El departamento de I+D+i de la empresa Agrobío asistirá a la reunión del grupo de trabajo "Integrated Control in Protected Crops, Mediterranean Climate", integrado en la organización OILB/WPRS. Ésta será celebrada en el Instituto de Agronomía Mediterráneo de Chania (MAICh) en Creta, entre los días 6-11 de septiembre. En esta reunión, entre otros temas, se presentarán nuevos protocolos de trabajo con diversos organismos de control biológico así como nuevos organismos de control para la presencia de nuevas plagas. En este sentido, será un punto de encuentro en el que expertos europeos hablarán de la plaga que más preocupa en el sector agrícola, la polilla Tuta absoluta.
The damsel bug Nabis pseudoferus (Hem.: Nabidae) as a new biological control agent of the South American Tomato Pinkworm, Tuta absoluta (Lep.: Gelechiidae), in tomato crops of Spain
T. Cabello1, J.R. Gallego1, F.J. Fernandez-Maldonado1, A. Soler2, D. Beltran3, A. Parra2 and E. Vila2
1Dpto. Biologia Aplicada, Universidad de Almeria. Ctra. Sacramento s/n, E-04120 Almeria, Spain. E-mail: tcabello@ual.e
2,3Agrobío S.L. and Agrocontrol 2007 S.L., Ctra Nacional 340, Km. 419, 04745-La Mojonera, Almería, Spain.
Abstract: The use of a new agent for biological pest control, the damsel bug Nabis pseudoferus, is being studied to be applied in Spanish greenhouses. This strict zoophagous has been described as an effective predator against aphids and lepidopteran eggs and larvae, although it can feed also on other preys. Preliminary results identify it as a candidate for biological control of the South American Tomato Pinworm, Tuta absoluta. Two semi field bioassays on tomato plants, under controlled conditions, have shown an important reduction in the number of eggs of T. absoluta, between 92 and 96 %, when releasing 8 or 12 first stage nymphs of N. pseudoferus per plant. An already known predator of T. absoluta, the mirid Nesidiocoris tenuis Reuter, was also tested, but the young nymphs did not show any mortality on the pest.
Key words: greenhouse, lepidopteran pests, predator, biological control, efficiency, Nabis pseudoferus.
Introduction
Recently, Almeria (southeast of Spain) has suffered a big change from the traditional chemical control of pests to Integrated Pest Management methods (IPM). This region is one of the major greenhouse horticultural production areas in Europe. The IPM surface has increased in Almeria from 1,400 ha in the season 2006-07 to more than 18,000 ha in 2008-09. However, the success achieved on the different protected crops is not the same; the biological control is still being applied on a rather low percentage of tomato crops. Moreover, several new problems must be faced like the increasing incidence of secondary and new exotic pests, specially the South American Tomato Pinkworm, Tuta absoluta (Meyrick), a quarantine specie. For the time being, there is no successful biological solution available for this pest in Spain.
So, new natural enemies are being studied to give solutions to this pest under Spanish greenhouses conditions. Recently, some promising results have been described with the autochthonous damsel bug Nabis pseudoferus ibericus Remane (Cabello, 2009).
The biology and ecology of some species of Nabidae have been studied, although not very extensively (Blatchley, 1926; Pericart, 1987; Lattin, 1989; Clements & Yeargan, 1997; Chiappinim & Reguzzi, 1998, 1999; Braman, 2000; Roth & Remane, 2003; Roth et al., 2008; Roth & Reinhardt, 2009), and few trials have been conducted about their use as biological control agents (Guppy, 1986; Elliott et al., 1998; Braman, 2000; Cardinale et al., 2003).
The general objective of this work is to evaluate the efficacy of N. pseudoferus to control lepidopteran and aphid pests in horticultural crops of Spain. The preliminary results about predation of T. absoluta on tomato plants are presented.
