Engineering multifunctional rhizosphere probiotics using consortia of Bacillus amyloliquefaciens transposon insertion mutants.

While bacterial diversity is beneficial for the functioning of rhizosphere microbiomes, multi-species bioinoculants often fail to promote plant growth. One potential reason for this is that competition between different species of inoculated consortia members creates conflicts for their survival and functioning. To circumvent this, we used transposon insertion mutagenesis to increase the functional diversity within Bacillus amyloliquefaciens bacterial species and tested if we could improve plant growth promotion by assembling consortia of highly clonal but phenotypically dissimilar mutants. While most insertion mutations were harmful, some significantly improved B. amyloliquefaciens plant growth promotion traits relative to the wild-type strain. Eight phenotypically distinct mutants were selected to test if their functioning could be improved by applying them as multifunctional consortia. We found that B. amyloliquefaciens consortium richness correlated positively with plant root colonization and protection from Ralstonia solanacearum phytopathogenic bacterium. Crucially, 8-mutant consortium consisting of phenotypically dissimilar mutants performed better than randomly assembled 8-mutant consortia, suggesting that improvements were likely driven by consortia multifunctionality instead of consortia richness. Together, our results suggest that increasing intra-species phenotypic diversity could be an effective way to improve probiotic consortium functioning and plant growth promotion in agricultural systems.

[Effects of partial substitution of chemical fertilizer with organic fertilizer on arbuscular mycorrhizal fungal community of Mangifera indica]. / æœ‰æœºè‚¥æ›¿ä»£éƒ¨åˆ†åŒ–è‚¥å¯¹èŠ’æžœä¸›æžèŒæ ¹çœŸèŒç¾¤è½çš„å½±å“.

Nutrient enrichment caused by fertilization would reduce the diversity of arbuscular mycorrhizal fungi (AMF). To explore whether partial substitution of chemical fertilizer with organic fertilizer would alleviate the negative effects of nutrient enrichment on AMF, we conducted a two-year mango (Mangifera indica) field experiment to examine the effects of different fertilization regimes on AMF communities in roots and rhizospheric soils by using high-throughput sequencing. The treatments included chemical-only fertilization (control), and two kinds of organic fertilizer (commercial organic fertilizer and bio-organic fertilizer) with replacing 12% (low) and 38% (high) chemical fertilizer. The results showed that under equivalent nutrient input, partial substitution of chemical fertilizer with organic fertilizer had positive effects on the yield and quality of mango. The application of organic fertilizer could effectively increase AMF richness. AMF diversity was significantly positively correlated with some indices of fruit quality. Compared with chemical-only fertilization, high replacement ratio of organic fertilizer could significantly change root AMF community, but did not affect AMF community in the rhizospheric soil. Bio-organic fertilizer could enrich more AMF species and form a more complex AMF co-occurrence network than commercial organic fertilizer. In all, replacing chemical fertilizer with a high proportion of organic fertilizer could improve the yield and quality of mango while maintain AMF richness. The changes of AMF community caused by organic fertilizer substitution pre-ferably occurred in roots rather than soils.

Efficient potassium (K) recycling and root carbon (C) metabolism improve K use efficiency in pear rootstock genotypes.

To investigate K absorption and transport mechanisms by which pear rootstock genotypes respond to low-K stress, seedlings of a potassium-efficient pear rootstock, Pyrus ussuriensis, and a potassium-sensitive rootstock, Pyrus betulifolia, were supplied with different K concentrations in solution culture. Significant differences in the absorption rate, Vmax and Km between the genotypes indicate that P. ussuriensis acclimatizes more readily to low-K stress by regulating its absorption and internal cycling. We also found that the K content in the leaves of P. betulifolia was significantly lower than that of P. ussuriensis, and the proportion of K that was returned to root from shoot, relative to K that was transported from root to shoot, was greater in P. ussuriensis, which suggests that P. ussuriensis more efficiently recycles and reuses K. When the transcriptomes of the two genotypes were compared, we found that photosynthetic genes such as CABs (Chlorophyll a/b-binding proteins), Lhcbs (Photosystem II-related proteins), and Psas (Photosystem Ⅰ associated proteins) displayed lower expression in leaves of P. betulifolia under no-K conditions, but not in P. ussuriensis. However, in the root of P. ussuriensis, carbon metabolism-related genes SS (Sucrose Synthase), HK (HexoKinase) and SDH (Sorbitol Dehydrogenase) and components of the TCA cycle (Tricarboxylic Acid cycle) were differentially expressed, indicating that changes in C metabolism may provide energy for increased K+ cycling in these plants, thereby allowing it to better adapt to the low-K environment. In addition, exogenous supply of various sugars to the roots influenced K+ influx, supporting the conclusion that sugar metabolism in roots significantly affects K+ absorption in pear.

