Methionine inducing carbohydrate esterase secretion of Trichoderma harzianum enhances the accessibility of substrate glycosidic bonds.

BACKGROUND: The conversion of plant biomass into biochemicals is a promising way to alleviate energy shortage, which depends on efficient microbial saccharification and cellular metabolism. Trichoderma spp. have plentiful CAZymes systems that can utilize all-components of lignocellulose. Acetylation of polysaccharides causes nanostructure densification and hydrophobicity enhancement, which is an obstacle for glycoside hydrolases to hydrolyze glycosidic bonds. The improvement of deacetylation ability can effectively release the potential for polysaccharide degradation. RESULTS: Ammonium sulfate addition facilitated the deacetylation of xylan by inducing the up-regulation of multiple carbohydrate esterases (CE3/CE4/CE15/CE16) of Trichoderma harzianum. Mainly, the pathway of ammonium-sulfate's cellular assimilates inducing up-regulation of the deacetylase gene (Thce3) was revealed. The intracellular metabolite changes were revealed through metabonomic analysis. Whole genome bisulfite sequencing identified a novel differentially methylated region (DMR) that existed in the ThgsfR2 promoter, and the DMR was closely related to lignocellulolytic response. ThGsfR2 was identified as a negative regulatory factor of Thce3, and methylation in ThgsfR2 promoter released the expression of Thce3. The up-regulation of CEs facilitated the substrate deacetylation. CONCLUSION: Ammonium sulfate increased the polysaccharide deacetylation capacity by inducing the up-regulation of multiple carbohydrate esterases of T. harzianum, which removed the spatial barrier of the glycosidic bond and improved hydrophilicity, and ultimately increased the accessibility of glycosidic bond to glycoside hydrolases.

Exploring the multi-level regulation of lignocellulases in the filamentous fungus Trichoderma guizhouense NJAU4742 from an omics perspective.

BACKGROUND: Filamentous fungi are highly efficient at deconstructing plant biomass by secreting a variety of enzymes, but the complex enzymatic regulation underlying this process is not conserved and remains unclear. RESULTS: In this study, cellulases and xylanases could specifically respond to Avicel- and xylan-induction, respectively, in lignocellulose-degrading strain Trichoderma guizhouense NJAU4742, however, the differentially regulated cellulases and xylanases were both under the absolute control of the same TgXyr1-mediated pathway. Further analysis showed that Avicel could specifically induce cellulase expression, which supported the existence of an unknown specific regulator of cellulases in strain NJAU4742. The xylanase secretion is very complex, GH10 endoxylanases could only be induced by Avicel, while, other major xylanases were significantly induced by both Avicel and xylan. For GH10 xylanases, an unknown specific regulator was also deduced to exist. Meanwhile, the post-transcriptional inhibition was subsequently suggested to stop the Avicel-induced xylanases secretion, which explained the specifically high xylanase activities when induced by xylan in strain NJAU4742. Additionally, an economical strategy used by strain NJAU4742 was proposed to sense the environmental lignocellulose under the carbon starvation condition, that only slightly activating 4 lignocellulose-degrading genes before largely secreting all 33 TgXyr1-controlled lignocellulases if confirming the existence of lignocellulose components. CONCLUSIONS: This study, aiming to explore the unknown mechanisms of plant biomass-degrading enzymes regulation through the combined omics analysis, will open directions for in-depth understanding the complex carbon utilization in filamentous fungi.

Evolution and comparative genomics of the most common Trichoderma species.

BACKGROUND: The growing importance of the ubiquitous fungal genus Trichoderma (Hypocreales, Ascomycota) requires understanding of its biology and evolution. Many Trichoderma species are used as biofertilizers and biofungicides and T. reesei is the model organism for industrial production of cellulolytic enzymes. In addition, some highly opportunistic species devastate mushroom farms and can become pathogens of humans. A comparative analysis of the first three whole genomes revealed mycoparasitism as the innate feature of Trichoderma. However, the evolution of these traits is not yet understood. RESULTS: We selected 12 most commonly occurring Trichoderma species and studied the evolution of their genome sequences. Trichoderma evolved in the time of the Cretaceous-Palaeogene extinction event 66 (±15) mya, but the formation of extant sections (Longibrachiatum, Trichoderma) or clades (Harzianum/Virens) happened in Oligocene. The evolution of the Harzianum clade and section Trichoderma was accompanied by significant gene gain, but the ancestor of section Longibrachiatum experienced rapid gene loss. The highest number of genes gained encoded ankyrins, HET domain proteins and transcription factors. We also identified the Trichoderma core genome, completely curated its annotation, investigated several gene families in detail and compared the results to those of other fungi. Eighty percent of those genes for which a function could be predicted were also found in other fungi, but only 67% of those without a predictable function. CONCLUSIONS: Our study presents a time scaled pattern of genome evolution in 12 Trichoderma species from three phylogenetically distant clades/sections and a comprehensive analysis of their genes. The data offer insights in the evolution of a mycoparasite towards a generalist.