Material and methods
Insects rearing
Although it has been mentioned that the rearing of the Nabis species is difficult (Roth et al., 2008), a reliable system for mass production was developed and N. pseudoferus is currently commercially available (NabisControl®, Agrobio S.L., Almería, Spain). The aphid Rophalosiphum padi (L.), reared on wheat plants following the same methodology described for banker plants production (Weintraub, 2007), is used in this system to feed N. pseudoferus. All the individuals of N. pseudoferus used in the bioassays came from this commercial rearing.
The specimens of T. absoluta used in all the bioassays were obtained from populations reared under laboratory conditions, on tomato leaves, and individuals of Nesidiocoris tenuis Reuter came from a commercial rearing (NesidioControl ®, Agrobio S.L., Almería, Spain).
Bioassays
Two trials were carried out to evaluate the efficacy of N. pseudoferus to control T. absoluta, both under controlled conditions (25±3º C, 60-80% R.H. and natural photoperiod). The first trial was conducted in April and the second from May to June of 2008. Cages (1.5 x 1.5 x 1.5 m) closed with a fine mesh were used in each trial, each one with 5 tomato plants arranged on pots, Raf variety (1 m height at the start).
One week before starting the trials, all the cages were infested with T. absoluta (4 adults per plant). In the first bioassay the treatment consisted in releasing 12 nymphs (first instar, N1) of N. pseudoferus per plant. In the second trial the release rate was reduced to 8 nymphs (N1) of N. pseudoferus per plant, and an additional treatment using Nesidicoris tenuis was evaluated (8 N1 per plant). In both trials, 3 replications of each treatment were carried out, and 3 cages without predators were left as control.
The total number of eggs and larvae of T. absoluta per plant were counted just before releasing the natural enemies and 7 days after. The predators were released when the eggs of T. absoluta had not hatched yet. The data were analyzed using ANOVA and means were compared with LSD, SPSS software v. 15 (SPSS, 2006).
Results and discussion
The number of hatched eggs of T. absoluta per plant at the end of the trials 1 and 2, depending on the treatment, are presented in figures 1 and 2, respectively.
In both bioassays, highly significant effects (P N. pseudoferus consumed an important amount of pest eggs, showing a reduction in the number of hatched eggs compared to the control: 91.4 % in the first and 99.45 % in the second bioassay. However, young nymphs of N. tenuis (figure 2) did not kill any egg of the pest. It is necessary to remark that N. tenuis has been described as a good predator of eggs and larvae of this pest, but only the adult stage (Urbaneja et al., 2008).
According to these results, N. pseudoferus is a promising candidate as a biological control agent against Tuta absoluta. Adults and last instar nymphs have been observed on crops preying larvae of Tuta absoluta even when those are hidden inside the mines.
Figure 1. Number of T. absoluta eggs hatched per plant in tomato plots 7 days after releases of the predator N. pseudoferus, or without releases, in the first trial, under controlled conditions (25±3º C, 60-80% R.H. and natural photoperiod).
Figure 2. Number of hatched eggs of T. absoluta on tomato plants, 7 days after releases of N. pseudoferus, N. tenuis or without predators, in the second trial, under controlled conditions (25±3º C, 60-80% R.H. and natural photoperiod).
This predator has also been described as an excellent weapon to control bigger larvae of other Lepidoptera species, like Spodoptera exigua (Hübner) in commercial protected pepper crops (unpublished data). Spontaneous populations of N. pseudoferus have appeared in greenhouse crops in Almeria, although highest populations have been found in open field crops, like artichoke. These results confirm previous works that describe different species of Nabis as important natural control agents on crops (Butcher et al., 1988; Lattin, 1989; Duran et al., 1998; Fauvel, 1999; Braman, 2000, Altieri et al., 2005). However, due to the scarce knowledge about the biology and ecology of this specie (now in course), it is necessary to carry out more studies in order to develop practical advices for using this natural enemy on different crops.
