Proteomic Analysis of Fusarium solani Isolated from the Asian Longhorned Beetle, Anoplophora glabripennis

Wood is a highly intractable food source, yet many insects successfully colonize and thrive in this challenging niche. Overcoming the lignin barrier of wood is a key challenge in nutrient acquisition, but full depolymerization of intact lignin polymers has only been conclusively demonstrated in fungi and is not known to occur by enzymes produced by insects or bacteria. Previous research validated that lignocellulose and hemicellulose degradation occur within the gut of the wood boring insect, Anoplophora glabripennis (Asian longhorned beetle), and that a fungal species, Fusarium solani (ATCC MYA 4552), is consistently associated with the larval stage. While the nature of this relationship is unresolved, we sought to assess this fungal isolate's ability to degrade lignocellulose and cell wall polysaccharides and to extract nutrients from woody tissue. This gut-derived fungal isolate was inoculated onto a wood-based substrate and shotgun proteomics using Multidimensional Protein Identification Technology (MudPIT) was employed to identify 400 expressed proteins. Through this approach, we detected proteins responsible for plant cell wall polysaccharide degradation, including proteins belonging to 28 glycosyl hydrolase families and several cutinases, esterases, lipases, pectate lyases, and polysaccharide deacetylases. Proteinases with broad substrate specificities and ureases were observed, indicating that this isolate has the capability to digest plant cell wall proteins and recycle nitrogenous waste under periods of nutrient limitation. Additionally, several laccases, peroxidases, and enzymes involved in extracellular hydrogen peroxide production previously implicated in lignin depolymerization were detected. In vitro biochemical assays were conducted to corroborate MudPIT results and confirmed that cellulases, glycosyl hydrolases, xylanases, laccases, and Mn- independent peroxidases were active in culture; however, lignin- and Mn- dependent peroxidase activities were not detected While little is known about the role of filamentous fungi and their associations with insects, these findings suggest that this isolate has the endogenous potential to degrade lignocellulose and extract nutrients from woody tissue

PAH biodegradation by telluric saprotrophic fungi isolated from aged PAH-contaminated soils in mineral medium and historically contaminated soil microcosms.

Purpose: Remediation of contaminated soils is of high relevance considering losses of this limited resource in most countries through erosion or through destruction for societal purposes. Most physicochemical remediation techniques lead to soil destruction avoiding reuse of the sites and loss of soil functions including ecological services like water retention/filtration, plant needs, carbon sequestration, or atmosphere restoration. This study aims to find out efficient telluric PAH-degrading fungi to both remediate soils and preserve their functioning. Materials and methods: Fifty telluric saprotrophic fungi were thus isolated from different aged PAH-contaminated soils sampled from four brownfields in the North of France. Thirty of these isolates were screened in a mineral medium for their ability to degrade benzo[a]pyrene (BaP) used as a model of HMW PAH. After 9 days of incubation, the remaining BaP was quantified through HPLC analysis. Then, a set of microcosms was performed with an aged PAH-contaminated non-sterile soil encompassing different approaches with bioaugmentation using mycelia of three strains pre-established on expanded clay particles and, for one of these strains, biostimulation strategies including nitrogen or nitrogen/phosphorous supplementations or aeration by stirring. After an incubation time of 30 days, remaining PAH were quantified through GC-MS and evolution of fungal populations evaluated through qPCR. Results and discussion: Isolated Penicillium canescens, Cladosporium cladosporioides, Fusarium solani, and Talaromyces helicus degraded more than 30% of the initial 500 μg of BaP after 9 days of incubation. The three latest strains were thus inoculated in aged PAH-contaminated soil microcosms. After 30 days, PAH quantitative analyses showed that the highest degradation was obtained by bioaugmentation with T. helicus (26% of the initial total PAH content: 321.7 mg kg −1 ), which seems high considering an aged industrial contamination. Biostimulation approaches coupled to the inoculation of this strain did not improve the degradation. DNA quantification of the inoculated species confirmed their enrichment in the soil revealing the interest of the used inoculation strategy. We discuss bioaugmentation and biostimulation approaches in the case of the considered pollutants and soil. Conclusions: A strain identified as T. helicus appears interesting for its HMW PAH degradation capacities both in mineral medium with pure BaP and in industrial non-sterile soil microcosms contaminated with a hydrocarbon complex mixture. This study also confirms the efficiency of a well-studied BaP-degrading strain of F. solani. These results indicate the potentialities of the used bioaugmentation approach and underline the necessity of scale-up strategies to apply this kind of technique on site

Energy-dependent uptake of benzo[a]pyrene and its cytoskeleton-dependent intracellular transport by the telluric fungus <em>Fusarium solani.</em>

In screening indigenous soil filamentous fungi for polycyclic aromatic hydrocarbons (PAHs) degradation, an isolate of the Fusarium solani was found to incorporate benzo[a]pyrene (BaP) into fungal hyphae before degradation and mineralization. The mechanisms involved in BaP uptake and intracellular transport remain unresolved. To address this, the incorporation of two PAHs, BaP, and phenanthrene (PHE) were studied in this fungus. The fungus incorporated more BaP into cells than PHE, despite the 400-fold higher aqueous solubility of PHE compared with BaP, indicating that PAH incorporation is not based on a simple diffusion mechanism. To identify the mechanism of BaP incorporation and transport, microscopic studies were undertaken with the fluorescence probes Congo Red, BODIPY&reg;493/503, and FM&reg;4-64, targeting different cell compartments respectively fungal cell walls, lipids, and endocytosis. The metabolic inhibitor sodium azide at 100&nbsp;mM totally blocked BaP incorporation into fungal cells indicating an energy-requirement for PAH uptake into the mycelium. Cytochalasins also inhibited BaP uptake by the fungus and probably its intracellular transport into fungal hyphae. The perfect co-localization of BaP and BODIPY reveals that lipid bodies constitute the intracellular storage sites of BaP in F. solani. Our results demonstrate an energy-dependent uptake of BaP and its cytoskeleton-dependent intracellular transport by F. solani

Ability of filamentous fungi to degrade four emergent water priority pollutants

This study was conducted to investigate biodegradation of four emergent water priority pollutants, di(2-ethylhexyl)phthalate (DEHP), fluoranthene, aminomethylphosphonic acid (AMPA), and estrone (EST), by filamentous fungi (Fusarium oxysporum, Geotrichum galactomyces, Trichoderma harzianum, and Fusarium solani). These pollutants are commonly found at high occurrence in French wastewater treatment plants. In acute toxicity tests, a weak sensitivity of fungal growth to the pollutants was observed with F. oxysporum showing the greatest growth inhibition (19.3%) in the presence of DEHP after four days of incubation. In addition, degradation experiments were conducted in mineral medium for each pollutant incubated with each filamentous fungus for 10 d. With the exception of EST, which was not degraded by any fungal isolate tested, the fungi degraded these emergent water priority pollutants, with F. solani and T. harzianum degrading 100% of DEHP and 69% of AMPA, respectively