Wide interguild relationships among entomopathogenic and free-living nematodes in soil as measured by real time qPCR

Abstract

Entomopathogenic nematodes (EPNs) are promising biological control agents of soil-dwelling insect pests of many crops. These nematodes are ubiquitous in both natural and agricultural areas. Their efficacy against arthropods is affected directly and indirectly by food webs and edaphic conditions. It has long been suggested that a greater understanding of EPN ecology is needed to achieve consistent biological control by these nematodes and the development of molecular tools is helping to overcome obstacles to the study of cryptic organisms and complex interactions. Here we extend the repertoire of molecular tools to characterize soil food webs by describing primers/probe set to quantify certain free-living, bactivorous nematodes (FLBNs) that interact with EPNs in soil. Three FLBN isolates were recovered from soil baited with insect larvae. Morphological and molecular characterization confirmed their identities as Acrobeloides maximum (RT-1-R15C and RT-2-R25A) and Rhabditis rainai (PT-R14B). Laboratory experiments demonstrated the ability of these FLBNs to interfere with the development of Steinernema diaprepesi, Steinernema riobrave and Heterorhabditis indica parasitizing the weevil Diaprepes abbreviatus (P<0.001), perhaps due to resource competition. A molecular probe was developed for the strongest competitor, A. maximum. We selected the highly conserved SSU rDNA sequence to design the primers/probe, because these sequences are more abundantly available for free-living nematodes than ITS sequences that can likely provide better taxonomic resolution. Our molecular probe can identify organisms that share ⩾98% similarity at this locus. The use of this molecular probe to characterize soil communities from samples of nematode DNA collected within a citrus orchard revealed positive correlations (P<0.01) between Acrobeloides-group nematodes and total numbers of EPNs (S. diaprepesi, H. indica and Heterorhabditis zealandica) as well as a complex of nematophagous fungi comprising Catenaria sp. and Monachrosporium gephyropagum that are natural enemies of EPNs. These relationships can be broadly interpreted as supporting Linford's hypothesis, i.e., decomposition of organic matter (here, insect cadavers) greatly increases bactivorous nematodes and their natural enemies.

Prospects for Using Entomopathogenic Nematodes to Control the Vine Mealybug, Planococcus ficus, in South African Vineyards

“…The use of quantitative real-time PCR (qPCR) can be used in the field and in the laboratory for identifying and quantifying EPN species (Campos-Herrera et al, 2012). Conventional insect baiting techniques can now be replaced by the qPCR method, which is faster, more accurate and more affordable (Campos-Herrera et al, 2011a;2011b).…”

Phylogenetic Relationships of Insect-Associated Free-Living Rhabditid Nematodes From Eastern Mediterranean Region of Turkey

“…: Pyralidae) larvae (Bedding and Akhurst, 1975) for screening entomopathogenic nematodes (EPN) in our previous study (Karabörklü, 2012;Karabörklü et al, 2015a). However, many rhabditid nematodes also detected during these procedures (Duncan et al, 2003, Campos-Herrera et al, 2012. These rhabditid nematodes harvested from dead G. mellonella larvae were placed individually into modified White traps (Kaya and Stock 1997) at room temperature (~25 ºC).…”

Analyzing spatial patterns linked to the ecology of herbivores and their natural enemies in the soil

“…The advantage of qPCR compared to conventional PCR is that the order of magnitude increase in the amplification potential of tiny quantities or even degraded material. This method has been successfully employed to detect cryptic organisms and assessed diversity and interactions among bacteria, fungi, nematodes, insect and plants from above and belowground systems (Atkins et al, 2005; Jones et al, 2006a,b; Zhang et al, 2006; Lundgren et al, 2009; King et al, 2010; Campos-Herrera et al, 2011a,b, 2012; Heidemann et al, 2011; Lundgren and Fergen, 2011; Pathak et al, 2012; Vervoort et al, 2012). Nevertheless, qPCR also has important limitations.…”

“…Some groups, such as free living nematodes, frequently lack adequate numbers of published sequences for comparison when designing molecular probes, so this method can finally be used for some undescribed, regional species (Jones et al, 2006a,b). In such cases, it may still be useful to use qPCR to measure broader taxa with similar ancestry/behavior rather than individual species (Campos-Herrera et al, 2012). The worldwide tendency to increasingly publish sequences in public domains is rapidly increasing the opportunity to design species-specific primers/probe combinations for a diverse array of organisms.…”