Comparative Study of Secreted Proteins, Enzymatic Activities of Wood Degradation and Stilbene Metabolization in Grapevine Botryosphaeria Dieback Fungi

Botryosphaeriaceae fungi are plant pathogens associated with Botryosphaeria dieback. To better understand the virulence factors of these fungi, we investigated the diversity of secreted proteins and extracellular enzyme activities involved in wood degradation and stilbene metabolization in Neofusicoccum parvum and Diplodia seriata, which are two major fungi associated with grapevine B. dieback. Regarding the analysis of proteins secreted by the two fungi, our study revealed that N. parvum, known to be more aggressive than D. seriata, was characterized by a higher quantity and diversity of secreted proteins, especially hydrolases and oxidoreductases that are likely involved in cell wall and lignin degradation. In addition, when fungi were grown with wood powder, the extracellular laccase and Mn peroxidase enzyme activities were significantly higher in D. seriata compared to N.parvum. Importantly, our work also showed that secreted Botryosphaeriaceae proteins produced after grapevine wood addition are able to rapidly metabolize the grapevine stilbenes. Overall, a higher diversity of resveratrol and piceatannol metabolization products was found with enzymes of N. parvum compared to D. seriata. This study emphasizes the diversity of secreted virulence factors found in B. dieback fungi and suggests that some resveratrol oligomers produced in grapevine wood after pathogen attack could be formed via pathogenic fungal oxidases.

Mycoremediation of Soils Polluted with Trichloroethylene: First Evidence of Pleurotus Genus Effectiveness

Trichloroethylene (TCE) is a proven carcinogenic chlorinated organic compound widely used as a solvent in industrial cleaning solutions; it is easily found in the soil, air, and water and is a hazardous environmental pollutant. Most studies have attempted to remove TCE from air and water using different anaerobic bacteria species. In addition, a few have used white-rot fungi, although there are hardly any in soil. The objective of the present work is to assess TCE removal efficiency using two species of the genus Pleurotus that have not been tested before: Pleurotus ostreatus and Pleurotus eryngii, growing on a sandy loam soil. These fungi presented different intra- and extracellular enzymatic systems (chytochrome P450 (CYP450), laccase, Mn peroxidase (MnP)) capable of aerobically degrading TCE to less harmful compounds. The potential toxicity of TCE to P. ostreatus and P. eryngii was firstly tested in a TCE-spiked liquid broth (70 mg L−1 and 140 mg L−1) for 14 days. Then, both fungi were assessed for their ability to degrade the pollutant in sandy loam soil spiked with 140 mg kg−1 of TCE. P. ostreatus and P. eryngii improved the natural dissipation of TCE from soil by 44%. Extracellular enzymes were poorly expressed, but mainly in the presence of the contaminant, in accordance with the hypothesis of the involvement of CYP450.

Ligninolytic Enzyme Production and Decolorization Capacity of Synthetic Dyes by Saprotrophic White Rot, Brown Rot, and Litter Decomposing Basidiomycetes

An extensive screening of saprotrophic Basidiomycetes causing white rot (WR), brown rot (BR), or litter decomposition (LD) for the production of laccase and Mn-peroxidase (MnP) and decolorization of the synthetic dyes Orange G and Remazol Brilliant Blue R (RBBR) was performed. The study considered in total 150 strains belonging to 77 species. The aim of this work was to compare the decolorization and ligninolytic capacity among different ecophysiological and taxonomic groups of Basidiomycetes. WR strains decolorized both dyes most efficiently; high decolorization capacity was also found in some LD fungi. The enzyme production was recorded in all three ecophysiology groups, but to a different extent. All WR and LD fungi produced laccase, and the majority of them also produced MnP. The strains belonging to BR lacked decolorization capabilities. None of them produced MnP and the production of laccase was either very low or absent. The most efficient decolorization of both dyes and the highest laccase production was found among the members of the orders Polyporales and Agaricales. The strains with high MnP activity occurred across almost all fungal orders (Polyporales, Agaricales, Hymenochaetales, and Russulales). Synthetic dye decolorization by fungal strains was clearly related to their production of ligninolytic enzymes and both properties were determined by the interaction of their ecophysiology and taxonomy, with a more relevant role of ecophysiology. Our screening revealed 12 strains with high decolorization capacity (9 WR and 3 LD), which could be promising for further biotechnological utilization.

