BIOLOGICAL AND MICROBIAL CONTROL
Beauveria bassiana (Ascomycota: Hypocreales) Wound Dressing for the Control of Euzophera pinguis (Lepidoptera: Pyralidae) E. QUESADA-MORAGA,1,2 M. YOUSEF,1 A. ORTIZ,3 M. RUI´Z-TORRES,4 I. GARRIDO-JURADO,1 5 AND A. ESTÉVEZ
J. Econ. Entomol. 106(4): 1602Ð1607 (2013); DOI: http://dx.doi.org/10.1603/EC13203
ABSTRACT Injury to olive tree trunks and branches because of biotic and abiotic factors, such as pruning and mechanical harvesting, attracts the olive pyralid moth Euzophera pinguis Haworth (Lepidoptera: Pyralidae). This moth has become increasingly important in the Mediterranean region during recent years. The use of an entomopathogenic fungus for wound dressing for pest control is reported for the Þrst time in this study. Beauveria bassiana (Ascomycota: Hypocreales) strain EABb 08/04-Ep was originally obtained from a diseased E. pinguis larva and has shown effective E. pinguis control in an olive crop in Jae´ n, Andalusia, Spain, under Þeld conditions during the spring and fall of 2008 and 2009 and the spring of 2011. Experimental artiÞcial 30 by 30-mm square wound cages were large enough to allow the E. pinguis females to oviposit. Approximately 80 and 40 Ð 60% of the control wounds contained live larvae in the experiments that occurred during the spring and fall, respectively. The B. bassiana wound dressing gave similar results as the chlorpyrifos wound dressing throughout the experiment, with efÞcacies reaching 80 Ð 85% in the spring and 90 Ð95% in the autumn. The B. bassiana fungus was recovered from 60 Ð90% of the wounds at the completion of the experiments and after 60 d of treatment. These data indicate that strain EABb 08/04-Ep applied to the pruning wounds can be an effective tool for the microbial control of E. pinguis in olive crops. Moreover, B. bassiana may be used within integrated pest management strategies to minimize chemicals, depending on the population density of the pyralid moth. KEY WORDS branch protection zone, natural target pruning, olive pyralid moth, boring larva, pruning paint
The olive pyralid moth Euzophera pinguis Haworth (Lepidoptera: Pyralidae) is a serious bark and woodborer olive pest in most Mediterranean regions and has become increasingly important during recent years (Dura´n et al. 1998, IOOC 2012). E. pinguis has two overlapping generations each year, a spring-summer generation and a winter generation, with adult ßight activity that may extend for 10 mo and with larvae present throughout the year (Navarro and Campos 2004, Tzanakakis 2006, Dura´n et al. 2010, Patanita 2010). The adult moths emerge in early spring and continue their Þrst ßight period until mid-June (De Andre´ s 1991). The female moths lay eggs in unprotected areas on the trunks of trees with injuries, such as tumors caused by the bacteria Pseudomonas syringae subsp. savastanoi (Smith) Janse and wounds caused by frost, wind, hail, or the fusion of branches. 1 Department of Agricultural and Forestry Sciences, ETSIAM, University of Cordoba, Campus de Rabanales, EdiÞcio C4 Celestino Mutis, 14071, Co´ rdoba, Spain. 2 Corresponding author, e-mail:
[email protected]. 3 Department of Inorganic and Organic Chemistry, University of Jaen, Campus Las Lagunillas, EdiÞcio B3, 23071 Jae´ n, Spain. 4 Laboratorio de Produccio ´ n y Sanidad Vegetal. Parque Tecnolo´ gico GEOLIT Jae´ n (Spain). C/ Sierra Morena, Manzana 11, 23620, Mengõ´bar, Jae´ n, Spain. 5 NUTESCA S.L. C/ Cuba, 3, 23400 Baeza, Jae ´ n, Spain.
