Restoring a maize root signal that attracts insect-killing nematodes to control a major pest

Abstract

When attacked by herbivorous insects, plants emit volatile compounds that attract natural enemies of the insects. It has been proposed that these volatile signals can be manipulated to improve crop protection. Here, we demonstrate the full potential of this strategy by restoring the emission of a specific belowground signal emitted by insect-damaged maize roots. The western corn rootworm induces the roots of many maize varieties to emit (E)-beta-caryophyllene, which attracts entomopathogenic nematodes that infect and kill the voracious root pest. However, most North American maize varieties have lost the ability to emit (E)-beta-caryophyllene and may therefore receive little protection from the nematodes. To restore the signal, a nonemitting maize line was transformed with a (E)-beta-caryophyllene synthase gene from oregano, resulting in constitutive emissions of this sesquiterpene. In rootworm-infested field plots in which nematodes were released, the (E)-beta-caryophyllene-emitting plants suffered significantly less root damage and had 60% fewer adult beetles emerge than untransformed, nonemitting lines. This demonstration that plant volatile emissions can be manipulated to enhance the effectiveness of biological control agents opens the way for novel and ecologically sound strategies to fight a variety of insect pests.

Fig. 1.

Insertion of an EβC synthase gene from Origanum vulgare L. in maize variety HiII results in a constitutive production of EβC. (A) Terpene synthase overexpression construct (for details, see Materials and Methods). (B) A typical chromatogram obtained for the volatiles emitted by roots of the hybrid variety HiII line alongside a chromatogram for one of the transformed lines. Peak 1 is EβC and peak 2 is α-humulene, a side-product of EβC synthase. (C) Average quantities of EβC present in the roots of the untransformed HiII and the 3 transformed lines that were used in the field experiments (n = 8).

Fig. 2.

Transformants releasing EβC suffered less damage than control lines when EPNs were present. (A) Root damage measured on plants that had received neither WCR eggs nor nematodes was minimal, and there was no difference between transformed and nontransformed plants (n = 5, P = 0.87). (B) Root damage on plants that received only WCR eggs, but no nematodes, was substantial. Again, no significant difference was found between the transformed and nontransformed plants (n = 5, P = 0.18). (C) In plots that received WCR eggs and H. megidis, roots from transformed plants (pooled) had significantly less damage than roots from control lines (n = 30, P = 0.007). Approximately one-quarter of the transformed plants were found not to emit EβC. Removing these plants from the statistical analysis did not significantly affect the results. The letters above the bars indicate significant differences within a graph. Error bars indicate standard errors.

Fig. 3.

Fewer adult WCR beetles emerged near EβC-releasing transformants when nematodes were applied. (A) Adult emergence for plants from the plots that received WCR eggs only, but no nematodes. No significant difference was found between the transformed and nontransformed plants (n = 5, P = 0.47). (B) Adult emergence for plants that received both WCR eggs and nematodes was significantly different between transformed plants (pooled) and control plants (n = 30, P < 0.0001). Approximately one-quarter of the transformed plants were found not to emit EβC. Removing these plants from the statistical analysis slightly reduced the average emergence near the transformed plants (black bar) and increased the difference with untransformed control plants to 60%. (C) Significantly fewer adults emerged near EβC-producing transformed plants (black bar) than near transformed plants that were not emitting EβC (gray bar; P = 0.023). The letters above the bars indicate significant differences within a graph. Error bars indicate standard errors. No WCR adults were recovered from plots that did not receive WCR eggs.

Fig. 4.

Confirmation of nematode attraction to transformed maize lines. H. megidis nematodes were more strongly attracted to transformed plants than to untransformed controls in 6-arm olfactometers. The graph depicts the average number of nematodes recovered from olfactometer arms connected to pots containing either a WCR-infested transformed plant (line 202 L2; black bar), a WCR-infested control plant (gray bar), or only moist sand (white bar). The plants were each infested with 5 second-instar WCR larvae. For each replicate, the total number of nematodes found in the 4 moist sand-only control pots (white bar) were summed and divided by 4. The attraction to transformed plants was significantly higher than to the control plants (n = 11, P < 0.001). The letters above the bars indicate significant differences. Error bars indicate standard errors.

Fig. 5.

There was no direct effect of the transformation on WCR performance. (A) Average weight gain of WCR larvae fed for 5 days on transformed (line 202 L2) or control plants. No statistical difference was found (n = 13, P = 0.75). (B) Survival of WCR larvae after 5 days on transformed or nontransformed plants. No statistical difference was found (n = 13, P = 0.18). Error bars indicate standard errors.