African Journal of Agricultural Research Vol. 7(23), pp. 3473-3484, 19 June, 2012 Available online at http://www.academicjournals.org/AJAR DOI: 10.5897/AJAR11.2073 ISSN 1991-637X ©2012 Academic Journals Full Length Research Paper Detection of predators within Brassica crops: A search for predators of diamondback moth (Plutella xylostella) and other important pests Reza Hosseini1*, Otto Schmidt2 and Michael A. Keller2 1Department of Plant Protection, College of Agriculture, University of Guilan, P. O. Box 41635-1314, Rasht, Iran. 2Discipline of Plant and Food Science, School of Agriculture, Food and Wine, Waite Campus, University of Adelaide, SA 5005, Australia. Accepted 28 March, 2012 Techniques based on the Polymerase Chain Reaction (PCR) have been shown to be powerful tools for ecological studies of predator-prey interactions. By using developed species-specific primers from the cytochrome oxidase subunit I (COI) gene for six insect pests of Brassica crops (Plutella xylostella, Pieris rapae, Hellula hydralis, Helicoverpa punctigera, Brevicoryne brassicae, and Myzus persicae) trophic relationships of selected predators and their prey in Brassica fields demonstrated the potential of DNA-based techniques to screen predator communities and to identify their prey. In this investigation, all examined predators including Nabis kinbergii (Heimptera: Nabidae), Oechalia schellenbergii (Hemiptera: Pentatomidae), Micromus tasmaniae (Neuroptera: Hemerobiidae), Hippodamia variegata, Coccinella transversalis (Coleoptera: Coccinellidae) and four wolf spider species including Trochosa expolita, Venatrix pseudospeciosa, Venator spenceri and Hogna kuyani (Araneae: Lycosidae) showed polyphagy to some extent. All tested positive for P. xylostella, hence can be considered as predators of this pest. Although, the records of DNA from the guts of predators probably represent instances of real predation, but interpretation of predation data because of some errors is very difficult. There are limitations for this type of interpretation which has been comprehensively discussed in this paper. Key words: Molecular markers, predator, gut contents, Brassica crop, biological control. INTRODUCTION The value of predators in the biological control of insect of predators on pest populations and especially critical in pests in integrated pest management programs has been evaluating the effectiveness of a predator as a biological highlighted by many investigators (Hagler and Durand, control agent (Hayes and Lockley, 1990). There are 1994; Symondson et al., 2002). Identification of trophic inherent difficulties associated with the study of the diet relationships between predators and particular pests is breath of predators in nature because of the relatively one of the first important steps in determining the impact small size of both predator and prey, and their often elusive and nocturnal activity (Greenstone, 1996; Hoogendoorn and Heimpel, 2001). Consequently, there is a lack of knowledge about predators’ feeding behaviour *Corresponding author. E-mail: [email protected], in the natural environment; because of this difficulty, little [email protected]. Fax: +98-131-6690281. is known about predator-prey trophic interactions and the 3474 Afr. J. Agric. Res. effects of predators on pests of Brassica crops. However, Studies showed that Brassica crops have a rich fauna of to elucidate the role of predators in the control of these predators. Schmaedick and Shelton (2000) have pests, some experiments have been done under documented a list of predators associated with P. rapae laboratory conditions or in field cages (Schmaedick and in cabbage fields of New York State. Hosseini et al. Shelton, 1999), but these studies do not necessarily (2006b) reported a range of predators associated with accurately simulate field conditions. So far, the most Brassica pests in South Australia. The impact of effective and least disruptive method for studying predators on pests of Brassica crops has not been predation has been the development of biochemical thoroughly studied and therefore their potential in diagnostic technologies, monoclonal and polyclonal suppression of major pests of Brassica has not been antibodies (Symondson et al., 2002) and enzyme- elucidated. Hooks et al. (2003) found broccoli plants electrophoresis (Traugott, 2003) can be used to protected by birds and spiders as predators sustained determine which prey has been consumed by a predator. less damage from caterpillars and the plants had greater However, amplification of specific prey DNA using the productivity compared to control plants. Cage exclusion polymerase chain reaction has proven to be more experiments have shown that predation varies practical and cost-effective in detecting prey