Acknowledgements
This publication has been done with the support of the Ministry of Sciences and Innovation (of Spain): CDTI Programme, CENIT-MEDIODIA Project. We thank Ana Roldan for her comments on this paper.
References
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Selection of refuges for Nesidiocoris tenuis (Reuter) (Het.: Miridae) and Orius laevigatus (Fieber) (Het.: Anthocoridae). Virus reservoir risk assessment
M. Cano1, E. Vila2, D. Janssen1, G. Bretones1, E. Salvador1, L. Lara1, MM. Tellez1
1IFAPA, Centro La Mojonera, Autovía del Mediterráneo, km 420, 04745 La Mojonera, Almería, Spain. E-mail: monserrat.cano.ext@juntadeandalucia.es
2Agrobío, S.L. Ctra. Nacional 340, km. 419, 04745 La Mojonera, Almería, Spain.
Abstract: Preliminary results have identified Mentha suaveolens Ehrhard and Dittrichia viscosa (L.) Greuter as promising candidates to be used as refuges for enhancing the activity of predators on protected crops. The first one as a refuge of the flower bug Orius laevigatus and the second as a refuge of the mirid bug Nesidiocoris tenuis, in the southeast of Spain. Augmentative releases of O. laevigatus and N. tenuis are commonly used on protected peppers and tomatoes, respectively, under IPM programs. However, establishment of these natural enemies is sometimes too slow to avoid pest problems in this area. Samplings of the native vegetal species surrounding the greenhouses were conducted to identify the natural host species of these predators. A risk assessment of the identified refuges as possible hosts of pests and viruses that can damage the crops was also completed. Results showed that M. suaveolens and D. viscosa host high populations of O. laevigatus and N. tenuis, respectively. No viruses were detected on both refuges and none were able to be infected, neither by mechanical inoculation nor by whitefly transmission.
Key words: native host plants, integrated pest management, refuges, Nesidiocoris tenuis, Orius laevigatus, protected crops, virus reservoir.
Introduction
The application of Integrated Pest Management methods (IPM) has recently increased very fast in Almería (southeast of Spain), one of the major areas of greenhouse production in Europe. The IPM surface has augmented in this area from 1,400 ha in the season 2006-07 to more than 18,000 ha in 2008-09. Biological control is based on augmentative releases of natural enemies on tomatoes and peppers, the main crops of the area. However, the success achieved on the different protected vegetables is not the same; the biological control is still being applied on a rather low percentage of tomato crops (approx. 21 % in the season 2008-09) while the percentage is higher on peppers (73 %).
There are two main natural enemies commonly used on tomatoes, the mirid bug Nesidiocoris tenuis and the parasitoid Eretmocerus mundus (Mercet), which offer a good control of the main pest, the whitefly Bemisia tabaci (Gennadius). However, biological control is sometimes disrupted in this crop, especially because the beneficial insects have a slow establishment, and farmers begin chemical treatments. On one hand, it is a common practice in the area to regularly defoliate the basal leaves, then eliminating parasitized larvae. On the other hand, tomatoes are mainly planted at the end of summer, so temperature is dropping when N. tenuis is released, and most of the greenhouses are unheated, which makes the establishment and reproduction of the mirid very slow.
On pepper crops, there is also a need to improve the early establishment of natural enemies, despite the higher success achieved. The main pests, the Western flower thrip Frankliniella occidentalis, (Pergande) and B. tabaci, are effectively controlled releasing the phytoseid mite Amblyseius swirskii Athias-henriot and the flower bug Orius laevigatus. However, natural enemies are released when plants open the first flowers, approximately four weeks after transplanting. In Almeria, the season of this crop begins in summer, so releases are done when temperatures are very warm and there is a high pest pressure. Moreover, due to problems with virus transmitted by F. occidentalis the level of acceptance of pest is very low. Then, preventive chemical treatments are always advised in the first weeks. The entomophagous, especially Orius laevigatus, are not well established since 7 or 8 weeks after planting.
