Bioturbation by endogeic earthworms facilitates entomopathogenic nematode movement toward herbivore-damaged maize roots

Abstract

Entomopathogenic nematodes (EPNs) have been extensively studied as potential biological control agents against root-feeding crop pests. Maize roots under rootworm attack have been shown to release volatile organic compounds, such as (E)-β-caryophyllene (Eβc) that guide EPNs toward the damaging larvae. As yet, it is unknown how belowground ecosystems engineers, such as earthworms, affect the biological control capacity of EPNs by altering the root Eβc-mediated tritrophic interactions. We here asked whether and how, the presence of endogeic earthworms affects the ability of EPNs to find root-feeding larvae of the beetle Diabrotica balteata. First, we performed a field mesocosm experiment with two diverse cropping systems, and revealed that the presence of earthworms increased the EPN infection potential of larvae near maize roots. Subsequently, using climate-controlled, olfactometer-based bioassays, we confirmed that EPNs response to Eβc alone (released from dispensers) was two-fold higher in earthworm-worked soil than in earthworm-free soil. Together our results indicate that endogeic earthworms, through burrowing and casting activities, not only change soil properties in a way that improves soil fertility but may also enhance the biocontrol potential of EPNs against root feeding pests. For an ecologically-sound pest reduction in crop fields, we advocate agricultural practices that favour earthworm community structure and diversity.

Figure 1

Graphical abstract. A semi-field experiment and several olfactometer-based experiments were conducted to address the effect, and the mechanisms, respectively, of earthworms on belowground tritrophic interactions between maize plants, rootworm larvae and entomopathogenic nematodes (EPNs). Particularly, we hypothesized that the borrowing and casting activity of earthworms would promote soil fertility, and thus plant growth (1), and also enhance chemical defence production (2), which in turn would stimulate EPNs recruitment (3). Finally, we hypothesized that soil labouring by earthworms would generate a more favourable soil environment for EPN movement and host seeking behaviour (4).

Figure 2

Probability of infection by EPNs. Solid black lines show the probability of a Galleria mellonella larva being infected by Heterorhabditis megidis EPNs when placed close to a healthy root system (Control), or a mechanically-damaged root system of maize (Zea mays var. Delprim) plants and induced with JA. Dashed and solid grey lines show the same effect, but the dataset is divided between the polyculture (maize grown with squash and bean plants), or the monoculture (only maize) treatments, respectively. Panel (A) shows the overall effect, while panels (B) and (C) show the probabilities of EPN infection in mesocosms with (dark grey boxes), or without (open boxes) Allolobophora icterica earthworms, respectively.

Figure 3

Four-arm olfactometer bioassay. (A) Schematic design of a four-arm olfactometer that fully-factorially manipulated the presence or the absence of Allolobophora icterica earthworms, and the presence or the absence of Diabrotica balteata larvae around seedlings of maize plants (Zea mays var. Delprim). The center of the arena, where Heterorhabditis megidis EPNs were released contained sand, whereas the side pots contained a mixture of sand and soil. (B) The number of H. megidis nematodes found in the arms of a four-arm olfactometer. (C) The amount of (E)-β-caryophyllene produced by the roots of the corn plants in the four treatments (n = 20). Letters above bars show significant differences among treatments obtained from contrasting marginal means after generalized linear model and linear model for panels (B) and (C), respectively.

Figure 4

Earthworms-EPNs direct interaction bioassay. (A) Schematic design of a two-arm olfactometer that manipulated the presence or absence of A. icterica in the central soil where also EPNs are present. The side pots were inoculated with a mixture of synthetic CO2 and (E)-β-caryophyllene (Eβc) for stimulating EPN directional movement. (B) Number of EPNs that were found in either sides of the two-arm olfactometer at the end of the bioassay (n = 10). Letters above bars show significant differences between treatments (linear model; p < 0.05).