collected in the experimental area and evaluated in the laboratory by the insect-trap method (Bedding and Arhust, 1975) to verify the absence of EPNs. No nematodes were found in these soil samples. Experimental units were open soil trenches (55 29 17 cm) under the tree canopy, according to methodology modified from Toledo et al. (2006a). To prevent interference from natural enemies, especially ants, the soil from the trenches was contained in wooden boxes coated at the base and sides with black plastic canvas, and covered at the top with thin cloth (voile) attached with metal clips, allowing aeration, and preventing escape of emerged adults. A manual motorized sprayer (10 L, model 428-01, GuaranyÒ) with a calibrated Teejet fan-type nozzle was used to apply aqueous suspensions of 250 IJs/cm2 of S. riobrave and H. bacteriophora. Air temperature and relative humidity records were obtained from a meteorological station at EMBRAPA/CPACT, and soil moisture was kept near ideal to enhance EPN action. Naturally and artificially infested peach fruit were used to evaluate control of A. fraterculus pre-pupae by S. riobrave and H. bacteriophora. Naturally infested fruits were collected in the field, under the tree canopy, selecting those showing injuries by the fruit-fly (fruit peel perforation and necrosis). Artificially infested fruits were placed individually in cylindrical plastic cages (10.5 cm diameter 9 cm high) with thin cloth (voile), and fixed on branches with strings. Each cage contained one fruit at the early development stage (Raseira and Centellas-Quezada, 2003) and one A. fraterculus female ready for oviposition. After 3 days, females and fruits were taken from cages. Thirteen days after fruit was exposed to females, larvae became pre-pupae, a stage in which there is no more feeding, and this fruit was used in the experiments. Dead pre-pupae and pupae were dissected under stereo-microscope to verify the presence of EPNs and bacteria. 2.5. Efficacy of S. riobrave and H. bacteriophora on A. fraterculus prepupae in peaches in the field The efficacy of EPNs to control A. fraterculus pre-pupae was tested in peach fruit at the ripening stage during the period of January 7th–14th, 2008. Six artificially infested fruit and 10 fruit collected in the field (average weight of 70–80 g), were distributed in each box, sprayed with an aqueous suspension of both nematodes and closed with voile fixed by metal clips. After 7 days, fruit from each box was removed, conditioned in plastic bags and transported to the laboratory to verify pre-pupae mortality with and without the presence of EPNs. Two field tests were performed, one after another. 2.6. Efficacy of two inoculation techniques of S. riobrave and H. bacteriophora on A. fraterculus pupae in peach fruit in the field Two inoculation methods were tested with the same nematodes previously selected, during February 30th–March 27th, 2008. An
average relative humidity of 79 ± 6%, air temperature of 21 ± 2 °C, soil temperature of 25 ± 2 °C, and total rain of 137 mm were registered. Ten A. fraterculus 1-day-old pupae were placed in each plot (2 cm deep and between them), around insect cadavers of G. mellonella infected 4 days before with S. riobrave and H. bacteriophora. Nematode application with aqueous suspensions was linear in each plot. Adult emergence was evaluated 12 days after treatment, as previously described for the greenhouse experiment. To determine the quantity of the cadavers to be used in the field, repeated bioassays determined the production of both nematodes on G. mellonella larvae. Thus, 100 G. mellonella larvae were inoculated with 2000 IJs of each EPN species, in plastic boxes (24 15 5 cm) containing two filter papers. After 72 h, insect cadavers were transferred to modified White traps (White, 1927) and emerged IJs were collected for 5 days to establish the average production per larvae to determine the number of cadavers to be used in the field. A completely randomized design was used, and pupal mortality data was subjected to ANOVA, and differences between treatment means were determined by Tukey’s HDS test at P < 0.05 probability.