Probing active microbes involved in Bt-containing rice straw decomposition.

Transgenic Bacillus thuringiensis (Bt) rice extends significant protection against insect pests and meets the increasing demands for food and energy. Many studies have been conducted investigating the impacts of Bt rice to the agricultural ecosystem, but much less attention has been given to efforts attempting to determine how the presence of Bt rice influences and shapes the microbial community, especially the active microbes. Stable isotope probing and high-throughput sequencing were employed to explore the active microbes involved in Bt-containing straw decomposition. Compared to its near isoline, the Bt straw contained higher contents of total N, total P, total K, lignin, cellulose, and Cry1Ab toxin protein. These chemical differences did not affect the decomposition rate but significantly changed the active microbial decomposer communities. During the decomposition of Bt-containing straw, fungi were more affected than bacteria. Agromyces, Terrabacter, Microbacterium, Glycomyces, and Kribbella were the most representative unique (existed only in the Bt treatments and appeared at the early stage) bacterial genera, and Trichoderma was the most representative unique fungal genus in the Bt straw decomposition. By using similarity index calculation and function prediction, the significant differences between Bt straw and non-Bt straw treatments were found to be transient for both microbial taxa and functional traits. These results suggested that Bt rice has a significant but transient impact on soil microbes in terms of microbial straw decomposition.

Two degradation strategies for overcoming the recalcitrance of natural lignocellulosic xylan by polysaccharides-binding GH10 and GH11 xylanases of filamentous fungi.

The recalcitrance of lignocellulose forms a strong barrier for the bioconversion of lignocellulosic biomass in chemical or biofuel industries. Filamentous fungi are major plant biomass decomposer, and capable of forming all the required enzymes. Here, they characterized the GH10 and GH11 endo-xylanases and a CE1 acetyl-xylan esterase (Axe1) from a superior biomass-degrading strain, Aspergillus fumigatus Z5, and examined how they interact in xylan degradation. Cellulose-binding (CBM1) domain inhibited GH10 xylanase activities for pure xylan, but afforded them an ability to hydrolyze washed corncob particles (WCCP). CBM1-containing GH10 xylanases also showed synergism with CBM1-containing Axe1 in WCCP hydrolysis, and this synergy was strictly dependent on the presence of their CBM1 domains. In contrast, GH11 xylanases had no CBM1, but still could bind xylan and hydrolyzed WCCP; however, no synergism displayed with Axe1. GH10 xylanases and GH11 xylanases showed a pronounced synergism in WCCP hydrolysis, which was dependent on the presence of the CBM1 in GH10 xylanases and absence from GH11 xylanases. They exhibit different mechanisms to bind to cellulose and xylan, and act in synergy when these two structures are intact. These findings will be helpful for the further development of highly efficient enzyme mixtures for lignocellulosic biomass conversion.

Characterization of extracellular polymeric substances of Bacillus amyloliquefaciens SQR9 induced by root exudates of cucumber.

Bacillus amyloliquefaciens SQR9 is a plant growth-promoting rhizobacterium (PGPRs) that forms biofilm on the roots of plants and protects them from a variety of pathogens. In this study, we reported the effect of root exudates produced by cucumber (Cucumis sativus L.) at different developmental stages on the biochemical composition of the biofilm matrix of SQR9. The results showed that the amino acids present in the root exudates of cucumber were responsible for triggering biofilm formation of SQR9. In addition, when root exudates harvested at different growth phases of cucumber were used as carbon sources for biofilm formation, the resulting biofilm matrixes differed both quantitatively and qualitatively. The biofilm matrix was mostly composed of amino groups observed by confocal laser scanning microscope (CLSM) hence the proteins formed the major component of the resulting extracellular polymeric substances (EPS). The potential use of amino acid-based dietary supplements to control biofilm formation in the plants may be a viable option to improve agricultural productivity by recruiting beneficial association with PGPRs in the manufacture of bio fertilizers or bio controls.

Genome-wide transcriptomic analysis of a superior biomass-degrading strain of A. fumigatus revealed active lignocellulose-degrading genes.