After hatching, the larvae feed endophytically into the main trunk and secondary branches, causing the interruption of sap ßow, which leads to branch drying, the yellowing of leaves, the defoliation of branches, and even the loss of young olives trees (Grouard 2001, Navarro and Campos 2004). Similarly, female egglaying activity is favored when olive tree trunks and branches are injured during pruning and mechanical harvesting, which occurs in the new intensive and super-intensive olive groves. Because of the endophytic behavior of the larvae, E. pinguis control is extremely difÞcult through application of the organophosphate insecticide chlorpyrifos (48%), which is the only method used by farmers for the control of this moth ( Ministerio de Agricultura, Alimentacio´ n, y Medio Ambiente [MAGRAMA] 2012). However, because of the ecological and healthrelated problems of insecticide application, alternative systems based on pheromone mating disruption have been recommended for the control of E. pinguis (Ortiz et al. 2004, 2007). Dura´n et al. (2010) reported that the efÞcacy of using of a synthetic pheromone for mating disruption of the olive pyralid moth is insufÞcient to prevent an increase in both the percentage of trees affected and the number of active galleries per tree. Biological control of E. pinguis is not widespread.
0022-0493/13/1602Ð1607$04.00/0 䉷 2013 Entomological Society of America
August 2013
QUESADA-MORAGA ET AL.: CONTROL OF E. pinguis WITH FUNGUS
Rodrõ´guez et al. (2005) has shown that spiders are the main predator of E. pinguis, followed by Scymnus suturalis Thunberg (Coleoptera: Coccinellidae) and Brachinotocoris ferreri n. sp. Baena (in litteris) (Heteroptera: Miridae). Neuroptera and ants are less important predators of E. pinguis, and it has been reported that two braconids, Iconella myelolenta (Wilkinson) and Phanerotoma ocularis Kohl, have some impact on populations of closely related species of E. pinguis. In a survey of the natural enemies of E. pinguis in Jae´ n, Andalusia in southern Spain, during 2007, a strain of the entomopathogenic, mitosporic, ascomycete Beauveria bassiana (Balsamo) Vuill. (Ascomycota: Hypocreales) was found infecting E. pinguis larvae within the branches of several olive trees (QuesadaÐ Moraga 2011). The objective of this study was to evaluate wound dressing with this strain of B. bassiana for the microbial control of the olive pyralid moth under Þeld conditions in an olive crop in Jae´ n, Andalusia, Spain. Materials and Methods Fungal Strain and Cultivation. The B. bassiana strain EABb 08/04-Ep (C.R.A.F., University of Cordoba Entomopathogenic Fungi Collection, Co´ rdoba, Spain) was isolated from a dead E. pinguis larva from an olive crop in Jaen, Spain. This strain was deposited under the Budapest Treaty in the Spanish Type Culture Collection located at the University of Valencia under accession number CECT 20746 on 2 February 2009. Monosporic cultures of this strain were grown on malt agar slants at 25⬚C in the dark and stored at 4⬚C. To obtain a spore suspension, slant cultures of the isolates were subcultured in petri plates containing malt agar (BioCult B. Laboratories, Madrid, Spain) for 15 d at 25⬚C in the dark. The petri plates were sealed with ParaÞlm. Freshly harvested conidia from 15-d-old cultures were used in the experiments. Conidial suspensions were prepared by scraping the conidia from the petri plates into an aqueous sterile solution of 0.1% Tween 80. This initial suspension was sonicated for 10 min (P-Selecta ultrasounds, Barcelona, Spain) to homogenize the inoculum and then Þltered through several layers of cheesecloth to remove mycelial mats and collect the pure conidia. The concentration of conidia was assessed by colony-forming unit assay (Goettel and Inglis 1997). The