3. Results and discussion 3.1. Selection of entomopathogenic nematode strains to control prepupae and pupae of A. fraterculus Pre-pupae of A. fraterculus exposed to EPNs for 72 h were susceptible to all 19 EPNs isolates tested, when compared to the control (F = 263; df = 19, 18; P = 0.01) (Fig. 1). While isolates of S. rarum RS47 and RS106 were not statistically different from the control, isolates of H. bacteriophora RS88 (55% and 51%) and S. riobrave RS59 (58% and 53%) resulted in the highest mortality rates in both bioassays. Lezama-Gutierrez et al. (1996), when evaluating third instar A. ludens (Loew) susceptibility to various EPNs species in pots containing sterile sandy soil, found higher larval and pupal mortality (90%) when pre-pupae were exposed to S. riobrave and S. carpocapsae All. However, Hernández (2003) observed mortality of just 23%, 18%, 17%, 17% and 16% of A. ludens larvae following treatment with 100 IJs/larvae of H. indica, S. carpocapsae All, H. bacteriophora HP88 and Mexican, S. riobrave, and S. carpocapsae under laboratory conditions. His mortality from H. bacteriophora and S. riobrave was lower than the present work. Despite the same concentration in both works (100 IJs/larvae), the methodology may have influenced the nematode’s efficiency. In the present work, IJs were applied to individual larvae, while Hernández (2003) applied 2000 IJs on 20 larvae (equivalent to 100 IJs/ larvae). As with pre-pupae, pupae of A. fraterculus exposed to the 19 EPNs isolates for 12 days also were susceptible to all nematodes tested (F = 564; df = 19, 18; P = 0.01) (Fig. 2A and B). Mortality caused by the isolates was statistically different from the control and as with pre-pupae, isolates of H. bacteriophora RS88 (92% and 92%) and S. riobrave RS59 (92% and 94.5%), resulted in the highest pupal mortality. Hernández (2003) observed only 30% and 21% mortality in the laboratory of A. ludens pupae inoculated with 100 IJs/larvae of H. bacteriophora HP88 and S. riobrave, respectively. This may be due to differences in methodology, since his IJ inoculation was made on larvae and ours directly on the pupae. This is the first record of high susceptibility of A. fraterculus pupae to EPNs. Stark and Lacey (1999) found that H. bacteriophora and S. riobrave resulted in 62.5% and 40% mortality of Rhagoletis indifferens Curran pupae, at 318 IJs/pupae. However, Yee and Lacey (2003) observed no pupal mortality of the same insect when inoculated with S. intermedium (Poinar), S. feltiae and S. carpocapsae Sal. Our results show EPN isolates
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Fig. 1. (A and B). Two bioassays showing percentage mortality (average + standard error) of Anastrepha fraterculus larvae inoculated with 100 infective juveniles of different entomopathogenic nematode isolates from Rio Grande do Sul, Brazil. Means with the same letter are not statistically different by Tukey’s test (P < 0.05).
caused higher mortality of A. fraterculus in the pupal stage, in agreement with Hernández (2003). However, Lezama-Gutierrez et al. (1996) observed higher EPN pathogenicity to the larval stage of A. ludens, which agrees with Beaver and Calkins (1984) work with in A. suspensa. Entomopathogenic nematodes enter the host through natural openings, such as the oral and anal openings (Salame and Glazer, 2000), spiracle (Triggiani and Poinar, 1976), or directly through the soft parts of the tegument as observed by Koppenhofer et al. (2000). Fraenkel and Bhaskaran (1973) suggested that EPNs pathogenicity to pupae results from nematode penetration in the inter-segmental membranes before cuticle development is finished. This may be seen in 1-day-old pupal mortality in the present work. On the other hand, despite pre-pupae not showing tegument schlerotization, the proportion of those with presence of EPNs inside was low, indicating there was nematode penetration and liberation of bacteria in these insects without development of the nematodes. The percentage of dead insects with presence of nematodes at different stages was relatively low in relation to total mortality, with higher percentage only in isolates of H. bacteriophora RS88 and S. riobrave RS59, with 4.8% and 5.8% of pre-pupae and 12.5% of pupae for both nematodes. The low development rate may be due to the suitability of A. fraterculus as a host, or factors, including host density, nematode density and environmental conditions, as well as internal factors such as the physiological status or age of the nematode (Shapiro and Lewis, 1999).
3.2. Determination of lethal dose (LD50) and (LD90) of S. riobrave and H. bacteriophora on A. fraterculus Lethal doses, LD50 and LD90 of S. riobrave on A. fraterculus prepupae and pupae were 382 (347 IJs/cm2) and 2851 IJs/larvae (2.592 IJs/cm2), respectively, and 112 (102 IJs/cm2) and 423 IJs/ pupae (384 IJs/cm2), respectively (Table 1). LD50 and LD90, of H. bacteriophora on A. fraterculus pre-pupae and pupae were 252 (229 IJs/cm2) and 1630 IJs/larvae (1.482 IJs/cm2), respectively, and 120 (109 IJs/cm2) and 457 IJs/pupae (415 IJs/cm2), respectively. Consequently, comparing EPNs virulence, H. bacteriophora had a lower LD50 and LD90 on A. fraterculus pre-pupae than S. riobrave, with similar results for pupae. Lethal doses of H. bacteriophora Costa Rica on third instar A. obliqua (Macquart), at the same soil depth (2 cm), were lower, LD50 (49 IJs/cm2) and LD95 (1.294 IJs/cm2) Toledo et al. (2005a) than the doses in the present study. In addition, Toledo et al. (2006b), while evaluating lethal concentration of H. bacteriophora Costa Rica on third instar A. serpentina, observed lower LD50 (36 IJs/cm2) and LD95 (686 IJs/cm2) than those found in the present study with A. fraterculus. 3.3. Efficacy of S. riobrave and H. bacteriophora on A. fraterculus pupae in a greenhouse Pupae of A. fraterculus were susceptible to the isolates of S. riobrave and H. bacteriophora in soil under semi-field conditions.