Biodegradation of nitroaromatic compounds in Red Water by white rot fungi Pleurotus ostreatus and floridae

Pleurotus fungi are basidiomycetes that stand out in the degradation of recalcitrant organic compounds such as lignin derivatives and phenolic compounds. The aim of this study was to make a comparative evaluation of the capacity of the Pleurotus ostreatus POS 560 and Pleurotus floridae PSP1 fungi in the degradation of 2,4 and 2,6-dinitrotoluene (DNTs) in effluent from an explosive factory. The characterization of the effluent indicated 318 mg L-1 of DNTs, 246 mg L-1 of COD and toxicity factor for Daphnia magna corresponding to 8. The conduct of a multivariate study estimated the influence of the variables pH (5.0 and 6.0), co-substrate concentration (10 and 20 g L-1 of glucose) and species of the fungus Pleurotus (ostreatus and floridae) on the degradation of DNTs, indicating that the variables Fungus and [Glucose] were significant (p <0.05) presenting effects in the order of + 4.45 ± 0.26 and -1.14 ± 0.26, respectively. The reproduction of the best efficiency conditions (P. floridae; pH 6.0 and 10 g L-1 of glucose) in agitated flasks (100 rpm, 26oC) was able to carry out, within 14 days of treatment, the removal of organic matter and toxicity factor in levels on the order of 55 and 50%, respectively, in addition to the complete degradation of DNTs which occurred in the first 120 hours of treatment. In this period, the maximum activity of the peroxidase and Mn-peroxidase enzymes was also characterized, suggesting high potential of the bioprocess under study for remediation of effluents contaminated with nitroaromatic compounds.

An Italian Research Culture Collection of Wood Decay Fungi

One of the main aims of the University of Pavia mycology laboratory was to collect wood decay fungal (WDF) strains in order to deepen taxonomic studies, species distribution, officinal properties or to investigate potential applications such as biocomposite material production based on fungi. The Italian Alps, Apennines and wood plains were investigated to collect Basidiomycota basidiomata from living or dead trees. The purpose of this study was to investigate the wood decay strains of the Mediterranean area, selecting sampling sites in North and Central Italy, including forests near the Ligurian and Adriatic seas, or near the Lombardy lakes. The isolation of mycelia in pure culture was performed according to the current methodology and the identity of the strains was confirmed by molecular analyses. The strains are maintained in the Research Culture Collection MicUNIPV of Pavia University (Italy). Among the 500 WDF strains in the collection, the most interesting isolates from the Mediterranean area are: Dichomitus squalens (basidioma collected from Pinus pinea), Hericium erinaceus (medicinal mushroom), Inocutis tamaricis (white-rot agent on Tamarix trees), Perenniporia meridionalis (wood degrader through Mn peroxidase) and P. ochroleuca. In addition, strains of species related to the Mediterranean climate (e.g., Fomitiporia mediterranea and Cellulariella warnieri) were obtained from sites with a continental-temperate climate.

Degradation of Aflatoxin B1 by a Sustainable Enzymatic Extract from Spent Mushroom Substrate of Pleurotus eryngii

Ligninolytic enzymes from white-rot fungi, such as laccase (Lac) and Mn-peroxidase (MnP), are able to degrade aflatoxin B1 (AFB1), the most harmful among the known mycotoxins. The high cost of purification of these enzymes has limited their implementation into practical technologies. Every year, tons of spent mushroom substrate (SMS) are produced as a by-product of edible mushroom cultivation, such as Pleurotus spp., and disposed at a cost for farmers. SMS may still bea source of ligninolytic enzymes useful for AFB1 degradation. The in vitro AFB1-degradative activity of an SMS crude extract (SMSE) was investigated. Results show that: (1) in SMSE, high Lac activity (4 U g−1 dry matter) and low MnP activity (0.4 U g−1 dry matter) were present; (2) after 1 d of incubation at 25 °C, the SMSE was able to degrade more than 50% of AFB1, whereas after 3 and 7 d of incubation, the percentage of degradation reached the values of 75% and 90%, respectively; (3) with increasing pH values, the degradation percentage increased, reaching 90% after 3 d at pH 8. Based on these results, SMS proved to be a suitable source of AFB1 degrading enzymes and the use of SMSE to detoxify AFB1 contaminated commodities appears conceivable.