BACKGROUND: Various saprotrophic microorganisms, especially filamentous fungi, can efficiently degrade lignocellulose that is one of the most abundant natural materials on earth. It consists of complex carbohydrates and aromatic polymers found in the plant cell wall and thus in plant debris. Aspergillus fumigatus Z5 was isolated from compost heaps and showed highly efficient plant biomass-degradation capability. RESULTS: The 29-million base-pair genome of Z5 was sequenced and 9540 protein-coding genes were predicted and annotated. Genome analysis revealed an impressive array of genes encoding cellulases, hemicellulases and pectinases involved in lignocellulosic biomass degradation. Transcriptional responses of A. fumigatus Z5 induced by sucrose, oat spelt xylan, Avicel PH-101 and rice straw were compared. There were 444, 1711 and 1386 significantly differently expressed genes in xylan, cellulose and rice straw, respectively, when compared to sucrose as a control condition. CONCLUSIONS: Combined analysis of the genomic and transcriptomic data provides a comprehensive understanding of the responding mechanisms to the most abundant natural polysaccharides in A. fumigatus. This study provides a basis for further analysis of genes shown to be highly induced in the presence of polysaccharide substrates and also the information which could prove useful for biomass degradation and heterologous protein expression.

The distinct plastisphere microbiome in the terrestrial-marine ecotone is a reservoir for putative degraders of petroleum-based polymers.

Research into plastic-degrading bacteria and fungi is important for understanding how microorganisms can be used to address the problem of plastic pollution and for developing new approaches to sustainable waste management and bioplastic production. In the present study, we isolated 55 bacterial and 184 fungal strains degrading polycaprolactone (PCL) in plastic waste samples from Dafeng coastal salt marshes, Jiangsu, China. Of these, Jonesia and Streptomyces bacteria also showed potential to degrade other types of petroleum-based polymers. The metabarcoding results proved the existence of plastisphere as a distinct ecological niche regardless of the plastic types where 27 bacterial and 29 fungal amplicon sequence variants (ASVs) were found to be significantly (p < 0.05) enriched, including some belonging to Alternaria (Ascomycota, Fungi) and Pseudomonas (Gammaproteobacteria, Bacteria) that were also mined out by the method of cultivation. Further assembly analyses demonstrated the importance of deterministic processes especially the environmental filtering effect of carbon content and pH on bacteria as well as the carbon and cation content on fungi in shaping the plastisphere communities in this ecosystem. Thus, the unique microbiome of the plastisphere in the terrestrial-marine ecotone is enriched with microorganisms that are potentially capable of utilizing petroleum-based polymers, making it a valuable resource for screening plastic biodegraders.

Expression, purification and characterization of two thermostable endoglucanases cloned from a lignocellulosic decomposing fungi Aspergillus fumigatus Z5 isolated from compost.

Two genes encoding endoglucanase, designated as egl2 and egl3, were cloned from a lignocellulosic decomposing fungus Aspergillus fumigatus Z5 and were successfully expressed in Pichia pastoris X33. The deduced amino acid sequences encoded by egl2 and egl3 showed strong similarity with the sequence of glycoside hydrolase family 5. SDS-PAGE and western blot assays indicated that the recombinant enzymes were secreted into the culture medium and the zymogram analysis confirmed that both recombinant enzymes had endoglucanase activity. Several biochemical properties of the two recombinant enzymes were studied: Egl2 and Egl3 showed optimal activity at pH 5.0 and 4.0, respectively, and at 50 and 60°C, respectively. Egl2 and Egl3 showed good pH stability in the range of 4-7, and both enzymes demonstrated good thermostability ranging from 30 to 60°C. The K(m) and V(max) values using carboxymethyl cellulose (CMC, soluble cellulose, polymerized by ß-1, 4-linked glucose residues) as the substrate at optimal conditions were determined. The activities of the enzymes on a variety of cello-oligosaccharide substrates were investigated, and Egl2 can hydrolyze cellotetraose and cellopentaose but not cellobiose and cellotriose, whereas Egl3 can hydrolyze all cello-oligosaccharides, except cellobiose.

Multiple fluorescence labeling and two dimensional FTIR-13C NMR heterospectral correlation spectroscopy to characterize extracellular polymeric substances in biofilms produced during composting.