Bio-Organic Fertilizer Promotes Pear Yield by Shaping the Rhizosphere Microbiome Composition and Functions.

Bio-organic fertilizers (BOF) containing both organic amendments and beneficial microorganisms have been consistently shown to improve soils fertility and yield. However, the exact mechanisms which link amendments and yields remain disputed, and the complexity of bio-organic fertilizers may work in parallel in several ways. BOF may directly improve yield by replenishing soil nutrients or introducing beneficial microbial genes or indirectly by altering the soil microbiome to enrich native beneficial microorganisms. In this work, we aim to disentangle the relative contributions of direct and indirect effects on pear yield. We treated pear trees with either chemical fertilizer or organic fertilizer with/without the plant-beneficial bacterium Bacillus velezensis SQR9. We then assessed, in detail, soil physicochemical and biological properties (metagenome sequencing) as well as pear yield. We then evaluated the relative importance of direct and indirect effects of soil amendments on pear yield. Both organic treatments increased plant yield by up to 20%, with the addition of bacteria tripling the increase driven by organic fertilizer alone. This increase could be linked to alterations in soil physicochemical properties, bacterial community function, and metabolism. Supplementation of organic fertilizer SQR9 increased rhizosphere microbiome richness and functional diversity. Fertilizer-sensitive microbes and functions responded as whole guilds. Pear yield was most positively associated with the Mitsuaria- and Actinoplanes-dominated ecological clusters and with gene clusters involved in ion transport and secondary metabolite biosynthesis. Together, these results suggested that bio-organic fertilizers mainly act indirectly on plant yield by creating soil chemical properties which promote a plant-beneficial microbiome. IMPORTANCE Bio-organic fertilization is a widely used, eco-friendly, sustainable approach to increasing plant productivity in the agriculture and fruit industries. However, it remains unclear whether the promotion of fruit productivity is related to specific changes in microbial inoculants, the resident microbiome, and/or the physicochemical properties of rhizosphere soils. We found that bio-organic fertilizers alter soil chemical properties, thus manipulating specific microbial taxa and functions within the rhizosphere microbiome of pear plants to promote yield. Our work unveils the ecological mechanisms which underlie the beneficial impacts of bio-organic fertilizers on yield promotion in fruit orchards, which may help in the design of more efficient biofertilizers to promote sustainable fruit production.

WGCNA analysis revealing molecular mechanism that bio-organic fertilizer improves pear fruit quality by increasing sucrose accumulation and reducing citric acid metabolism.

It's been long known that the application of organic fertilizer (OF) and bio-organic fertilizer (BF) which containing beneficial microorganisms to pear trees can both significantly improve fruit quality and yield. In order to reveal the mechanism of BF and OF regulating fruit growth and quality in pear, the effects of BF and OF on the photosynthetic characteristics and the accumulation of major sugars and organic acids of the pear fruit were quantified compared with chemical fertilizer (CF). Additionally, the molecular mechanisms regulating pear fruit development and quality were studied through transcriptome analysis. The three treatments were conducted based on the same amounts of nitrogen supply. The results showed that compared with CF, BF and OF treatments increased the fruit yield, and also significantly improved the photosynthesis efficiency in pear. BF and OF both significantly increased the sucrose content but significantly decreased the fructose and glucose content within the pear fruit. The amount of malic acid was significantly higher in OF treatment. Compared with CF and OF, BF significantly increased the sugar-acid ratio and thus improved the fruit quality. Transcriptome analysis and weighted correlation network analysis (WGCNA) revealed that the sugar metabolism of fruits applied with the BF was enhanced compared with those applied with CF or OF. More specifically, the expression of SDH (Sorbitol dehydrogenase) was higher in BF, which converts sorbitol into fructose. For both of the OF and BF, the transcript abundance of sugar transporter genes was significantly increased, such as SOT (Sorbitol transporter), SUT14 (Sugar transport 14), UDP-GLUT4 (UDP-glucose transporter 4), UDP-SUT (UDP-sugar transporter), SUC4 (Sucrose transport 4), SUT7 (Sugar transporter 7), SWEET10 and SWEET15 (Bidirectional sugar transporter), which ensures sugar transportation. The genes involved in organic acid metabolism showed decreased transcripts abundance in both BF and OF treatments, such as VAP (Vesicle-associated protein) and cyACO (Cytosolic aconitase), which reduce the conversion from succinate to citric acid, and decrease the conversion from citric acid to malic acid in the TCA cycle (Tricarboxylic Acid cycle) through Pept6 (Oligopeptide transporter). In conclusion, the application of BF and OF improved fruit quality by regulating the expression of sugar and organic acid metabolism-related genes and thus altering the sugar acid metabolism. Both BF and OF promote sucrose accumulation and citric acid degradation in fruits, which may be an important reason for improving pear fruit quality. The possible mechanism of bio-organic fertilizer to improve fruit quality was discussed.