Microbial Degradation of Azo Dyes

Azo dyes are considered as xenobiotic compounds, which are often recalcitrant to be biodegraded. Many dyes are relevant for staining in industries, especially textile and food related. Among other environmental problems, one obvious issue is the coloring effect on effluents from industrial sites and thus the release of xenobiotics into nature. Microorganisms (algae, fungi, yeast, and bacteria) have been found to decolorize a number of azo dyes. Decolorization of azo dyes by microbial cultures is summarized and this is mostly linked to initial activation or even cleavage of the azo bond (e.g., by azoreductases). However, it does not necessarily mean that the compound is degraded to non-toxic products. Various mechanisms of microbial decolorization processes were discovered, including adsorption, enzymatic degradation, or a combination of both. Oxidases and reductases were found to be involved, which contain azoreductase, lignin peroxidase, Mn peroxidase, laccase, tyrosinase, and so on. A focus is on the azoreductases including classification, activity, and applicability.

Microbial Degradation of Azo Dyes

Azo dyes are considered as xenobiotic compounds, which are often recalcitrant to be biodegraded. Many dyes are relevant for staining in industries, especially textile and food related. Among other environmental problems, one obvious issue is the coloring effect on effluents from industrial sites and thus the release of xenobiotics into nature. Microorganisms (algae, fungi, yeast, and bacteria) have been found to decolorize a number of azo dyes. Decolorization of azo dyes by microbial cultures is summarized and this is mostly linked to initial activation or even cleavage of the azo bond (e.g., by azoreductases). However, it does not necessarily mean that the compound is degraded to non-toxic products. Various mechanisms of microbial decolorization processes were discovered, including adsorption, enzymatic degradation, or a combination of both. Oxidases and reductases were found to be involved, which contain azoreductase, lignin peroxidase, Mn peroxidase, laccase, tyrosinase, and so on. A focus is on the azoreductases including classification, activity, and applicability.

Optimasi pertumbuhan dan aktivitas enzim ligninolitik Omphalina sp. dan Pleurotus ostreatus pada fermentasi padat Optimization of growth and ligninolytic enzymes activity of Omphalina sp. and Pleurotus ostreatus using solid state fermentation