Knowledge on the structure and function of extracellular polymeric substances (EPS) in biofilms is essential for understanding biodegradation processes. Herein, a novel method based on multiple fluorescence labeling and two-dimensional (2D) FTIR-(13)C NMR heterospectral correlation spectroscopy was developed to gain insight on the composition, architecture, and function of EPS in biofilms during composting. Compared to other environmental biofilms, biofilms in the thermophilic (>55 °C) and cooling (mature) stage of composting have distinct characteristics. The results of multiple fluorescence labeling demonstrated that biofilms were distributed in clusters during the thermophilic stage (day 14), and dead cells were detected. In the mature stage (day 26), the biofilm formed a continuous layer with a thickness of approximately 20-100 µm around the compost, and recolonization of cells at the surface of the compost was easily observed. Through 2D FTIR-(13)C NMR correlation heterospectral spectroscopy, the following trend in the ease of the degradation of organic compounds was observed: heteropolysaccharides > cellulose > amide I in proteins. And proteins and cellulose showed significantly more degradation than heteropolysaccharides. In summary, the combination of multiple fluorescence labeling and 2D correlation spectroscopy is a promising approach for the characterization of EPS in biofilms.

The functioning of a novel protein, Swollenin, in promoting the lignocellulose degradation capacity of Trichoderma guizhouense NJAU4742 from a proteomic perspective.

The functioning of a novel auxiliary enzyme, TgSWO from Trichoderma guizhouense NJAU4742, was investigated based on the proteomic analysis of wild-type (WT), knockout (KO) and overexpression (OE) treatments. The results showed that the cellulase and hemicellulase activities of OE and WT were significantly higher than those of KO. Simultaneously, tandem mass tag (TMT) analysis results indicated that cellulases and hemicellulases were significantly upregulated in OE, especially hydrophobin (HFB, A1A105805.1) and endo-ß-1,4-glucanases (A1A101831.1), with ratios of 43.73 and 9.88, respectively, compared with WT. The synergistic effect of TgSWO on cellulases increased the reducing sugar content by 1.45 times in KO + TgSWO (1.8 mg) compared with KO, and there was no significant difference between KO + TgSWO (1.2 mg) and WT. This study elucidated the function of TgSWO in promoting the lignocellulose degradation capacity of NAJU4742, which provides new insights into the efficient conversion of lignocellulose.

Key extracellular enzymes triggered high-efficiency composting associated with bacterial community succession.

A consortium of key bacterial taxa plays critical roles in the composting process. In order to elucidate the identity and mechanisms by which specific bacterial species drive high-efficiency composting, the succession of key bacterial consortia and extracellular enzymes produced during the composting process were monitored in composting piles with varying initial C/N ratios. Results showed that C/N ratios of 25 and 35 enhanced composting efficiency through elevated temperatures, higher germination indices, enhanced cellulose and hemicellulose degradation, and higher cellulase and dehydrogenase activities. The activities of cellulase and ß-glucosidase, cellulase and protease, and cellulase and ß-glucosidase exhibited significant relationships with bacterial community composition within the mesophilic, thermophilic, and mature phases, respectively. Putative key taxa, linked to a higher composting efficiency, such as Nonomuraea, Desemzia, Cellulosimicrobium, Virgibacillus, Clostridium, and Achromobacter, exhibited significantly positive relationships with extracellular enzyme activities, suggesting a significant contribution to these taxa to the development of composting maturity.

Redox imbalance contributed differently to membrane damage of cucumber leaves under water stress and Fusarium infection.

Redox-associated events are important in plant development and responses to environmental stresses. In this study, we investigated spatial redox responses of cucumber (Cucumis sativus L.) leaves to biotic stress (Fusarium infection) or abiotic stress (water stress). Plants were grown under hydroponic conditions and either treated with polyethylene glycol to simulate drought or infected with Fusarium oxysporum f. sp. cucumerinum. Both water stress and Fusarium infection restricted cucumber growth and were associated with cellular plasma-membrane damage, reactive oxygen species accumulation, and changes in antioxidants; however, the responses to each stress were distinctive. Under water stress, H2O2 generation at the leaf edge increased 29.7% compared with that at the centre but with Fusarium infection there was a relative 10.4% decrease at the edge. These changes correlated with changes in antioxidants and linked enzyme activities. The key sources of variation in oxidative events were defined by principal component analysis of all of the data and redox balance evaluations. We suggest that these spatial differences under water stress and Fusarium infection arise from discrete regulatory mechanisms, reflecting either developmental effect over the leaf regions or systemic anti-oxidative events occurred following infection.

Effect of organic fertilizers prepared from organic waste materials on the production of antibacterial volatile organic compounds by two biocontrol Bacillus amyloliquefaciens strains.

