Enzyme activity profiles of four different decay-strategy fungi cultivated on birch wood and barley straw

Fungal wood-decayers play an important role in the recycling of biomass and circulation of nutrients in nature. Fungi are capable to convert cellulose, hemicellulose, pectin and lignin, by the action of carbohydrate-acting enzymes (CAZymes) secreted and also by non-enzymatic reactions, depending on the ecology and decay strategy of the fungus. In the present study, four Basidiomycota fungi with different decay strategies were studied to compare their enzyme activity profiles. The white rot fungus Phlebia radiata, brown rot fungus Fomitopsis pinicola and “grey rot” fungus Schizophyllum commune were cultivated on birch (Betula pendula) wood pieces for twelve weeks, whereas the litter-decomposing fungus Coprinopsis cinerea was cultivated on cut barley (Hordeum vulgare) straw for six weeks. All fungi were also cultivated on liquid medium (malt extract 2%) for four weeks. Laccase, manganese peroxidase (MnP), β-glucosidase, xylanase and endoglucanase activities were followed weekly by measuring the absorbances on 96-well plates. The pH and the production of organic acids at each time point were also followed. The results showed that P. radiata produced high laccase and MnP activities. Additionally, high amounts of succinic acid in the aqueous phase of the solid-state cultivations were detected. F. pinicola had a notable production of xylanase activity on birch, in contrast to the moderate β-glucosidase and endoglucanase activities observed on the same substrate. S. commune was a strong producer of β-glucosidase, but especially xylanase activity on solid substrate. Lastly, the litter-decomposer C. cinerea seemed to have a poor performance in enzymatically decomposing the lignin portion from barley straw, whereas a preference on hemicellulose decomposition was observed. Overall, the results indicated the ability of the studied fungi in decomposing the components of the plant cell wall to different extents according to their decay strategy, which is key in the understanding of the ecophysiology of wood-decay and litter-decomposing fungi, and the potential of fungal enzymes for biotechnological applications

Evaluación de la capacidad antibiótica y antifúngica de extractos orgánicos y peptídicos de hongos endófitos provenientes de la colección de endófitos, Quito-Católica (CEQCA)

Se evaluó la capacidad inhibitoria de extractos orgánicos y peptídicos de doce hongos endófitos de la Colección de Endófitos Quito-Católica (CEQCA) contra las bacterias S. aureus ATCC® 25923TM, P. aeruginosa ATCC® 27853TM, S. marcescens ATCC® 13880TM, E. coli ATCC® 25922TM y S. enterica ATCC® 13076TM, y contra el hongo Pythium ultimum. Los hongos endófitos fueron elegidos de acuerdo a la información registrada en la base de datos de la CEQCA sobre su bioactividad y al considerarse algunos de ellos como especies nuevas. Se trabajó con 34 extractos orgánicos, de los cuales 16 fueron obtenidos por primera vez, y 18 se encontraban en existencia en la Colección CEQCA. Adicionalmente se estandarizó la metodología para obtener extractos peptídicos, de estos se obtuvieron y evaluaron 14 extractos. Los extractos se ensayaron con el método por difusión directa en el agar como primer paso para evaluar su bioactividad; de ellos, seis inhibieron totalmente el crecimiento de S. aureus ATCC® 25923TM (O1113 DCM.1, O1113 EA.1, O1399 EA.2, O1393 EA.2, O1394 EA.2 y O1400 P.2), mientras que el extracto O1113 EA.2 presentó inhibición total contra E. coli ATCC® 25922TM. Estos extractos también se evaluaron por el método de inhibición por difusión con discos, obteniéndose el halo de inhibición de mayor diámetro con el extracto O1113 DCM.1. Adicionalmente, se determinaron las concentraciones mínimas inhibitorias (CMIs) de estos extractos utilizando el método de microdilución. Se purificó el extracto orgánico O1113 DCM.1 mediante cromatografía líquida de alta eficiencia (HPLC), recuperándose la bioactividad contra S. aureus ATCC® 25923TM en dos fracciones. En el análisis por espectrometría de masas de una de ellas se identificaron, según su fórmula molecular, 11 posibles compuestos. En cuanto a los ensayos antifúngicos contra P. ultimum, los extractos O1113 DCM.1 y O1399 P.1 inhibieron totalmente al hongo (crecimiento del 0% al tercer día de ensayo), encontrándose un efecto fungicida del primer extracto y uno fungistático del segundo extracto, dejando abierta la posibilidad de continuar con ensayos con líneas celulares de cáncer debido a su acción citotóxica hacia el hongo