conidial suspension used for the bioassay was adjusted by diluting the conidia with 0.1% Tween 80 to a Þnal concentration of 4.1 ⫻ 107 conidia/ml. Experimental Site, Design, and Wound Dressing. The experiments were performed from 2008 to 2011 in a 20-ha olive crop with 15Ð20-yr-old trees (ÔPicualÕ) planted 8 by 6 m at Mancha Real, Jaen, in southern Spain (37⬚ 56.176⬘⬘ N, 03⬚ 36.709⬘⬘ W; 303 m above sea level). This area was selected because of a large E. pinguis population and because of the pest incidence registered during the past 10 yr by the regional crop protection authorities of the Andalusia Region (M.R.-T., personal communication). Only olive crops
1603
surrounded the experimental area, and the crop was cultivated by a grower using integrated pest management (IPM). The plants were irrigated and fertilized, as necessary, by drip irrigation. A randomized complete block design was used for each experiment on each date that included three blocks with three treatments. A block consisted of 20 olive trees; each with an artiÞcial 30 by 30-mm2 wound, 17 mm in depth, performed 1.7 m above the ground with a cutter. The following three treatments were evaluated: 1) topical application of chlorpyrifos to the wound surface, 2) topical application of B. bassiana to the wound surface, and 3) untreated control. Chlorpyrifos was selected for comparison, as it is the only active ingredient authorized in Spain for using against olive pyralid moth. The fungal suspension was applied using a manual sprayer (Bellota, Spain). The spray deposition at the level of the target wound surface was ⬇0.1 liter/m2 (l/mm2), resulting in a deposition from ⬇1,00,000 to 10,00,000 viable conidia/mm2. The chlorpyrifos commercial product was Pyrinex 48 EC (Aragonesas Agro) that was sprayed using the same hand sprayer at the recommend rate of 150 cc of active ingredient per hectare. The experiment was repeated Þve times, twice in 2008 (spring and autumn), twice in 2009 (spring and autumn), and once in 2011 (spring). Sex pheromone funnel traps were used to measure the population dynamics of E. pinguis each year. Presence of B. bassiana in the Wounds at the Completion of the Experiment. Two methods were used to evaluate the presence of fungus in the wounds at the completion of each experiment (2 mo). Several pieces of bark were removed from the spring of 2008 and were stored in plastic bags until transportation to the laboratory. In addition, the surface of each wound was cleaned with a sterile cotton swab, which was also transported to the laboratory. A water suspension was prepared from the pieces of bark and the sterile cotton swabs. This suspension was then stirred with a rotary shaker (P-Selecta shakers, Barcelona, Spain) at 12 rpm for 60 min. The presence or absence of B. bassiana in the pieces of bark and cotton swabs was determined using Sabouraud chloramphenicol agar culture medium in the colony-forming unit assay. Statistical Analysis. The data were analyzed using a one-way analysis of variance (Statistix 9.0, Analytical Software, Tallahassee, FL). TukeyÕs honestly signiÞcant difference test was used to compare the means. Results E. pinguis Flight Activity in the Study Area. E. pinguis ßight activity was monitored with pheromone traps. As shown in Fig. 1, there was similar ßight behavior of the adult populations in the successive monitoring campaigns. There were two E. pinguis ßights or population peaks per year, with a high level of activity during spring-summer and a low level of activity during autumn (Fig. 1). Evaluation of the B. bassiana Wound Dressing Treatments for E. pinguis Control. In 2008, the spring B. bassiana wound dressing had a signiÞcant effect on
1604
JOURNAL OF ECONOMIC ENTOMOLOGY
Vol. 106, no. 4
Fig. 1. Annual E. pinguis ßight curves for 3 yr.
