When Competitors Join Forces: Consortia of Entomopathogenic Microorganisms Increase Killing Speed and Mortality in Leaf- and Root-Feeding Insect Hosts

Abstract

Combining different biocontrol agents (BCA) is an approach to increase efficacy and reliability of biological control. If several BCA are applied together, they have to be compatible and ideally work together. We studied the interaction of a previously selected BCA consortium of entomopathogenic pseudomonads (Pseudomonas chlororaphis), nematodes (Steinernema feltiae associated with Xenorhabdus bovienii), and fungi (Metarhizium brunneum). We monitored the infection course in a leaf- (Pieris brassicae) and a root-feeding (Diabrotica balteata) pest insect after simultaneous application of the three BCA as well as their interactions inside the larvae in a laboratory setting. The triple combination caused the highest mortality and increased killing speed compared to single applications against both pests. Improved efficacy against P. brassicae was mainly caused by the pseudomonad-nematode combination, whereas the nematode-fungus combination accelerated killing of D. balteata. Co-monitoring of the three BCA and the nematode-associated Xenorhabdus symbionts revealed that the four organisms are able to co-infect the same larva. However, with advancing decay of the cadaver there is increasing competition and cadaver colonization is clearly dominated by the pseudomonads, which are known for their high competitivity in the plant rhizosphere. Altogether, the combination of the three BCA increased killing efficacy against a Coleopteran and a Lepidopteran pest which indicates that this consortium could be applied successfully against a variety of insect pests. Graphical Abstract

Entomopathogenic pseudomonads can share an insect host with entomopathogenic nematodes and their mutualistic bacteria

“…Nematodes ( Table 1 ) were propagated in G. mellonella larvae using kanamycin (10 μg/larva) injections as a pretreatment as previously described [ 12 ]. IJs were stored at 15°C in tap water in tissue culture flasks and adjusted to the desired concentrations before use.…”

“…Larvae were homogenized in 1 ml LB as previously described [ 46 ] resulting in 1250 μl homogenate for an average larva. Alive larvae were additionally surface-disinfected before homogenization as previously described [ 12 ]. The homogenate was plated directly or frozen for later qPCR analysis.…”

“…One way to improve biocontrol performance is the combined application of several BCAs [ 9 , 10 ]. Some studies have already shown an improved plant protection [ 11 ], increased killing speed, or insect mortality [ 12-15 ] with the use of BCA combinations. However, depending on the BCA combinations and application techniques used, negative effects on insect control were observed, which several authors attributed to negative interactions between the BCAs [ 10 , 13 , 16 ].…”

“…In this study, we investigated the interactions between entomopathogenic pseudomonads (EPPs) and entomopathogenic nematodes (EPNs) with their mutualistically associated bacteria (NABs), both long-studied and promising BCAs. In previous studies, the combination of these two BCAs showed increased protection of radish against the cabbage root fly in lab and semi-field experiments [ 11 ] as well as increased killing efficacy against two different insect species [ 12 ]. The EPPs Pseudomonas protegens and Pseudomonas chlororaphis are aggressive root colonizers with plant growth-promoting and disease-suppressing abilities [ 21-24 ].…”

“…The second hypothesis is supported by the findings of co-occurrence and association of EPPs with EPNs in nature [ 38-40 ]. In addition, our previous work showed that a P. chlororaphis strain and a Xenorhabdus bovienii strain can co-colonize larvae of two insect species [ 12 ]. What is lacking is a detailed understanding of the interaction dynamics between EPPs and NABs inside insects and the effect of EPPs on EPN fitness.…”