Non-specific transient mutualism between the plant parasitic nematode, Bursaphelenchus xylophilus , and the opportunistic bacterium Serratia quinivorans BXF1, a plant-growth promoting pine endophyte with antagonistic effects

The aim of this study is to understand the biological role of Serratia quinivorans BXF1, a bacterium commonly found associated with Bursaphelenchus xylophilus, the plant parasitic nematode responsible for pine wilt disease. Therefore, we studied strain BXF1 effect in pine wilt disease. We found that strain BXF1 promoted in vitro nematode reproduction. Moreover, the presence of bacteria led to the absence of nematode chitinase gene (Bxcht-1) expression, suggesting an effect for bacterial chitinase in nematode reproduction. Nevertheless, strain BXF1 was unable to colonize the nematode interior, bind to its cuticle with high affinity or protect the nematode from xenobiotic stress. Interestingly, strain BXF1 was able to promote tomato and pine plant-growth, as well as to colonize its interior, thus, acting like a plant-growth promoting endophyte. Consequently, strain BXF1 failed to induce wilting symptoms when inoculated in pine shoot artificial incisions. This bacterium also presented strong antagonistic activities against fungi and bacteria isolated from Pinus pinaster. Our results suggest that B. xylophilus does not possess a strict symbiotic community capable of inducing pine wilt disease symptoms as previously hypothesized. We show that bacteria like BXF1, which possess plant-growth promoting and antagonistic effects, may be opportunistically associated with B. xylophilus, possibly acquired from the bacterial endophytic community of the host pine.

Novos desenvolvimentos para a deteção de leveduras e bactérias presentes em argamassas

The development of simple and rapid new approaches for analysing microbial communities colonising Cultural Heritage materials is pivotal for its safeguard. Fluorescence in situ hybridisation technique using ribosomal RNA directed probes (RNA-FISH) has demonstrated a great potential for this purpose. A protocol for analysing filamentous fungi in mortars has been already developed in previous studies. In this work this protocol has been adapted for detecting bacteria and yeasts. Good results have been obtained for the analysis of suspensions of isolates. In this way, the optimized protocol was applied in microsamples from synthetic mortar artificially inoculated with yeast and bacterial isolates. Promising results have been obtained for the ex situ analysis of yeast and bacteria thriving in mortar microsamples.

A SHORTENED RNA-FISH PROTOCOL FOR ANALYZING ARTWORKS’ MICROCOLONIZERS

Microorganisms are involved in the deterioration of Cultural Heritage. Thus, there is a need to enhance the techniques used for their detection and identification. RNA Fluorescent In Situ Hybridization (RNA-FISH) has been successfully applied for phylogenetic identification of the viable components of the microbial communities colonizing artworks both in situ and ex situ. Until recently, it was time-consuming, taking not less than 6 h for the analysis. We have developed an RNA-FISH in suspension protocol that allowed ex situ analysis of microorganisms involved in artworks’ biodeterioration in 5 h. In this work, three modified protocols, involving microwave heating, were evaluated for further shortening two of the four main critical steps in RNA-FISH: hybridization and washing. The original and modified protocols were applied in cellular suspensions of bacteria and yeast isolates. The results obtained were evaluated and compared in terms of detectability and specificity of the signals detected by epifluorescence microscopy. One of the methods tested showed good and specific FISH signals for all the microorganisms selected and did not produce signals evidencing non-specific or fixation-induced fluorescence. This 3 h protocol allows a remarkable reduction of the time usually required for performing RNA-FISH analysis in Cultural Heritage samples. Thus, a rapid alternative for analyzing yeast and bacteria cells colonizing artworks’ surfaces by RNA-FISH is presented in this work.