the percentage of wounds without larval infestation (WWI) (F2,8 ⫽ 117.4; P ⬍ 0.001). The WWI was signiÞcantly reduced after wound dressing with B. bassiana and chlorpyrifos compared with the controls, whereas the WWI was signiÞcantly higher (93.3%) for chlorpyrifos than B. bassiana (48.3%) (Table 1). There was also a signiÞcant reduction in the percentage of wounds that contained live larvae (WLL) in the chlorpyrifos and B. bassiana wound dressing treatments (F2,8 ⫽ 37.5; P ⬍ 0.001). The percentage of wounds without E. pinguis larvae but with signs of larval boring as evidenced by the presence of frass accumulations (WLB) was only signiÞcant for the B. bassiana wound dressing (F2,8 ⫽ 139.7; P ⬍ 0.001) (Table 1). During the autumn of 2008, the B. bassiana treatment had a signiÞcant effect on both the WWI (F2,8 ⫽ 10.7; P ⫽ 0.01) and WLL (F2,8 ⫽ 223.2; P ⬍ 0.001) (Table 1). Although the WWI in the control was higher in the autumn than spring, it was ⬇50% higher in the chlorpyrifos and B. bassiana treatments (Table 1). No live larvae were observed in the chlorpyrifos and B. bassiana treatments, whereas 60% of the control wounds presented with live larvae (Table 1). The WLB were found only in wounds dressed with the fungus (8.3%) (Table 1). During 2009, the spring B. bassiana wound dressing had a signiÞcant effect on the percentage of wounds without larval infestation (WWI) (F2,8 ⫽ 81.5; P ⬍ 0.001). The WWI was signiÞcantly reduced after wound dressing with chlorpyrifos (93.3%) and B. bassiana (20.0%) compared with the controls without WWI (Table 1). In this experiment, the chemical and microbial treatments again resulted in a signiÞcant (F2,8 ⫽ 318.4; P ⬍ 0.001) reduction in the presence live larvae in the wounds, which was zero in both treatments and 83.3% in the controls (Table 1). Similarly,
the B. bassiana wound dressing was the only treatment with a signiÞcant percentage of wounds without E. pinguis larvae but with signs of larval boring (F2,8 ⫽ 985.6; P ⬍ 0.001). During the autumn of 2009, the B. bassiana treatment had a signiÞcant effect on the percentage of wounds without larval infestation (F2,8 ⫽ 52.19; P ⬍ 0.001), which was signiÞcantly increased by the chlorpyrifos and B. bassiana wound treatments (Table 1). There was also a signiÞcant reduction in the percentage of WLL (F2,8 ⫽ 585.7; P ⬍ 0.001). There were no wounds with live larvae in the chemical and fungal treatments, whereas 21.1% of the control wounds presented with live larvae (Table 1). SigniÞcant differences were also detected between the treatments for wounds with no E. pinguis larvae but signs of larval boring (F2,8 ⫽ 985.6; P ⬍ 0.001), which were observed only in the controls and fungal treatment (Table 1). During the spring of 2011, the treatment signiÞcantly impacted the percentage of wounds without larval infestation (F2,8 ⫽ 84.9; P ⬍ 0.001), whereas this number was signiÞcantly reduced only for the chemical treatment (Table 1). Notably, the wounds dressed with B. bassiana showed the highest (43.3%) percentage of wounds with no larvae but signs of larval boring (Table 1) during this spring. There were also signiÞcant differences between the treatments for the percentage of wounds that contained live larvae (F2,8 ⫽ 7.6; P ⫽ 0.022), which was reduced by both the chemical and microbial treatments (Table 1). Presence of B. bassiana in the Wounds at the Completion of the Experiment. The presence of B. bassiana was detected at the time of wound opening. Based on the results, no B. bassiana was detected
WLB WLL WWI WLB WLL WWI WLB WLL WWI
C, control experimental wound; CL, chlorpyrifos-treated artiÞcial wounds; BBD, B. bassiana-dressed artiÞcial wound; WWI, wounds with no attack by E. pinguis larvae; WLL, wounds with E. pinguis live larvae; WLB, wounds with no E. pinguis larvae but signs of larval boring, as evidenced by the presence of frass accumulations. a The numbers in the columns followed by the same letter are not signiÞcantly different, as determined by the Tukey test (P ⱕ 0.05).
WLB WLL WWI
WLL
WLB
WWI
2011
Spring Autumn
2009 Spring Autumn 2008 Spring Wound treatment
Percentage of wounds attacked by the olive pyralid moth of olive bark with artificial wounds per treatment type Table 1.