In this situation, the use of plant refuges inside the greenhouses could improve the early establishment of natural enemies released on these crops, as it is already developed, for instance, with banker plants for multiplying parasitoids of aphids (Weintraub, 2007). Studies about the seasonal abundance of Orius spp. on refuges conducted in the northeast of Spain showed that Vicia sativa L. and Lupinus hispanicus Boiss & Reuter were good candidates to maintain the populations of this predator during winter and to increase its populations in spring (Alomar et al., 2006). Other studies in Spain have shown that, common plant species such as Diplotaxis erucoides L., Sonchus spp and other species of the families Asteraceae and Umbeliferae host high populations of Orius spp. (Ferragut & Gonzalez-Zamora, 1994). N. tenuis has been collected on Geranium spp. L., Calendula arvensis L., Dittrichia viscosa, Cistus salviifolius L., Plantago sp. L. and Mentha sp. L. in the northeast of Spain (Vila, 2004).
It is very important to select refuges that can not cause problems as a source of pest species or reservoirs of virus. Several virus transmitted by insect vectors are causing important economical losses in Almería. F. occidentalis transmits Tomato spotted wilt virus (TSWV) in tomato and pepper (Cuadrado, 1996). B. tabaci transmits Tomato yellow leaf curl virus (TYLCV and TYLCSV) (Morilla et al., 2003; Morilla et al., 2005) and Tomato chlorosis virus (ToCV) in tomato and pepper (Navas-Castillo et al., 2000; Lozano et al., 2003).
The company Agrobío S.L. on collaboration with the Andalusian Institute for Research and Training in Agriculture and Fisheries (IFAPA Centro La Mojonera) is studying which are the native host plants of N. tenuis and O. laevigatus in Almeria, in order to develop refuges for these predators in the greenhouses. The general aim is to guarantee an earlier establishment of these beneficial insects on tomato and pepper crops. Preliminary prospects to identify the hosts and the evaluation of risks associated with the refuges, as possible sources of pest species or virus, are presented in this work.
Materials and methods
Insects sampling
Native vegetal species adjacent to greenhouse crops or within 50 meters were sampled to identify the hosts of N. tenuis and O. laevigatus. Samples were collected fortnightly from September of 2007 to April of 2008 in the most important greenhouse areas of Almería. Especial attention was devoted on surroundings of greenhouses where IPM methods were applied. A vacuum sampler (KPC®) adapted for collections of insects (Stewart & Wright, 1995) (30 seconds vacuum time/sample) and also visual inspections were used. All samples were stored in mesh bags and transferred to 70% ethanol in the laboratory to identify the species. All the individuals of Miridae and Anthocoridae were identified and quantified under a binocular.
Evaluation of virus epidemic risk
The virus host properties of all the prospected plants were studied in order to estimate the possible role of these species within the framework of a general risk assessment procedure. The presence of viruses was evaluated in the case of insect-transmitted Tomato spotted wilt virus (TSWV), Tomato yellow leaf curl virus (TYLCV/TYLCSV) and Tomato chlorosis virus (ToCV), as well as for Tomato mosaic virus (ToMV) and Pepper mild mottle virus (PMMoV), the latter two being easily transmitted by contact to tomato and pepper, respectively.
The evaluation was done at two levels. First, several leaves from plants which hosted interesting predators were analysed by PCR (TYLCV/TYLCSV) and RT-PCR (TSWV, ToCV, ToMV and PMMoV) using virus-specific primers (Cuadrado & Janssen, 2002; Janssen & Cuadrado, 2004).
Second, groups of 10 individuals from each of the selected plant species were inoculated with each of the virus species: ToMV, PMMoV and TSWV by mechanic inoculation; and TYLCV/TYLCSV and ToCV by viruliferous B. tabaci adults. The B. tabaci individuals were collected in a greenhouse with high presence of both viruses. All the infections were carried out under controlled conditions. Three weeks after the inoculation the plants were analysed by PCR and RT-PCR.