remains. considerably in space and time. Once it is developed, this assay can be used to Wang et al. (2004) estimated between 7 and 81% of efficiently and sensitively test large numbers of field- immature stages of P. xylostella were lost due to collected predators for evidence of feeding on a particular predation on farms in Queensland, Australia. prey species, such as key agricultural pest (Symondon, Subsequently, more extensive research (Furlong et al., 2002). PCR-based techniques are rapidly replacing other 2004) confirmed estimated losses of P. xylostella to molecular techniques because molecular biology facilities predation of the same magnitude, between 2 and 85%. A are widely available and prey-specific primers can be better understanding of the identities of key predators used in different contexts such as ecological, taxono- and factors that affect their feeding activities could enable mical, behavioral study of insects (Hoy, 2003) once they farmers and pest managers to conserve key predators. have been designed. This technique has been This would ensure that they more consistently kill 80% or successfully used for detection of a variety of prey more of immature diamondback moths. Hence, for this remains in predators’ gut contents (Zaidi et al., 1999; one insect, predators are known to cause considerable Chen et al., 2000; Hoogendoorn and Heimpel, 2001; mortality at times. It is essential to develop a reliable Agustí et al., 2003a, 2003b; Harper et al., 2005; Juen and technique to evaluate the diets of key predatory species Traugott, 2005; Read et al., 2006; Harwood et al., 2007; in order to understand their role in suppressing pests like Zhang et al., 2007; Kuusk and Agusti, 2008; Schmidt et P. xylostella. Ma et al. (2005) developed a species- al., 2009; Monzó et al., 2010). Brassica vegetables and specific marker for P. xylostella based on the internal oilseeds are economically important crops; approximately transcribed spacer (ITS-1) of the ribosomal gene. This 3.1 and 26.1 million ha respectively were grown specific primer pair was used to detect prey in the gut worldwide in 2004 (Food and Agriculture Organization of contents of two polyphagous predators, Nabis kinbergii the United Nations, 2009). These crops are associated and Trochosa expolita (reported as Lycosa sp.). In a pilot with several destructive and widespread insect pests. study of predation of diamondback moth on cauliflower Total damage caused by these pests is substantial; for and broccoli farms near Virginia, South Australia, the example, management costs for Plutella xylostella alone remains of P. xylostella were detected in the gut contents were estimated at US$ 1 billion annually in 1997 (Shelton of both species of field-collected predators. In the current et al., 1997). In Australia, the pests of Brassica crops study, cytochrome oxidase subunit I (COI) was selected include the lepidopterans P. xylostella, Pieris rapae, as a potentially diagnostic gene. As a mitochondrial gene, Hellula hydralis and Helicoverpa punctigera, and two it occurs as multiple copies in each cell (Hoy, 2003), aphids, Brevicoryne brassicae and Myzus persicae which increases the likelihood of successful amplification (Hosseini et al., 2006a). Integrated pest management of prey residues in the predators’ gut contents. This gene (IPM) systems and the use of biological control methods is a protein-coding gene that has a high level of are preferred approaches to controlling these pests over interspecific variability (Zhang and Hewitt, 1996), which insecticides due in part to the prevalence of insecticide allows closely related species to be separated. resistance in diamondback moth (Shelton et al., 1997). In the present study, a PCR-based technique was used Generalist predators can play a major role in the control for the first time to study predator-prey interactions of agricultural pests (Symondson et al., 2002); but this is extensively in Brassica crops. The aim of this research considered controversial (Rosenheim et al., 1993). reported in this paper was to determine the trophic Hosseini et al. 3475 relationships among predators and prey, mainly focusing primer pairs and 4 μl of DNA extract. The thermocycling program on the identification of predators of Brassica crops key consisted of an initial step of 2 min at 95°C, followed by 35 cycles of pest “diamondback moth” and other common pests. 30 s at 94°C, 30 s at the specific annealing temperature for each prey specific primer (Table 1), 1 min at 72°C and a final elongation step of 5 min at 72°C. PCR products were visualised following electrophoresis on 1.8% agarose gel in TAE containing 0.5 μl/ml MATERIALS AND METHODS ethidium bromide for DNA staining and then photographed. Each PCR assay had a positive (DNA