3.3 ⫾ 1.6aa 83.3 ⫾ 3.3c 0a 35.0 ⫾ 7.6a 60.0 ⫾ 5.7b 0a 0a 88.3 ⫾ 4.4a 0a 52.6 ⫾ 1.5a 21.1 ⫾ 1.6b 16.6 ⫾ 1.6c 39.0 ⫾ 2.3a 43.3 ⫾ 3.3b 5.0 ⫾ 2.8a 93.3 ⫾ 1.6b 5.0 ⫾ 2.8a 0a 68.3 ⫾ 4.4b 0a 0a 93.3 ⫾ 2.4b 0b 0a 98.3 ⫾ 1.6c 0a 0b 90.0 ⫾ 2.8b 5.0 ⫾ 2.1a 5.0 ⫾ 2.8a 48.3 ⫾ 4.4c 6.6 ⫾ 4.4a 43.3 ⫾ 6.0b 70.0 ⫾ 5.0b 0a 8.3 ⫾ 1.6b 20.0 ⫾ 2.8c 0b 70.0 ⫾ 2.8b 86.6 ⫾ 2.0b 0a 6.6 ⫾ 1.6a 33.3 ⫾ 3.5a 10.0 ⫾ 4.3ab 43.3 ⫾ 3.3b
QUESADA-MORAGA ET AL.: CONTROL OF E. pinguis WITH FUNGUS
C CL BBD
August 2013
1605
from the control treatment for all years, whereas B. bassiana was variably detected from the treated wounds according to the two evaluation methods used during the experiment. No signiÞcant differences were found for the presence of B. bassiana between the two methods for all years, except for the treatment during the spring of 2008, in which B. bassiana was detected in 93.3% of the bark pieces removed from the wounds and in 63.3% of the sterile cotton swabs pieces (Table 2). Discussion Pheromone trap monitoring indicated that olive pyralid moth activity occurred in the study area throughout the entire three growing seasons. The annual ßight curves of E. pinguis largely coincide with those published by Dura´n et al. (1998, 2010), who used light and pheromone traps in the province of Seville in Andalusia in southwestern Spain, and with those recorded by Olivero et al. (2005), who used pheromone traps in the Andalusia province of Malaga in South Spain. Consistent with Olivero et al. (2005), this similarity in ßight curve shapes among the olive crops from different regions of Andalusia highly support a similar behavior of the pest in geographically distant olive regions, most likely conditioned by macro-ambient variable conditions. Although there were slight differences in the start dates of the adult ßight in 2008, 2009, and 2011, in general, the adult activity of E. pinguis begins with the onset of spring, with the optimum temperature for emergence in the range of 20 Ð25⬚C (Dura´n et al. 1998). The daily evolution of adult E. pinguis is highly affected by rainfall (i.e., several raining days negatively inßuence adult ßight) and wind (Patanita 2010). Our experimental artiÞcial 30 by 30-mm square wound cages allowed the E. pinguis females to oviposit, as demonstrated by the high percentage of wounds showing live larvae in the controls, with ⬇80% in the spring experiments and 40 Ð 60% in the fall experiments. The results of this study indicate that the B. bassiana EABb 08/04-Ep strain provided effective E. pinguis control by reducing both the percentage of wounds with live larvae and the percentage of wounds with no live larvae but signs of larval boring. Notably, we did not observe dead larvae in the wounds with signs of larval boring, which may be because the majority of larval death occurred in the Þrst-instar neonate larvae, which according to our previous study, are the most susceptible to EABb 07/06-Ep infection (QuesadaÐMoraga 2011). The fungal treatment gave similar results as the chlorpyrifos treatment throughout the experiment, with efÞcacies reaching 80 Ð 85% during spring and 90 Ð95% during autumn. However, wounds with signs of larval boring were not observed in the chemical treatment, indicating either an excellent ovicidal or repellent activity (Nazir et al. 2001), whereas these two activities were not detected in the B. bassiana dressed wounds. Therefore, the use of the fungus either alone or within an IPM strategy to replace or
1606 Table 2. Treatmenta 1 2 3 a b
JOURNAL OF ECONOMIC ENTOMOLOGY
Vol. 106, no. 4
Presence of B. bassiana in the wounds at the time of wound opening % of B. bassiana recovered from the woundsb Spring 2008
Autumn 2008
Spring 2009
Autumn 2009
Spring 2011
0c 63.3 ⫾ 3.3b 93.3 ⫾ 3.3a
0b 36.7 ⫾ 5.1a 40.0 ⫾ 3.2a
0b 60.0 ⫾ 10.0a 53.3 ⫾ 3.3a
0b 40.0 ⫾ 5.7a 33.3 ⫾ 3.3a
0b 60.0 ⫾ 6.7a 63.3 ⫾ 11.5a
1 ⫽ control, 2 ⫽ pieces of bark, 3 ⫽ sterile cotton swab. The means within the columns with the same letter are not signiÞcantly different (P ⱕ 0.05) according to the Tukey (HSD) test (P ⱕ 0.05).