Results and discussion
Predator refuge trait and virus reservoir risk assessment of spontaneous plant species
The identified native host plants of N. tenuis and O. laevigatus are shown on table 1. N. tenuis was collected on 9 species and O. laevigatus on 5 species. PCR analysis of leaf samples from the 31 collected plant species was positive only for Tomato chlorosis virus (ToCV) on Datura inoxia and Vitex agnus-castus (table 1).
The highest populations of O. laevigatus, both nymphs and adults, were collected on Mentha suaveolens Ehrhard and Thymelaea hirsuta (L.). M. suaveolens is an aromatic plant that is very common on wet areas. The population level of O. laevigatus was higher during the blooming period, between April and November. M. suaveolens was already described as a refuge of this predator in Almería (Pérez-Fuentes, 2007) and some farmers are currently using it to enhance the early establishment of O. laevigatus on crops. Some of them are collecting plants from the environment, removing completely natural patches, and distributing the cut stems in the greenhouses.
Collection of plants from the field is not an advisable practice, not only because it can damage the landscape, but also because it can increase the pest problems. M. suaveolens has not found to be a risk as a reservoir of virus that can damage crops (table 1). Samples of this plant collected directly from the field by Sánchez-Campos et al. (1999) had also given negative detections of the virus TYLCV-Sr and TYLCV-Is. However, this refuge hosts some pests that can be accidentally introduced to the protected crops if plants are collected from field. Surveys of this plant have shown that it hosts high whiteflies populations and also individuals of the plant bug Nezara viridula L. (Het.: Pentatomidae), an increasing pest problem on peppers.
Thymelaea hirsuta is a little shrub present in the Mediterranean area that flowers throughout the whole year, although its maximum bloom occurs between the end of summer and autumn. The highest populations of O. laevigatus, both nymphs and adults, were collected during this blooming period. However, this species has been discarded for future studies because it is very difficult to reproduce.
The highest populations of N. tenuis were collected on Dittrichia viscosa and Pelargonium spp. The specie D. viscosa is a little shrub blooming from May to autumn in Almería. This colonizing species is more abundant in abandoned lands and margins of paths. Description of the seasonal abundance of the predator on this refuge has not been possible because the short period sampled, although visual inspections have shown that the populations are lower during the blooming period. D. viscosa Greuter has also been studied in the northeast of Spain where it was found to be the best refuge of the mirid Macrolophus caliginosus (Wagner) [= M. melanotoma (Costa)] between April and June, coinciding with the vegetative growths of the plant, while populations of the mirid predator were lower after blooming (Vila & Alomar, 2000; Vila, 2004).
Table 1. List of vegetal species that hosted populations of Orius spp. and Nesidiocoris tenuis. The total number of samples (n) and the maximum number of nymphs and adults collected by 30 seconds of vacuum sampling is showed. (* = leaf samples that were positive for Tomato chlorosis virus)
D. viscosa does not host any of the main viruses that can damage crops. This agrees with other studies conducted both in southeast and northeast of Spain, where the viruses TYLCV-Sr and TYLCV-Is were not detected on specimens collected from the field (Sánchez-Campos et al., 1999; Morales et al., 2006). However, a search done with the aim of finding alternative hosts of TYLCV, concluded that D. viscosa can be also infected (Jordá et al., 2001). D. viscosa presented negative results for TSWV virus based on the study of Morales et al., 2006.
The ornamental species Pelargonium spp., even though it does not appear spontaneously, has been localized in flower beds next to greenhouses. It hosted high populations of N. tenuis by the end of summer. No viruses were detected on this plant, although it had been previously identified as a host species of TSWV (Cuadrado, 1996).