SummarySolid wastes of bagasse and empty fruitbunch (EFB) respectively from sugarcane andpalm oil mill in Indonesia are abundant. Nowdays, up to now these solid wastes have not yetbeen optimally utilized so that the added valueis still very low and even cause an environ-mental problem. Research on bioconversion ofbagasse and EFB with two culture of white-rotfungi (WRF) i.e., Omphalina sp. and Pleurotusostreatus to produce ligninolytic enzymes wasconducted to provide added value to thislignocellulosic waste. Production of extracellular enzymes from WRF was not onlydetermined by the type of isolate but also theculture condition. This research was aimed todetermine the optimum culture condition ofsolid state fermentation in producing lignino-lytic enzymes at laboratory scale. In thisresearch, WRF was examined for ligninolyticproducing enzymes (laccase, lignin peroxidase/ LiP and Mn-peroxidase / MnP), using mediaconsisting of bagasse and EFB separately asmain substrate with supplementation of ricebran, Cu 2+ with or without rice bran. Theobservation was based on their growth andligninolytic enzyme activities. Characteristicsof optimum pH of LiP, MnP and laccaseactivity were also determined. The resultsshowed that addition of supplement was notable to increase the Cu 2+ growth of myceliaespecially in the first and second months but inthe third month the addition of supplementenhanced the mycelia growth. The growth ofmycelia on the addition of Cu 2+ with or withoutrice bran significantly lower compared to thecontrols both of Omphalina sp. and P. ostreatusin bagasse and EFB. The optimum pH oflaccase, MnP, and LiP activities was five bothfor Omphalina sp. and P. ostreatus at EFBand bagasse. Omphalina sp. was better thanP. ostreatus in producing laccase on bagasseand EFB without any supplementations. Thehighest laccase activity showed by P. ostreatuswith bagasse and EFB media treated with Cuand Cu + rice bran. Supplementation withCu 2+ was more effective in increasing laccaseactivity than rice bran. Activities of Li-P onbagasse and EFB for the two WRF cultureswere significantly influenced by supple-mentation of both of rice bran and Cu 2+ . Li-Pactivity on EFB was slightly higher than thaton bagasse. Mn-P activity was not influencedby rice bran, Cu 2+ or the combination of both.However, these enzymes activities on EFBwere higher compared to bagasse especiallyfor P. ostreatus. Suplementation of Cu wasenhance the activity of laccase and LiP both ofP. ostreatus and Omphalina sp in baggasse andEFB though inhibited the growth of those fungiespecially in the initial growth.RingkasanLimbah padat bagas tebu dan tandankosong kelapa sawit (TKKS) masing-masingdari proses pengolahan gula tebu dan minyaksawit di Indonesia jumlahnya melimpah dansampai saat ini belum mendapat penangananyang efektif sehingga nilai tambahnya masihsangat rendah dan bahkan mengganggulingkungan. Penelitian biokonversi limbahpadat bagas tebu dan TKKS menggunakan duaisolat fungi pelapuk putih (FPP) yaituOmphalina sp. dan Pleurotus ostreatus untukproduksi enzim ligninolitik dilakukan untukmeningkatkan nilai tambah limbah ligno-selulosa tersebut. Penelitian ini, mengujiaktivitas enzim ekstraseluler dari FPP antaralain lakase, lignin peroksidase (LiP), dan Mn-peroksidase (MnP) dari dua spesies FPP yaituOmphalina sp. dan P. ostreatus. Penelitianbertujuan menetapkan kondisi optimum mediafermentasi untuk produksi enzim ligninolitikdari bagas tebu dan TKKS sebagai substrat dankarakterisasi pH optimum enzim ligninolitikdari dua FPP yaitu Omphalina sp. danP. ostreatus. Pengamatan dilakukan berdasar-kan laju pertumbuhan dan aktivitas enzimligninolitik. Enzim lakase, MnP, dan LiPdiekstraksi dan dikarakterisasi pH optimumaktivitasnya. Hasil penelitian menunjukkanbahwa pemberian suplemen menghambatpertumbuhan miselia pada satu dan dua bulanpertama inkubasi, namun laju pertumbuhanmiselium khususnya pada perlakuan pemberianCu 2+ dan Cu 2+ + dedak meningkat tajam padabulan ketiga setelah inkubasi. Pertumbuhanmiselium Omphalina sp dan P. ostreatus padamedium yang ditambah Cu 2+ dan Cu 2+ +dedaklebih rendah dibandingkan dengan kontrol.Pada inkubasi tiga bulan, aktivitas optimumlakase, MnP dan LiP diperoleh pada pH 5, baikuntuk Omphalina sp. maupun P. ostreatusyang diekstrak dari bahan lignoselulosa bagastebu dan TKKS. Aktivitas lakase dariOmphalina sp. lebih tinggi daripadaP. ostreatus pada substrat TKKS dan bagastebu tanpa suplementasi. Pemberian suplemenberupa Cu 2+ dan dedak atau kombinasinyameningkatkan aktivitas lakase baik pada bagastebu maupun pada TKKS. Aktivitas lakasetertinggi ditunjukkan oleh isolat P. ostreatuspada medium bagas tebu dan TKKS padaperlakuan pemberian Cu 2+ dengan atau tanpadedak. Aktivitas lakase nampaknya lebihdipengaruhi oleh penambahan Cu 2+ dibanding-kan dengan pemberian dedak. Aktivitas LiPbaik pada bagas tebu maupun TKKS untukkedua FPP yang diuji pada perlakuanpenambahan dedak dan Cu nyata lebih tinggidibandingkan dengan aktivitas LiP yangdiekstrak dari medium tanpa penambahansuplemen. Aktivitas LiP pada TKKS lebihtinggi dibandingkan dengan pada bagas tebukhususnya untuk P. ostreatus. Sedangkanaktivitas MnP tidak dipengaruhi penambahandedak dan Cu 2+ demikian pula kombinasikeduanya. Aktivitas MnP yang diekstrak dariTKKS lebih tinggi dibandingkan denganaktivitas MnP yang diekstrak dari bagas tebukhususnya untuk P. ostreatus. PenambahanCu 2+ meningkatkan aktivitas lakase dan LiPP. ostreatus dan Omphalina sp yang ditum-buhkan pada bagas dan TKKS walaupun ionlogam ini menghambat pertumbuhan keduaJPP ini khususnya pada awal pertumbuhan.