Three organic fertilizers made of different animal and plant waste materials (BOFs) were evaluated for their effects on the production of antibacterial volatile organic compounds (VOCs) by two Bacillus amyloliquefaciens strains SQR-9 and T-5 against the tomato wilt pathogen Ralstonia solanacearum (RS). Both strains could produce VOCs that inhibited the growth and virulence traits of RS; however, in the presence of BOFs, the production of antibacterial VOCs was significantly increased. The maximum inhibition of growth and virulence traits of RS by VOCs of T-5 and SQR-9 was determined at 1.5% BOF2 and 2% BOF3, respectively. In case of strain T-5, 2-nonanone, nonanal, xylene, benzothiazole, and butylated hydroxy toluene and in case of strain SQR-9, 2-nonanone, nonanal, xylene and 2-undecanone were the main antibacterial VOCs whose production was increased in the presence of BOFs. The results of this study reveal another significance of using organic fertilizers to improve the antagonistic activity of biocontrol agents against phytopathogens.

In situ visualisation and characterisation of the capacity of highly reactive minerals to preserve soil organic matter (SOM) in colloids at submicron scale.

Mineral-organo associations (MOAs) are a mixture of identifiable biopolymers associated with highly reactive minerals and microorganisms. However, the in situ characterization and correlation between soil organic matter (SOM) and highly reactive Al and Fe minerals are still unclear for the lack of technologies, particularly in the long-term agricultural soil colloids at submicron scale. We combined several novel techniques, including nano-scale secondary ion mass spectrometry (NanoSIMS), X-ray absorption near edge structure (XANES) and confocal laser scanning microscopy (CLSM) to characterise the capacity of highly reactive Al and Fe minerals to preserve SOM in Ferralic Cambisol in south China. Our results demonstrated that: (1) highly reactive minerals were strongly related to SOM preservation, while SOM had a more significant line correlation with the highly reactive Al minerals than the highly reactive Fe minerals, according to the regions of interest correlation analyses using NanoSIMS; (2) allophane and ferrihydrite were the potential mineral species to determine the SOM preservation capability, which was evaluated by the X-ray photoelectron spectroscopy (XPS) and Fe K-edge XANES spectroscopy techniques; and (3) soil organic biopolymers with dominant compounds, such as proteins, polysaccharides and lipids, were distributed at the rough and clustered surface of MOAs with high chemical and spatial heterogeneity according to the CLSM observation. Our results also promoted the understanding of the roles played by the highly reactive Al and Fe minerals in the spatial distribution of soil organic biopolymers and SOM sequestration.

[Studies on the changes in rice straw composition in relay treatment of chemical-microbial process by FTIR spectroscopy].

Direct burning of crop straw in the field has given or is giving rise to a serious pollution of atmosphere. The difficult decomposing of the crop straw by soil microorganisms is one of the reasons the crop straw is not popularly used in agriculture. Fourier transform infrared spectroscopy (FTIR) was used to study the changes in straw composition during the relay treatment of chemical-microbial process. The results showed that the method of FTIR spectra could indicate the changes in straw composition during the treatment processes. After the relay treatment of chemical-microbial process, the contents of cellulose, semi-cellulose, and silicon, and C/N ratio were decreased significantly, while the water soluble substances were increased, which was in accordance with the results of chemical analysis. The method to treat crop straw proposed in this paper could provide a practicable way in agricultural utilization of crop straw.

Fate of biopolymers during rapeseed meal and wheat bran composting as studied by two-dimensional correlation spectroscopy in combination with multiple fluorescence labeling techniques.

Detailed knowledge of the molecular events during composting is important in improving the efficiency of this process. By combining two-dimensional Fourier transform infrared (FTIR) correlation spectroscopy and multiple fluorescent labeling, it was possible to study the degradation of biopolymers during rapeseed meal and wheat bran composting. Two-dimensional FTIR correlation spectroscopy provided structural information and was used to deconvolute overlapping bands found in the compost FTIR spectra. The degradation of biopolymers in rapeseed meal and wheat bran composts followed the sequence: cellulose, heteropolysaccharides, and proteins. Fluorescent labeling suggested that cellulose formed an intact network-like structure and the other biopolymers were embedded in the core of this structure. The sequence of degradation of biopolymers during composting was related to their distribution patterns.

[Effect of combined application of bioorganic manure and inorganic nitrogen fertilizer on soil nitrogen supplying characteristics].

Pot experiment was carried out to study the effect of application bioorganic manure on soil nitrogen supplying characteristics. Compared with the application of raw rice straw, a combined application of bio-organic manure and inorganic nitrogen fertilizer could boost the content of microbial biomass carbon and nitrogen, and the increased percentage could be 300-400%. A strong immobilization of nitrogen by microorganisms was always followed by a net N mineralization, which was mostly favorable for the growth and development of plant, and improved the use efficiency of nitrogen fertilizer.