As a summary, we can conclude that Dittrichia viscosa and Mentha suaveolens are interesting refuge plants for N. tenuis and O. laevigatus, respectively. Both species are common in the crop production area of Almería and host high populations of the predators, both adults and nymphs. No viruses were detected on both refuges and none were able to be infected, neither by mechanical inoculation nor by whitefly transmission. More studies must be done about the biology of these predators on the refuges, evaluating his movement between the refuge and the crops (now in course) in order to establish practical advices about how to use the refuges on protected peppers and tomatoes.
Acknowledgements
This publication has been realized with the support of the Ministry of Sciences and Innovation (of Spain): CDTI Programme, CENIT-MEDIODIA Project. Thanks to David Beltrán for his comments.
References
Alomar, O., Gabarra, R., González, O. & Arnó, J. 2006: Selection of insectary plants for ecological infrastructure in Mediterranean vegetable crops. IOBC/WPRS Bull. 29 (6): 5-8.
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Jordá, C., Font, I., Martínez, P., Juarez, M., Ortega & Lacasa, A. 2001: Current Status and New Natural Hosts of Tomato yellow leaf curl virus (TYLCV) in Spain. Plant Disease, 85: 445.
Lozano, G., Moriones, E. & Navas-Castillo, J. 2003: First Report of Sweet Pepper (Capsicum annuum) as a Natural Host Plant for Tomato chlorosis virus. Plant Disease, 88:224.
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Morales, I., Sanchez, J.A., Alomar, O., Lacasa, A. & Fereres, A. 2006: Susceptibility to virus infection of candidate plants used to enhance predatory dicyphine (Heteroptera: Miridae). IOBC/WPRS Bull. 29 (4): 255-260.
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Biological control of the South American Tomato Pinworm, Tuta absoluta (Lep.: Gelechiidae), with releases of Trichogramma achaeae (Hym.: Trichogrammatidae) on tomato greenhouse of Spain
T. Cabello1, J.R. Gallego1, E. Vila2, A. Soler2, M. del Pino3, A. Carnero3, E. Hernández-Suárez3, A. Polaszek4
1Dpto. Biologia Aplicada, Universidad de Almeria. Ctra. Sacramento s/n, 04120- Almeria, Spain.E-mail: tcabello@ual.es
2Agrobío, Ctra Nacional 340, Km. 419, 04745-La Mojonera, Almería, Spain
3Instituto Canario de Investigaciones Agrarias ICIA, Valle de Guerra, P.O. 60, 38200-La Laguna, Tenerife, Spain
4Department of Entomology, Natural History Museum, Cromwell Road, London SW7 5BD, U.K.
Abstract: The egg parasitoid Trichogramma achaeae has been identified as a candidate for biological control of the South American Tomato Pinworm, Tuta absoluta. On laboratory conditions a marginal attack rate of 100 % was found and 83.3 % of the parasitized eggs developed until the blackhead stage (apparent parasitism). On greenhouse conditions a high efficacy (91.74 % of damage reduction) was obtained when releasing 30 adults/ plant (= 75 adults/ m2) every 3-4 days on August and September of 2008. This shows that this parasitoid can be a good weapon to control T. absoluta on greenhouses of the southeast of Spain.
Key words: greenhouse, tomato, exotic pest species, oophagous parasitoid, biological control, Tuta absoluta, Trichogramma achaeae.
Introduction
The South American Tomato Pinworm, Tuta absoluta (Meyrick), has spread from Central America to most of South America, and it is considered one of the major pests on tomato and other solanaceous crops (EPPO, 2005). Its chemical control is difficult, firstly due to its biology, because of the mine-feeding behaviour of larvae (Branco. & Franca, 1993; Urbaneja et al., 2007), which has originated an intensive use of chemical treatments. Secondly, it has developed insecticide resistance to the active ingredients used until now (Siqueira et al., 2000; EPPO, 2005; Lietti et al., 2005). Consequently, biological control has been developed and it is being applied on several South American countries, especially using the oophagous parasitoids Trichogramma pretiosum (Riley) and T. nerudai Pintureau and Gerding (Riquelme & Botto, 2003; Parra & Zucchim, 2004; Bueno, 2005; Faria et al., 2008).
