No abstract

A major theme in the history of life is the formation of beneficial associations between symbiotic microbes and plants and animals (1). The pervasiveness of these associations in every ecosystem illustrates how the acquisition of a microbe's physiological capacity confers substantial fitness benefits to hosts (1). However, dependence on mutualistic microbes becomes a liability if antagonistic microbes attack or out-compete beneficial ones (2). Therefore, mechanisms to preserve beneficial microbes must be a widespread, although poorly understood, component of host-microbe mutualisms. Here, we show that a beetle uses a bacterium to protect its fungal food source from a competitor fungus.Southern pine beetles (SPB, Dendroctonus frontalis) engage in a beneficial symbiosis with the fungus Entomocorticium sp. A (EsA), which provides nourishment for their developing larvae. Adult SPBs carry EsA in a specialized storage compartment called a mycangium (Fig. 1A); excavate ovipositional galleries within the inner bark and phloem of host pine trees; and inoculate these galleries with EsA (3,4). The success of the SPB-EsA mutualism is challenged by an antagonistic fungus, Ophiostoma minus (Om), which can out-compete EsA and thereby disrupt SPB larval development (3,4). Our results indicate that successful maintenance of the SPB-EsA mutualism is likely mediated by an actinomycetous bacterium that produces antibiotics which selectively inhibit Om.The presence of previously unknown actinomycetes within the SPB-EsA mutualisms was established by scanning electron microscopy (SEM) and enrichment culture isolations. SEM revealed unexpected and profuse growth of actinomycetes within the galleries of SPBs, as well as inside the mycangia (Fig. 1B, Fig S1A). Isolations from 110 beetle individuals yielded 846 colony forming units (CFUs) of actinomycetes, including at least one CFU from each of 92 individuals. Out of 164 actinomycete CFUs selected to be transferred to pure culture, 99 isolates had a red morphotype while 65 isolates had a white morphotype. DNA sequence analyses confirmed the visual morphotype distinction, and within each of the two morphotypes there was complete 16S rDNA sequence identity. The two morphotypes form a monophyletic clade closely related toStreptomyces thermosacchari. We also successfully isolated the same red morphotype from 5 of 10 mycangia sampled.