Antimicrobial activity of Xenorhabdus sp. RIO (Enterobacteriaceae), symbiont of the entomopathogenic nematode, Steinernema riobrave (Rhabditida: Steinernematidae)

doi:10.1016/S0022-2011(02)00019-8

Journal of Invertebrate Pathology

Volume 79, Issue 3, March 2002, Pages 146-153

https://doi.org/10.1016/S0022-2011(02)00019-8 Get rights and content

Abstract

Galleria mellonella larvae infected with Steinernema riobrave soon showed (after 24 h) the typical growth of its Xenorhabdus sp. RIO symbiont and, in parallel, the growth of another Gram negative bacterial species in the body cavity. A population of Entercoccus sp. in the nematode infected larvae collapsed to zero by 96 h. The level of antibiotic and antimycotic activity followed a pattern similar to that of the growth curve to stationary phase of the Xenorhabdus sp. RIO symbiont, over a period of 168 h. The antimycotic activity was composed of exo- and endochitinases as well as other proteinaceous and some small molecule compounds. The changing pH, relatively high growth rate of Xenorhabdus sp. RIO compared with that of other Gram negative bacterial species and of collapse of the Enterococcus sp. population enabled Xenorhabdus sp. RIO to out-compete other species.

Introduction

Nematodes of the family Steinernematidae are used as biological control agents for a variety of agricultural and horticultural insect pests. Upon infection of an insect host the nematode releases its bacterial symbiont, Xenorhabdus sp., into the insect's haemocoel resulting in a bacterial septicemia and death of the insect within 24–48 h. The secondary metabolites produced by the Xenorhadus sp. overcome the insect's immune system (Akhurst and Dunphy, 1993; Forst and Nealson, 1996), kill the insect, and inhibit the growth of various fungal and bacterial competitors (Akhurst, 1982; Chen et al., 1994; Chen et al., 1996). By doing so, the bacterial symbionts are believed to prevent putrefaction of the insect cadaver and establish conditions that favor the development of both the nematode and bacterial symbionts (Akhurst and Boemare, 1990). However, the nature of this bacterial nematode–insect relationship is not fully understood.

The production of secondary metabolites with antimicrobial properties is common to many Xenorhabdus species (Akhurst and Dunphy, 1993; Webster et al., 1998). In addition to small molecular weight antibiotics, Xenorhabdus species produce extracellular enzymes including proteases, lipases, phospholipases, and DNAses that are involved in breaking down insect tissues to provide nutrients for both the nematode and bacterial symbionts (Forst and Nealson, 1996). Chen et al. (1996) showed that Xenorhabdus nematophilus and Xenorhabdus bovienii produce both exo- and endochitinases, and suggested that these enzymes may play an important role in protecting the dead or dying insect from fungal invasion. It is widely believed that the antimicrobial metabolites help to maintain a relatively, competitor-free environment for Xenorhabdus and Steinernema within the host insect cadaver (Akhurst, 1982; Akhurst and Boemare, 1990; Boemare et al., 1997; Forst and Nealson, 1996). However, there are differing views as to the level of suppression of bacterial and fungal competitors, and whether Xenorhabdus alone is responsible for preventing putrefaction of the insect cadaver by other microorganisms (Jarosz, 1996a, Jarosz, 1996b; Maxwell et al., 1994).

Steinernema riobrave shows considerable promise in controlling insect pests (Cabanillas et al., 1994), but little is known of the properties of its Xenorhabdus sp. RIO symbiont. This study was done to determine some of the antimicrobial properties of the metabolites produced by Xenorhabdus sp. RIO. and in particular, to determine (i) whether these compounds prevent development of all secondary microorganisms within nematode infected Galleria mellonella larvae and (ii) the relative contribution of chitinase to the antifungal activity of the antimicrobial metabolites.

Section snippets

Growth and level of bioactivity of microflora in nematode infected larvae

Late instar G. mellonella larvae, obtained from the Department of Biological Sciences Insectary, were infected with S. riobrave infective juveniles (IJs), obtained from Dr. R. Gordon (Memorial University of Newfoundland), in 9-cm petri dishes, as described by Hui and Webster (2000). Sampling and monitoring of the resulting bacterial population were adapted from Hu and Webster (1998). Five larvae were added to each of the 15 petri dishes for a total of 75 larvae and incubated in the dark at 25

Growth and level of bioactivity of microflora in nematode infected larvae

Insect mortality was first observed at 48 h post exposure, and by 72 h, all of the insect larvae, except for those in the controls, had died. Xenorhabdus sp. RIO was first detected at 48 h, which was when the insects began to die. The number of bacteria increased rapidly from 1.0×10⁶ CFU/g to 1.1×10⁹ at 120-h post-infection and remained at about this level for at least 240 h (Fig. 1). At this early stage of infection, the only other bacterium isolated from the larvae was a Gram positive coccus,

Discussion

Xenorhabdus species are able to withstand the initial immune response of the G. mellonella larvae, rapidly colonize the insect, and produce antimicrobial compounds with both the antibacterial and antimycotic activities, as shown here for Xenohabdus sp. RIO. As the Xenorhabdus sp. RIO population increased rapidly to the stationary phase, the population of Enterococcus sp. collapsed. G. mellonella larvae infected by S. riobrave died within 72 h, presumably from the bacterial toxins released by the

Acknowledgements

We acknowledge with thanks the assistance of Bruce Leighton and the financial support of the Natural Sciences and Engineering Research Council of Canada.

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Antimicrobial activity of Xenorhabdus sp. RIO (Enterobacteriaceae), symbiont of the entomopathogenic nematode, Steinernema riobrave (Rhabditida: Steinernematidae)

Abstract

Introduction

Section snippets

Growth and level of bioactivity of microflora in nematode infected larvae

Growth and level of bioactivity of microflora in nematode infected larvae

Discussion

Acknowledgements

Biol. Control

J. Invert. Pathol.

J. Invert. Pathol.

J. Invert. Pathol.

Anal. Biochem.

Parasite Today

Antibiotic activity of Xenorhabdus spp., bacteria symbiotically associated with insect pathogenic nematodes of the families Heterorhabditidae and Steinernematidae

J. Gen. Microbiol.

Biology and taxonomy of Xenorhabdus

Tripartite interactions between symbiotically associated entomopathogenic bacteria, nematodes, and their insect hosts

Symbiosis and pathogenicity of nematode–bacterium complexes

Symbiosis

A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein–dye binding

Analytic. Biochem.

The microbial flora of laboratory cultures of the Greater Wax Moth and its effect on rearing parasites

J. Invert. Path.

Steinernema riobravis n. sp. (Rhabditida: Steinernematidae) from Texas.

Fundam. Appl. Nematol.