Selecting microbial strains from pine tree resin: biotechnological applications from a terpene world

Abstract

Resin is a chemical and physical defensive barrier secreted by many plants, especially coniferous trees, with insecticidal and antimicrobial properties. The degradation of terpenes, the main components accounting for the toxicity of resin, is highly relevant for a vast range of biotechnological processes, including bioremediation. In the present work, we used a resin-based selective medium in order to study the resin-tolerant microbial communities associated with the galls formed by the moth Retinia resinella; as well as resin from Pinus sylvestris forests, one of the largest ecosystems on Earth and a yet-unexplored source of terpene-degrading microorganisms. The taxonomic and functional diversity of the cultivated, resin-tolerant fraction of the whole microbiota were unveiled by high-throughput sequencing, which resulted in the detection of more than 40 bacterial genera among the terpene-degrading microorganisms, and a range of genes involved in the degradation of different terpene families. We further characterized through culture-based approaches and transcriptome sequencing selected microbial strains, including Pseudomonas sp., the most abundant species in both environmental resin and R. resinella resin-rich galls, and three fungal species, and experimentally confirmed their ability to degrade resin and also other terpene-based compounds and, thus, their potential use in biotechnological applications involving terpene catabolism.

Figure 1. Relative abundance of bacterial genera in the communities cultivated from environmental resin and galls.

Horizontal bars indicate the presence of a particular genus in environmental resin (red) or galls (blue).

Figure 2. Resin degradation by Pseudomonas abietaniphila strain PS and the fungal strains isolated from environmental resin.

A) Bacterial and total dry weight of RM cultures of strain PS and evolution of the estimated amount of resin in the medium. B) Variation in resin content in RM cultures of different fungi isolated from resin, estimated from optical density of the medium (OD₆₀₀), as described in Methods S1. C) Resin colloid removal by fungal isolates grown for 7 days compared to a non inoculated RM control (from left to right, control, F9, F8, and F1 cultures).

Figure 3. SEM images of latex and rubber degradation performed by fungi.

Latex (A and D) and rubber (B, C, E, and F) were used as the sole carbon source in the selective media. A, B and C show non-inoculated control media, whereas 15-day-old cultures of isolates F9 and F1 are shown in subfigures D and E–F, respectively. A, C, D and F scale bars = 50 µm; B, E scale bars = 500 µm.

Figure 4. SEM images of latex degradation performed by Pseudomonas abietaniphila strain PS.

Particles from (A) a non-inoculated latex-containing medium; and a 15-days (B) and a one-month culture (C and D) of strain PS in the same medium are shown. Arrows indicate the cell-shaped niches formed on the latex surface. A and B scale bars = 10 µm, C and D scale bars = 2 µm.

Figure 5. Colony size of three fungal isolates during confrontation assays with strain PS.

A and B, F1; C and D, F8; and E and F, F9. The experiments were carried out on RM (A, C, and E) and LB (B, D, and F) medium. Colony size measured in mm. Pictures of particular experiments were taken after 3 and 5 days in the case of LB and RM media, respectively.

Figure 6. Relative abundance of selected GO terms in the transcriptome of fungal isolate F1.

GO terms corresponding to molecular functions (A) and cellular processes (B) are shown for F1 confronted with PS (reference, purple bars), and for the subset of genes not detected in the transcriptome of F1 grown isolated (test, orange bars). Asterisks indicate statistically significant differences for p-value<0.05 (**) and p-value<0.1 (*).