Compositional and functional succession of bacterial and fungal communities is associated with changes in abiotic properties during pig manure composting.

While both bacteria and fungi are important for the degradation and humification of organic matter during composting, it is unclear to what extent their roles are associated with abiotic compost properties. This study evaluated changes in abiotic compost properties and the succession of bacterial and fungal communities during pig manure composting for 90 days. The compost rapidly reached thermophilic phase (>58 ℃), which lasted for 15 days. Both bacterial and fungal community compositions changed drastically during composting and while bacterial diversity increased, the fungal diversity decreased during the thermophilic phase of composting. Two taxa dominated both bacterial (Bacillales and Clostridiales) and fungal (Eurotiales and Glomerellales) communities and these showed alternating abundance fluctuations following different phases of composting. The abundance fluctuations of most dominant bacterial and fungal taxa could be further associated with decreases in the concentrations of fulvic acid, cellulose, hemicellulose and overall biodegradation potential in the compost. Moreover, bacterial predicted metabolic gene abundances dominated the first three phases of composting, while predicted fungal saprotrophic functional genes increased consistently, reaching highest abundances towards the end of composting. Finally, redundancy analysis (RDA) showed that changes in abiotic compost properties correlated with the bacterial community diversity and carbohydrate metabolism and fungal wood saprotrophic function. Together these results suggests that bacterial and fungal community succession was associated with temporal changes in abiotic compost properties, potentially explaining alternating taxa abundance patterns during pig manure composting.

Isolation of a novel psychrotrophic fungus for efficient low-temperature composting.

This study aimed to isolate psychrotrophic cellulose-degrading fungi and to investigate their application potential for composting in cold climate regions in China. One out of five psychrotrophic cellulose-degrading fungal isolates was identified as a novel fungal species, Aureobasidium paleasum sp. nov., with a strong straw degradation potential. Enzyme activity assays and FITR spectroscopy revealed high cellulolytic activities of this psychrotrophic fungus at lower temperatures, with high thermal adaptability from 5 °C to 50 °C (optimum at 10 °C). A. paleasum efficiently decomposed rice straws and cellulose at 10 °C compared to the common cellulose-degrading fungus Penicillium oxalicum. In comparison to P. oxalicum, A. paleasum shortened the thermophilic stage, enhanced compost maturity and improved compost quality. Our work suggests that the psychrotrophic fungus A. paleasum is efficient for rice straw degradation and composting at low temperatures, highlighting its application potential for composting in colder regions.

Livestock Manure Type Affects Microbial Community Composition and Assembly During Composting.

Composting is an environmentally friendly way to turn plant and animal wastes into organic fertilizers. However, it is unclear to what extent the source of animal waste products (such as manure) affects the physicochemical and microbiological properties of compost. Here, we experimentally tested how the type of livestock manure of herbivores (sheep and cattle) and omnivores (pig and chicken) influences the bacterial and fungal communities and physicochemical properties of compost. Higher pH, NO3-N, Total carbon (TC) content and C/N were found in sheep and cattle manure composts, while higher EC, NH4-N, Total nitrogen (TN) and total phosphorus (TP) content were measured in pig and chicken manure composts. Paired clustering between herbivore and omnivore manure compost metataxonomy composition was also observed at both initial and final phases of composting. Despite this clear clustering, all communities changed drastically during the composting leading to reduced bacterial and fungal diversity and large shifts in community composition and species dominance. While Proteobacteria and Chloroflexi were the major phyla in sheep and cattle manure composts, Firmicutes dominated in pig and chicken manure composts. Together, our results indicate that feeding habits of livestock can determine the biochemical and biological properties of manures, having predictable effects on microbial community composition and assembly during composting. Manure metataxonomy profiles could thus potentially be used to steer and manage composting processes.

Siderophore-Mediated Interactions Determine the Disease Suppressiveness of Microbial Consortia.

