Host-associated Intraspecific Phenotypic Variation in the Saprobic Fungus Phlebiopsis gigantea.

Whether intraspecific phenotypic variation in saprobic fungi may be driven by the host of origin has received little attention. We addressed this issue by testing hypotheses using the model system Phlebiopsis gigantea, a wood destroying fungus associated with Picea abies and Pinus sylvestris, among others, and widely employed in practical forestry as a biological control agent. By examining approximately 60 sympatric P. gigantea isolates from both P. abies and P. sylvestris, we showed that the former grew in vitro significantly (P < 0.05) slower than the latter (average 5.56 mm/day vs. 6.84) while producing 1.8-fold significantly higher number of mitospores. An overall significant trade-off between these two phenotypic traits was detected, in particular for isolates originating from P. abies. Comparative inoculation experiments of a subsample of isolates and the assessment of mycelial growth in logs of both hosts allowed to reject the hypothesis that isolates are equally fit in terms of growth rate in wood of both hosts regardless of the host of origin. Tree models revealed that the growth rate of isolates was associated not only with the wood species in which the isolates were inoculated (P < 0.001), P. sylvestris being more rapidly colonized than P. abies, but also with the host of origin of isolates (P < 0.001). Results showed that P. gigantea isolates originating from different hosts differ phenotypically in terms of some key phenotypic traits demonstrating that a host-driven intraspecific phenotypic variation may occur in saprobic fungi.

Pyoluteorin Produced by the Biocontrol Agent Pseudomonas protegens Is Involved in the Inhibition of Heterobasidion Species Present in Europe.

Pseudomonas protegens (strain DSMZ 13134) is a biocontrol agent with promising antagonistic activity hinging on antibiosis against the fungal forest pathogens Heterobasidion spp. Here, by using High-Performance Liquid Chromatography coupled to Mass Spectrometry (HPLC-MS), we assessed whether monocultures of P. protegens (strain DSMZ 13134) produce the three major determinants of biocontrol activity known for the genus Pseudomonas: 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin (PLT), and pyrrolnitrin (PRN). At the tested culture conditions, we observed the production of PLT at concentrations ranging from 0.01 to 10.21 mg/L and 2,4-DAPG at a concentration not exceeding 0.5 mg/L. Variations of culture conditions involving culture medium, incubation temperature, and incubation period had no consistent influence on PLT production by the bacterium. Assays using culture medium amended with PLT at the same concentration of that present in cell-free filtrate of the bacterium, i.e., 3.77 mg/L, previously documented as effective against Heterobasidion spp., showed a remarkable activity of PLT against genotypes of all the four Heterobasidion species present in Europe, including the non-native invasive H. irregulare. However, such antifungal activity decreased over time, and this may be a constraint for using this molecule as a pesticide against Heterobasidion spp. When the bacterium was co-cultured in liquid medium with genotypes of the different Heterobasidion species, an increased production of PLT was observed at 4 °C, suggesting the bacterium may perform better as a PLT producer in field applications under similar environmental conditions, i.e., at low temperatures. Our results demonstrated the role of PLT in the inhibition of Heterobasidion spp., although all lines of evidence suggest that antibiosis does not rely on a single constitutively produced metabolite, but rather on a plethora of secondary metabolites. Findings presented in this study will help to optimize treatments based on Pseudomonas protegens (strain DSMZ 13134) against Heterobasidion spp.

Comparative Efficacy of State-of-the-Art and New Biological Stump Treatments in Forests Infested by the Native and the Alien Invasive Heterobasidion Species Present in Europe.

The Heterobasidion annosum species complex includes major fungal pathogens of conifers worldwide. State-of-the-art preventative stump treatments with urea or with commercial formulations of the fungal biological control agent Phlebiopsis gigantea (i.e., Rotstop®) may become no longer available or are not approved for use in many areas of Europe infested by the three native Heterobasidion species and by the North American invasive H. irregulare, making the development of new treatments timely. The efficacy of Proradix® (based on Pseudomonas protegens strain DSMZ 13134), the cell-free filtrate (CFF) of the same bacterium, a strain of P. gigantea (MUT 6212) collected in the invasion area of H. irregulare in Italy, Rotstop®, and urea was comparatively investigated on a total of 542 stumps of Abies alba, Picea abies, Pinus pinea, and P. sylvestris in forest stands infested by the host-associated Heterobasidion species. Additionally, 139 logs of P. pinea were also treated. Results support the good performances of Rotstop®, and especially of urea against the native Heterobasidion species on stumps of their preferential hosts and, for the first time, towards the invasive North American H. irregulare on stumps of P. pinea. In some experiments, the effectiveness of Proradix® and of the strain of P. gigantea was weak, whereas the CFF of P. protegens strain DSMZ 13134 performed as a valid alternative to urea and Rotstop®. The mechanism of action of this treatment hinges on antibiosis; therefore, further improvements could be possible by identifying the active molecules and/or by optimizing their production. Generally, the performance of the tested treatments is not correlated with the stump size.