T. absoluta was accidentally introduced in Castellón, Spain, in 2006; it has spread from Comunidad Valenciana to Murcia and Ibiza (Urbaneja et al., 2007; EPPO, 2008) and first infestations were detected in the greenhouses of Almeria in 2008. This quarantine pest has caused severity damage on tomato crops in Murcia area (Lucas et al., 2009), although impact is still fairly low in Andalucía (Gonzalez-Garcia, 2009). Several species of autochthonous predators have been evaluated as biological control agents in Spain with promising results, mainly the mirid bugs Nesidiocoris tenuis (Reuter) and Macrolophus pygmaeus (Rambur) (Urbaneja et al., 2008) and the damsel bug Nabis pseudoferus Remane (Cabello, 2009). However, the establishment of these predators into the crops is often too slow to avoid damage of the pest, and consequently chemical treatments are needed, which disturbs biological control. Then, several works are being conducted to evaluate autochthonous parasitoids, using them alone or combined with predators, on tomato greenhouse crops from the southeast of Spain. Preliminary results with the specie Trichogramma achaeae Nagaraja & Nagarkatti are presented on this work.
T. achaeae has a world wide distribution; it has been mentioned in Asia (China, India and Russia), Europe (France, Russia), Africa (Cabo Verde) and the New World (Argentina, Barbados, Chile, Trinidad & Tobago, U.S.A.). Recently, it has been reported in Spain (unpublished data). It is an egg parasitoid of 26 Lepidoptera species belonging to 10 families: Gelechiidae, Geometridae, Noctuidae, Notodontidae, Oecophoridae, Pieridae, Pyralidae, Sphingidae, Tortricidae and Yponomeutidae (Polaszek, not publ.). T. achaeae has been evaluated as a biological control agent of different lepidopteran pests (Jalali et al., 2001; Jalali & Singh, 2002; Jalali et al., 2002; Chandrashekhar et al., 2003; Yadav & Anand, 2003) and is commercially available in India (Nagaraja et al., 2002).
Material and methods
Insects rearing
The individuals of T. absoluta used in the trials were obtained from a laboratory rearing on tomato leaves. The rearing of T. achaea, also in the laboratory, was conducted using eggs of Ephestia kuehniella Zeller as alternative host, according to the methodology described by Cabello (1985).
Laboratory bioassay
A preliminary test under laboratory conditions (25±1º C, 60-80 % RH and 16:8 hours L:D) was carried out to evaluate acceptance and parasitation of T. absoluta eggs by females of the parasitoid. Methodology was adapted from Brotodjojo & Walter (2006), with the exception that 10 eggs of T. absoluta (less than 24 hours aged) were offered to each female. 10 repetitions were conducted. All the parasitized eggs were developed until adult emergence.
Greenhouse bioassay
To evaluate the efficacy of T. achaea a test was conducted in a commercial greenhouse of Almeria (Spain), between August the 27th and September the 22th of 2008. Eight cages (8 m2) were placed into the greenhouse with 20 tomato plants (Cherry variety) per cage arranged on pots. Plants were infested with T. absoluta (4 adults/ plant) when reaching one meter height.
A total of seven releases of T. achaeae (30 adults/ plant) were conducted every three or four days on four cages. The other four cages were leaved as a control. The number of larves of T. absoluta, leaf mines and damaged fruits were counted alter 27 days. Data was analyzed using one-way ANOVA and means were compared with LSD, SPSS software v. 15 (SPSS, 2006).