Interactions between plant pathogens and root-associated microbes play an important role in determining disease outcomes. While several studies have suggested that steering these interactions may improve plant health, such approaches have remained challenging in practice. Because of low iron availability in most soils, competition for iron via secreted siderophore molecules might influence microbial interaction outcomes. Here, we tested if bacterial interactions mediated by iron-scavenging siderophores can be used to predict the disease suppressiveness of microbial consortia against soilborne Ralstonia solanacearum, a bacterial pathogen in the tomato rhizosphere. Iron availability significantly affected the interactions within inoculated consortia and between the consortia and the pathogen. We observed contrasting effects of siderophores and other nonsiderophore metabolites on the pathogen growth, while the siderophore effects were relatively much stronger. Specifically, disease incidence was reduced in vivo when the inoculated consortia produced siderophores that the pathogen could not use for its own growth. Employing siderophore-mediated interactions to engineer functionally robust microbial inoculants shows promise in protecting plants from soilborne pathogens.IMPORTANCE Soil-borne pathogens cause high losses in crop yields globally. The development of environmentally friendly approaches is urgently needed, but is often constrained by complex interactions between root-associated microbes and pathogens. Here, we demonstrate that the interactions within microbial consortia mediated by iron-scavenging siderophores play an important role in reducing pathogen infection and enhancing plant health. This study provides a promising and novel research direction for dealing with a wide range of microbial infections through iron exploitation, which is important for the colonization and infection of both plant and human hosts by pathogens.

Competition for iron drives phytopathogen control by natural rhizosphere microbiomes.

Plant pathogenic bacteria cause high crop and economic losses to human societies1-3. Infections by such pathogens are challenging to control as they often arise through complex interactions between plants, pathogens and the plant microbiome4,5. Experimental studies of this natural ecosystem at the microbiome-wide scale are rare, and consequently we have a poor understanding of how the taxonomic and functional microbiome composition and the resulting ecological interactions affect pathogen growth and disease outbreak. Here, we combine DNA-based soil microbiome analysis with in vitro and in planta bioassays to show that competition for iron via secreted siderophore molecules is a good predictor of microbe-pathogen interactions and plant protection. We examined the ability of 2,150 individual bacterial members of 80 rhizosphere microbiomes, covering all major phylogenetic lineages, to suppress the bacterium Ralstonia solanacearum, a global phytopathogen capable of infecting various crops6,7. We found that secreted siderophores altered microbiome-pathogen interactions from complete pathogen suppression to strong facilitation. Rhizosphere microbiome members with growth-inhibitory siderophores could often suppress the pathogen in vitro as well as in natural and greenhouse soils, and protect tomato plants from infection. Conversely, rhizosphere microbiome members with growth-promotive siderophores were often inferior in competition and facilitated plant infection by the pathogen. Because siderophores are a chemically diverse group of molecules, with each siderophore type relying on a compatible receptor for iron uptake8-12, our results suggest that pathogen-suppressive microbiome members produce siderophores that the pathogen cannot use. Our study establishes a causal mechanistic link between microbiome-level competition for iron and plant protection and opens promising avenues to use siderophore-mediated interactions as a tool for microbiome engineering and pathogen control.

Bacterial community richness shifts the balance between volatile organic compound-mediated microbe-pathogen and microbe-plant interactions.

Even though bacteria are important in determining plant growth and health via volatile organic compounds (VOCs), it is unclear how these beneficial effects emerge in multi-species microbiomes. Here we studied this using a model plant-bacteria system, where we manipulated bacterial community richness and composition and determined the subsequent effects on VOC production and VOC-mediated pathogen suppression and plant growth-promotion. We assembled VOC-producing bacterial communities in different richness levels ranging from one to 12 strains using three soil-dwelling bacterial genera (Bacillus, Paenibacillus and Pseudomonas) and investigated how the composition and richness of bacterial community affect the production and functioning of VOCs. We found that VOC production correlated positively with pathogen suppression and plant growth promotion and that all bacteria produced a diverse set of VOCs. However, while pathogen suppression was maximized at intermediate community richness levels when the relative amount and the number of VOCs were the highest, plant growth promotion was maximized at low richness levels and was only affected by the relative amount of plant growth-promoting VOCs. The contrasting effects of richness could be explained by differences in the amount and number of produced VOCs and by opposing effects of community productivity and evenness on pathogen suppression and plant-growth promotion along the richness gradient. Together, these results suggest that the number of interacting bacterial species and the structure of the rhizosphere microbiome drive the balance between VOC-mediated microbe-pathogen and microbe-plant interactions potentially affecting plant disease outcomes in natural and agricultural ecosystems.

A fast and easy method for predicting agricultural waste compost maturity by image-based deep learning.