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Fig. 2. (A and B). Two bioassays showing percentage mortality (average + standard error) of Anastrepha fraterculus pupae inoculated with 100 infective juveniles of different entomopathogenic nematode isolates from Rio Grande do Sul, Brazil. Means with the same letter are not statistically different by Tukey’s test (P < 0.05).
There were no statistical difference between the nematode concentrations tested (Fig. 3) (F = 21.6; df = 4, 3; P = 0.01). These results suggest that half of the lethal concentration (CL90) (250 IJs/cm2) was enough to produce high mortality of A. fraterculus pupae. This may be due to the characteristics of the soil interfering with in nematode efficiency. To determine the lethal concentration, sterilized sand was used, while in greenhouse, clay soil, collected in the field was used. Toledo et al. (2005a) recorded higher mortality of A. obliqua larvae in sandy-clay soil (85%), compared to sandy
soil (39%), at the same concentrations (~500 IJs/cm2) and depth (2 cm) used in the present study. 3.4. Efficacy of S. riobrave and H. bacteriophora on A. fraterculus larva, in peach fruit in the field During the experiment, relative humidity of 72 ± 8%, air temperature of 23 ± 2 °C, soil temperature of 25 ± 2 °C and total rain of 35.4 mm were registered. The infestation was 38% and
Fig. 3. Percentages mortality (mean + standard error) of Anastrepha fraterculus pupae inoculated with different concentrations of Heterorhabditis bacteriophora RS88 and Steinernema riobrave RS59 in greenhouse (T1 – S. riobrave RS59, 250 IJs/cm2; T2 – S. riobrave RS59, 500 IJs/cm2; T3 – H. bacteriophora RS88, 250 IJs/cm2; T4 – H. bacteriophora RS88, 500 IJs/cm2; T5 – control = water). Means with the same letter are not statistically different by Tukey’s test (P < 0.05).
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Fig. 4. Percentage mortality (mean + standard error) of pre-pupae of Anastrepha fraterculus in artificially and naturally infested fruits, sprayed with Heterorhabditis bacteriophora RS88 and Steinernema riobrave RS59 in the field (n = total number of larvae in each treatment) (T1 – S. riobrave RS59 + artificially infested fruit; T2 – S. riobrave + naturally infested fruit; T3 – H. bacteriophora RS88 + artificially infested fruit; T4 – H. bacteriophora RS88 + naturally infested fruit; T5 – water + artificially infested fruit; T6 – water + naturally infested fruit). Means with the same letter are not statistically different by Tukey’s test (P < 0.05).
52% in artificially and naturally infested fruit, respectively. Higher A. fraterculus larval mortality was observed when artificially and naturally infested fruit were sprayed with H. bacteriophora, 51.25% and 28.12%, respectively (F = 47.8; df = 5; P = 0.01) (Fig. 4). Treatments with S. riobrave were lower (20% and 24.3%). In a field test, Toledo et al. (2006a) observed 52.1% pupal mortality 8 days after application of H. bacteriophora Costa Rica on mango fruit infested with the third instar of A. ludens. In that study, pupae came from larvae that emerged from fruit, and 15% of remaining larvae were infected by the nematode. Yet Hernández (2003) found equal mortality (17%) for both species of nematodes tested in the present work, when applied on A. ludens larvae. The presence of nematodes inside the insect was much lower than total mortality, with the best results for artificially and naturally infested fruit sprayed with H. bacteriophora, showing 15.7% and 7.8% of larvae with nematodes, respectively. The difference A. fraterculus larval mortality, in artificially and naturally infested fruits, especially when exposed to H. bacteriophora, may have resulted from high fruit water content (observed visually), since artificially infested fruits showed higher water content (visually) than those selected from the soil. It is well known that EPNs are used especially for control of soil and cryptic pests (Grewal et al., 2001), and these organisms need a water film to move and search
efficiently for the host in these environments, with the possibility of inactivation under dehydration conditions (Griffin et al., 2005). Thus, although H. bacteriophora has ‘‘cruiser” foraging behavior, that is, active searching for a host, and despite having resulted in higher mortality independently of applied concentration, the lower water content inside fruits may have negatively affected the efficacy of this nematode (Griffin et al., 2005). The nematode H. bacteriophora has been studied for the control of different species of fruit-flies (Hernández, 2003; Toledo et al., 2005a,b; Toledo et al., 2006a,b). For efficient application in the field, three factors have to be considered, according to Toledo et al. (2006a): developmental stage of the insect; infectivity of nematode population after application; survival of nematodes in soils affected by biotic and non-biotic factors. Pérez (2000) found that H. bacteriophora remained infective for 18 days in laboratory soil with 15% humidity. However, infection was significantly higher during the first four days. Despite S. riobrave having an intermediary type of foraging (‘‘cruiser” and ‘‘ambusher”), it did not achieve the same results as H. bacteriophora, possibly due to factors beyond behavior, such as type of host invasion. In particular, IJs of Heterorhabditis are able to penetrate insect host through the cuticle (Bedding and Molyneux, 1982), due to the presence of a chitinous tooth at the
Fig. 5. Percentage mortality (mean + standard error) of Anastrepha fraterculus pupae, after spraying aqueous suspension and liberation of cadavers with Heterorhabditis bacteriophora RS88 and Steinernema riobrave RS59 in the field (T1 – S. riobrave RS59 + aqueous suspension; T2 – H. bacteriophora RS88 + aqueous suspension; T3 – control = water; T4 – S. riobrave RS59 + insect cadaver; T5 – H. bacteriophora RS88 + insect cadaver; T6 – control without water). Means with the same letter are not statistically different by Tukey’s test (P < 0.05).