Results and discussion
Laboratory bioassay
The eggs of T. absoluta were well accepted as hosts by T. achaea (100 % of the eggs received a laying of the parasitoid). However, only 83.3 % of the eggs developed to the blackhead stage (prepupa of the parasitoid) (apparent parasitism), while the rest did not and finally collapsed. The eggs of T. absoluta are smaller than the eggs of the rearing host, E. kuehniella. Adult females of T. achaeae, both reared on Chrysodeixis chalcites (Esper) or E. kuehniella, are larger than the eggs of Tuta absoluta but shorter than the eggs of E. kuehniella (fig.1). It has been described that the size of the rearing host does affect the parasitism rate of natural hosts (Brotodjojo & Walter, 2006); female parasitoids in these cases accept host eggs with about the same size as their natal host or larger ones (Salt, 1940; Nurindah et al., 1999). In the present work T. achaeae accepted all the offered eggs of T. absoluta, although the small size of the host may have affected the development of the parasitoid, which could explain why a percentage of the eggs collapsed.
Adults of Trichogramma also kill host eggs by feeding on them; the host egg is stung and the female feeds on the drop of liquid appearing at the site of the sting. The host egg dies, leaving no evidence of parasitism (Knutson, 1998). In some species of Trichogramma, host feeding contributes significantly to pest control (Vasquez et al., 1997). In the present work host feeding by female parasitoids was not observed. However, all the individuals had a supplier of food, a drop of honey:water (1:1), during the bioassay, and host feeding could happen under field conditions.
Figure 1. Comparative size of E. kuehniella eggs, T. absoluta eggs and adult females of the parasitoid (T. achaeae) when reared on eggs of Chryxodeixis chalcites or Ephestia kuehniella (C.L. at P=0.05).
This means that checking the colour as a way to evaluate the ratio of parasitism, both on field or laboratory conditions, can underestimate the real impact. For this reason, egg hatch should be recorded in addition to egg parasitism (Knutson, 1998).
It could also explain why data about the rate of parasitism, in this case of T. pretiosum, shows low values, among 1.5 % (Faria et al., 2008), 22.7-24.4 % (Villas-Boas & Franca, 1996) and 49.0 % as the maximum (Haji et al., 1995), although it is a real effective field method to control the pest on several countries of South American. Then, it is a subject to be considered when establishing the methodology to estimate the efficacy of Trichogramma to control this pest on field conditions.
Greenhouse bioassay
On figure 2 the number of larvae, leaf mines and damaged fruits on tomato plants after 27 days from the beginning of the trial are showed. Number of larvae and damaged leaves are significantly lower where parasitoids had been released (P . absoluta was 12 times lower on plots where T. achaeae was released compared with the control. This means a 91.74 % of efficacy.
On one hand, the doses of releases used in this bioassay are similar to that used commercially in Brasil with T. pretiosum (Freitas et al., 1994: Parra & Zucchim, 2004). Likewise, the levels of damaged fruits are similar to that described when using T. pretiosum, with cited values from 2.0 % (Villas-Boas & Franca, 1996) to 13.0 % (Domingues et al., 2003). On the other hand, the determined efficacy (91.7 %) is lightly higher than the cited when using T. pretiosum (87.7 %) on said country (Parra & Zucchim, 2004).
Figure 2. Number of Tuta absoluta larvae and damage on tomato plants counted in a greenhouse according to the treatment (with or without releases of T. achaeae).
Showed data are promising, but must be completed with other studies (now in course), mainly to establish the needed releases on greenhouses according to the density of the pest and the growth stage of the crop, in the protected crops of the southeast of Spain. A good possibility may be to combine releases of T. achaeae with the mirid predator N. tenuis. One of the major problems of using N. tenuis in Almeria is his slowly establishment, needing at least around 4 weeks. Tomato crop is mainly planted at the end of the summer, so releases are made in autumn, when temperature is dropping. During this period Tuta absoluta can cause heavy damage, and weekly releases of the parasitoid could help to guaranty an effective control of the pest.
Acknowledgements
This publication has been realized with the support of the Ministry of Sciences and Innovation (of Spain): CDTI Programme, CENIT-MEDIODIA Project.
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