minimize the use of chlorpyrifos, which has several nontarget effects and continues threat of removing from the list of authorized insecticides, seems to be justiÞed (Rafalimanana et al. 2002, Prischmann et al. 2005, Delpuech et al. 2009). To the best of our knowledge, no previous studies on the use of entomopathogenic fungi for wound dressing against insect pests have been reported. Wound dressing to protect olive crops against E. pinguis has been performed only with chemicals. Olivero et al. (2005) reported that wound dressing with chlorpyrifos (48%) and a mixture of fenitrothion and esfenvalerate provided effective control of E. pinguis larval boring. Connell et al. (2005) also used wound dressing with diazinon and copper oil for control of the American plum borer Euzophera semifuneralis, which is the major insect vector of the fungus Ceratocystis fimbriata. They found that both chemical treatments signiÞcantly reduced the number of plum borer strikes compared with the untreated control. The studies by Camilli et al. (2007) supported the need to apply pruning paints to all wounds on susceptible trees in areas with oak wilt. Our experiments highlight the potential for preventive E. pinguis control using B. bassiana for olive tree wound dressing; however, we are also interested in elucidating the activity of the fungus after borer feeding activity is observed in the wounds. The presence of fungus in the treated wounds after 60 d of treatment, which ranged between 36 and 63% for the bark pieces removed from the wounds and between 33 and 93% for the sterile cotton swabs, revealed the potential of the fungus for long-lasting control of the olive pyralid moth. Additional studies are required to clearly deÞne the number of fungal applications that are necessary for seasonal or annual control. Our studies indicate that B. bassiana strain EABb 08/04-Ep applied to pruning wounds can be an effective tool for the microbial control of E. pinguis in olive trees. Moreover, it can be used within IPM strategies to minimize chemicals, depending on the population density of the pyralid moth. Acknowledgments We thank Sandra Ma Castuera Santacruz and Carlos Campos Porcuna for excellent technical assistance. The results of this work are covered by a Spanish international patent application (assignation P201030539; ES2376444A1; WO2011/128473A1; PCT/ES2011/000115).