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anterior extremity, a fact that may favor H. bacteriophora to the detriment of S. riobrave.
control in the soil, independently of the application method (aqueous suspension or infected cadavers) in the field. Acknowledgments
3.5. Efficacy of two inoculation techniques of S. riobrave and H. bacteriophora on A. fraterculus pupae in peach fruit in the field Production of S. riobrave and H. bacteriophora in G. mellonella larvae was 50,000 and 100,000 IJs per larvae, respectively. Thus, eight and four larvae infected by S. riobrave and H. bacteriophora, respectively, were liberated, corresponding to a concentration of 250 IJs/cm2. There was no statistical difference on A. fraterculus pupae between nematodes following aqueous application (F = 25.4; df = 5; P = 0.01) (Fig. 5). However, there was a statistical difference between nematodes when application of cadavers was used, with H. bacteriophora being more efficient than S. riobrave. Independent of nematode species used, the aqueous application caused higher A. fraterculus pupal mortality than the use of infected cadavers. These results diverge from those of Shapiro and Lewis (1999), where the percentage of nematodes that invaded bait G. mellonella larvae were 4–10 higher when using cadavers infected by H. bacteriophora (isolate Hb), than with aqueous suspensions. Besides that, Shapiro and Glazer (1996) observed that IJs of H. bacteriophora HP88 that emerged from cadavers of G. mellonella larvae had higher dispersion ability than those applied in aqueous suspension. On the other hand, Pérez (2000) observed that no difference occurred in survival of H. bacteriophora (isolate Hb) between the treatments with aqueous suspension and cadavers. However, it was observed that S. riobrave Texas and S. carpocapsae All reduce in survival and infectivity exponentially over time, while H. bacteriophora Hb had higher survival and infectivity until 10 days after emergence. The increase in the infection of H. bacteriophora may be explained by the hypothesis of the ‘‘infectivity of phase”, that is, a great proportion of the IJs remain non-infectious until the gradual process of maturation over time (Shapiro and Lewis, 1999; Pérez et al. 2003), with such phenomena being observed only among heterorhabditids (Campbell et al., 1999). Shapiro and Glazer (1996) concluded that the quantity of nematodes emerged from cadavers is uncertain. Thus, although soil represents a relatively stable environment, IJs may experience stress conditions such as desiccation and high temperatures, especially in the soil surface, while in flooded soils they may experience lack of oxygen and influence of diseases and antagonistic organisms (Griffin et al., 2005). Del Valle et al. (2008a) verified the dispersion of H. baujardi LPP7 was delayed (fifth week after liberation) by the adverse conditions of temperature and soil moisture, when applying infected cadavers of G. mellonella. Besides these factors, time of exposure of A. fraterculus pupae to IJs that emerged from cadavers in this study was 6 days, lower than that observed by Del Valle et al. (2008b), thus this may have negatively affected the efficacy of both nematode species. The dose applied in the field here (250 IJs/cm2) was higher than that recommended for field applications of EPNs to control pests at any life stage in the soil (25 IJs/cm2) (Grewal et al., 1994), so these results would seem to make commercial use impractical. However, in areas of fruit cultivation, the quantities of nematodes may be reduced, by applying in rows, under the tree-canopy, concentrating application in places with higher density of the pest (Toledo et al., 2006a). Among 19 EPNs isolates indigenous to RS, Brazil, H. bacteriophora RS88 and S. riobrave RS59 are the most pathogenic to A. fraterculus pupae and pre-pupae. These isolates have shown similar virulence when applied on A. fraterculus pupae, with H. bacteriophora being more virulent in this insect’s larva. The EPN H. bacteriophora RS88 has better efficacy in host search inside infested fruit, and in pupal
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