Enzyme Activity Profiles Produced on Wood and Straw by Four Fungi of Different Decay Strategies

Four well-studied saprotrophic Basidiomycota Agaricomycetes species with different decay strategies were cultivated on solid lignocellulose substrates to compare their extracellular decomposing carbohydrate-active and lignin-attacking enzyme production profiles. Two Polyporales species, the white rot fungus Phlebia radiata and brown rot fungus Fomitopsis pinicola, as well as one Agaricales species, the intermediate “grey” rot fungus Schizophyllum commune, were cultivated on birch wood pieces for 12 weeks, whereas the second Agaricales species, the litter-decomposing fungus Coprinopsis cinerea was cultivated on barley straw for 6 weeks under laboratory conditions. During 3 months of growth on birch wood, only the white rot fungus P. radiata produced high laccase and MnP activities. The brown rot fungus F. pinicola demonstrated notable production of xylanase activity up to 43 nkat/mL on birch wood, together with moderate β-glucosidase and endoglucanase cellulolytic activities. The intermediate rot fungus S. commune was the strongest producer of β-glucosidase with activities up to 54 nkat/mL, and a notable producer of xylanase activity, even up to 620 nkat/mL, on birch wood. Low lignin-attacking but moderate activities against cellulose and hemicellulose were observed with the litter-decomposer C. cinerea on barley straw. Overall, our results imply that plant cell wall decomposition ability of taxonomically and ecologically divergent fungi is in line with their enzymatic decay strategy, which is fundamental in understanding their physiology and potential for biotechnological applications

Enzyme Activity Profiles Produced on Wood and Straw by Four Fungi of Different Decay Strategies

Four well-studied saprotrophic Basidiomycota Agaricomycetes species with different decay strategies were cultivated on solid lignocellulose substrates to compare their extracellular decomposing carbohydrate-active and lignin-attacking enzyme production profiles. Two Polyporales species, the white rot fungus Phlebia radiata and brown rot fungus Fomitopsis pinicola, as well as one Agaricales species, the intermediate “grey” rot fungus Schizophyllum commune, were cultivated on birch wood pieces for 12 weeks, whereas the second Agaricales species, the litter-decomposing fungus Coprinopsis cinerea was cultivated on barley straw for 6 weeks under laboratory conditions. During 3 months of growth on birch wood, only the white rot fungus P. radiata produced high laccase and MnP activities. The brown rot fungus F. pinicola demonstrated notable production of xylanase activity up to 43 nkat/mL on birch wood, together with moderate β-glucosidase and endoglucanase cellulolytic activities. The intermediate rot fungus S. commune was the strongest producer of β-glucosidase with activities up to 54 nkat/mL, and a notable producer of xylanase activity, even up to 620 nkat/mL, on birch wood. Low lignin-attacking but moderate activities against cellulose and hemicellulose were observed with the litter-decomposer C. cinerea on barley straw. Overall, our results imply that plant cell wall decomposition ability of taxonomically and ecologically divergent fungi is in line with their enzymatic decay strategy, which is fundamental in understanding their physiology and potential for biotechnological applications

Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application.

Continued waves, new variants, and limited vaccine deployment mean that SARS-CoV-2 tests remain vital to constrain the coronavirus disease 2019 (COVID-19) pandemic. Affordable, point-of-care (PoC) tests allow rapid screening in non-medical settings. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is an appealing approach. A crucial step is to optimize testing in low/medium resource settings. Here, we optimized RT-LAMP for SARS-CoV-2 and human β-actin, and tested clinical samples in multiple countries. "TTTT" linker primers did not improve performance, and while guanidine hydrochloride, betaine and/or Igepal-CA-630 enhanced detection of synthetic RNA, only the latter two improved direct assays on nasopharygeal samples. With extracted clinical RNA, a 20 min RT-LAMP assay was essentially as sensitive as RT-PCR. With raw Canadian nasopharygeal samples, sensitivity was 100% (95% CI: 67.6% - 100%) for those with RT-qPCR Ct values ≤ 25, and 80% (95% CI: 58.4% - 91.9%) for those with 25 < Ct ≤ 27.2. Highly infectious, high titer cases were also detected in Colombian and Ecuadorian labs. We further demonstrate the utility of replacing thermocyclers with a portable PoC device (FluoroPLUM). These combined PoC molecular and hardware tools may help to limit community transmission of SARS-CoV-2