Cellulose-degrading bacteria associated with the invasive woodwasp Sirex noctilio

Abstract

Sirex noctilio is an invasive wood-feeding wasp that threatens the world's commercial and natural pine forests. Successful tree colonization by this insect is contingent on the decline of host defenses and the ability to utilize the woody substrate as a source of energy. We explored its potential association with bacterial symbionts that may assist in nutrient acquisition via plant biomass deconstruction using growth assays, culture-dependent and -independent analysis of bacterial frequency of association and whole-genome analysis. We identified Streptomyces and γ-Proteobacteria that were each associated with 94% and 88% of wasps, respectively. Streptomyces isolates grew on all three cellulose substrates tested and across a range of pH 5.6 to 9. On the basis of whole-genome sequencing, three Streptomyces isolates have some of the highest proportions of genes predicted to encode for carbohydrate-active enzymes (CAZyme) of sequenced Actinobacteria. γ-Proteobacteria isolates grew on a cellulose derivative and a structurally diverse substrate, ammonia fiber explosion-treated corn stover, but not on microcrystalline cellulose. Analysis of the genome of a Pantoea isolate detected genes putatively encoding for CAZymes, the majority predicted to be active on hemicellulose and more simple sugars. We propose that a consortium of microorganisms, including the described bacteria and the fungal symbiont Amylostereum areolatum, has complementary functions for degrading woody substrates and that such degradation may assist in nutrient acquisition by S. noctilio, thus contributing to its ability to be established in forested habitats worldwide.

Figure 1

Neighbor-joining phylogenetic tree and frequency of association of cellulose-degrading bacteria associated with S. noctilio. The out-group Burkholderia multivorans and closely related type strains (denoted ‘T') are included, as determined by the Ribosomal Database Project (http://rdp.cme.msu.edu). Frequency of association of CMC-degrading taxa detected by DGGE and pooled class-level phylogenetic determination is represented by the pie charts (percentage of wasps associated with corresponding taxa in black; percentage of wasps with taxa not detected in white). Frequency of association was determined by DGGE, except when culturing was used (labeled ‘C'). Inset: number of cellulose-degrading taxa detected from individual wasps. ¹⁻⁵Enterobacteriales isolates used to estimate frequency of association using DGGE. Common numerals were indistinguishable. †Isolates from which draft genomes were analyzed.

Figure 2

Summary of bacterial performance on cellulolytic substrates, including degradation of CMC, growth on corn stover and cellulolytic enzyme production after growing on microcrystalline cellulose. Growth and enzymatic activity are categorically ranked, with no label corresponding to no activity, (+) detectable, (++) moderate and (+++) strong activity. Label in parentheses represents presence (+) or absence (−) of degradation of CMC from culture growing in the absence of microcrystalline cellulose. ¹Fungus.