References Cited Connell, J. H., W. D. Gubler, and R. A. Steenwyk. 2005. Almond trunk injury treatment following bark damage during shaker harvest, pp. 199 Ð202. In M. M. Oliveira and V. Cordeiro. (eds.), XIII GREMPA Meeting on Almonds and Pistachios, Zaragoza, Spain. Camilli, K., D. N. Appel, and W. T. Waston. 2007. Studies on pruning cuts and wound dressing for oak wilt control. Arboric. Urban For. 33: 132Ð139. De Andre´s, C. F. 1991. Enfermedades y Plagas del olivo. Riquelme y Vargas, Jae´ n. Spain. Delpuech, J., B. Froment, P. Fouillet, F. Pompanon, S. Janillon, and M. Boule´treau. 2009. Inhibition of sex pheromone communications of Trichogramma brassicae (Hymenoptera) by the insecticide chlorpyrifos. Environ. Toxicol. Chem. 17: 1107Ð1113. Dura´ n, J. M., M. Alvarado, A. Serran, and A. De la Rosa. 1998. Contribucio´ n al conocimiento de Euzophera pinguis (Haworth, 1811) (Lep.: Pyralidae), plaga del olivo. Bol. San. Veg. Plagas 24: 267Ð278. Dura´ n, J. M., M. I. Gonza´ lez, A. Sa´ nchez, and A. Serrano. 2010. Experiencia de control de Euzophera pinguis (Haworth, 1811) (Lepidoptera: Pyralidae) en olivar mediante confusio´ n sexual. Bol. San. Veg. Plagas. 36: 101Ð112. Goettel, M. S., and G. D. Inglis. 1997. Fungi: Hyphomycetes, pp. 213Ð249. In L. A. Lacey (ed.), Manual of Techniques in Insect Pathology. Academic, San Diego, CA. Grouard, B. C. 2001. Lepido´ pteros Xilo´ fagos en las plantaciones de olivo. Olient 4: 23Ð29. (IOOC) International Olive Oil Council. 2012. Olivae 117. International Olive Oil Council. (http://www. internationaloliveoil.org). (MAGRAMA) Ministerio de Agricultura, Alimentacio´ n y Medio Ambiente. 2012. Ministerio de Agricultura, Alimentacio´ n y Medio Ambiente. (http://www.magrama. gob.es/es/). Navarro, E. R., and M. Campos. 2004. Me´ todos de control inregrado de la Euzophera pinguis del olivo. Vida Rural 186: 50 Ð53. Nazir, A., I. Mukhopadhyay, D. K. Saxena, and D. Kar Chowdhuri. 2001. Chlorpyrifos-induced hsp70 expression and effect on reproductive performance in transgenic Drosophila melanogaster (hsp70-lacZ). Arch. Environ. Contam. Toxicol. 41: 443Ð 449. Olivero, J., E. J. Gracı´a, A. Ortiz, A. Quesada, and A. Sa´ nchez. 2005. Ensayo de estrategias quõ´micas de control de Euzophera pinguis (Haworth) (Lepidoptera: Pyralidae), apoyadas en el seguimiento del vuelo con feromona sexual. Bol. San. Veg. Plagas. 31: 459 Ð 469. Ortiz, A., A. Quesada, and A. Sanchez. 2004. Potential for use of synthetic sex pheromone for mating disruption of the olive Pyralid moth, Euzophera pinguis. J. Chem. Ecol. 30: 991Ð1000.
August 2013
QUESADA-MORAGA ET AL.: CONTROL OF E. pinguis WITH FUNGUS
Ortiz, A., A. Peraba´ , A. Quesada, and A. Sa´ nchez. 2007. Mating disruption of the olive Pyralid moth, Euzophera pinguis. IOBC WPRS Bull. 30: 26 Ð28. Patanita, M. I. 2010. Contribution to the knowledge of Euzophera pinguis Haworth biology in Alentejo (Portugal). IOBC WPRS Bull. 59: 47Ð50. Prischmann, D., D. James, L. Wright, R. Teneyck, and W. Snyder. 2005. Effects of chlorpyrifos and sulfur on spider mites (Acari: Tetranychidae) and their natural enemies. Biol. Control 33: 324 Ð334. Quesada–Moraga, E. 2011. Me´ todo para la proteccio´ n de plantas len˜ osas frente a Euzophera pinguis. Spanish International patent application (assignation P201030539; ES2376444A1; WO2011/128473A1; PCT/ES2011/000115).
1607
Rafalimanana, H., L. Kaiser, and J. M. Delpuech. 2002. Stimulating effects of the insecticide chlorpyrifos on host searching and infestation efÞcacy of a parasitoid wasp. Pest Manag. Sci. 58: 321Ð328. Rodrı´guez, E., C. Lozano, and M. Campos. 2005. Detection of predation on Euzophera pingu¨ is (Lepidoptera: Pyralidae) using an enzyme-linked immunosorbent assay. Eur. J. Entomol. 102: 793Ð796. Tzanakakis, M. E. 2006. Insects and mites feeding on olive. Distribution, importance, habits, seasonal development and dormancy.Applied entomology library. Brill, Leiden.
Received 30 April 2013; accepted 4 June 2013.