Large amounts of agricultural wastes are generated in agricultural production, and composting this waste is one of the best ways to recycle resources. Compost maturity is an important criterion for measuring the quality of compost-products. Biochemical tests are conventional methods to evaluate compost maturity, but they are time consuming and difficult to perform. Therefore, convolutional neural networks (CNNs) were introduced to realize fast evaluation of compost maturity by analyzing images of different composting stages. Images of 3 different composting materials were collected to build 4 data sets, which included nearly 30,000 images, and a series of experiments were performed on them. The accuracy of proposed method was 99.7%, 99.4%, 99.7% and 99.5% on the 4 test sets, respectively. Experimental results demonstrate that the proposed CNN-based prediction model produces state of the art results and can be used to predict compost maturity during the composting process.

The change in microstructure of petioles and peduncles and transporter gene expression by potassium influences the distribution of nutrients and sugars in pear leaves and fruit.

Potassium (K) is one of the most important mineral nutrients required for fruit growth and development and is known as a 'quality element'. To investigate the role of K in more detail, we performed experiments in which seven-year-old pot-grown 'Huangguan' pear trees were treated with three levels of K (0, 0.4, or 0.8 g K2O kg-1 soil). K supply improved the development of vascular bundles in pear petioles and fruit peduncles and enhanced expression of genes involved in nutrients and sugar transport. Different from K and calcium (Ca), magnesium (Mg) concentrations in the leaves, petioles, and fruit peduncles were significantly higher under low K but lower under high K. However, the concentrations of K, Ca, and Mg in fruit all increased as more K was applied. Correspondingly, the expression of leaf Mg transporters (MRS2-1 and MRS2-3) increased under low K, indicating that Mg had an obvious compensation effect on K, while their expression decreased under medium and high K, showing that K had an obvious antagonistic effect on Mg. Except for NIPA2, the expressions of fruit K, Ca, and Mg transporters increased under high K, implying a synergistic effect among them in fruit. The concentration of sorbitol, sucrose, and total sugar in leaves and fruit at maturity significantly increased in response to the supply of K. The increase in sugar concentration was closely related to the up-regulated expression of sucrose transporter (SUT) and sorbitol transporter (SOT) genes. Together, these effects may promote the transport of nutrients and sugar from sources (leaves) to sinks (fruit) and increase the accumulation of sugar in the fruit.

Profiling of soil volatile organic compounds after long-term application of inorganic, organic and organic-inorganic mixed fertilizers and their effect on plant growth.

The complexity of soil processes involved in the production, consumption and accumulation of volatile organic compounds (VOCs) makes hard to access the overall dynamics of VOCs in the soil. In this study, the field soil, applied with inorganic (CF), organic (OF) and inorganic-organic mixed (CFOF) fertilizers for ten years was evaluated for the emission of VOCs at different temperature and moisture levels. We identified 30-50 soil emitted VOCs representing the most common soil VOCs groups by using the solid-phase microextraction (SPME) fiber and gas chromatography-mass spectroscopy. The highest total emission of VOCs was found in OF treatment, but it was non-significantly different with CF treatment. The emission of VOCs was significantly increased with the decrease in moisture contents and increase in the temperature of the soil. Among different fertilizer treatments, the emission of VOCs was significantly higher in OF treatment at 5% moisture, and in CF and OF treatments at 35°C. Further, the VOCs emitted from soil treated with CFOF showed the highest increase in plant growth while CF and OF treatments showed similar results. The VOCs were also extracted from the soil using methanol to better understand the dynamics of VOCs. The abundance of VOCs extracted from the soil was 44-61%, while the richness was 65-70% higher than the VOCs emitted from the soil in different treatments. Taken together the results of emitted and extracted VOCs from the soil, we conclude that the fertilizers are able to discriminate among the VOC patterns of soil. In addition, most of the VOCs are retained in the soil and the emission of VOCs from soil depends on the type of VOCs, soil properties and environmental conditions; however, more research is required to find out better soil VOCs analysis methods.

Parasites and competitors suppress bacterial pathogen synergistically due to evolutionary trade-offs.

Parasites and competitors are important for regulating pathogen densities and subsequent disease dynamics. It is, however, unclear to what extent this is driven by ecological and evolutionary processes. Here, we used experimental evolution to study the eco-evolutionary feedbacks among Ralstonia solanacearum bacterial pathogen, Ralstonia-specific phage parasite, and Bacillus amyloliquefaciens competitor bacterium in the laboratory and plant rhizosphere. We found that while the phage had a small effect on pathogen densities on its own, it considerably increased the R. solanacearum sensitivity to antibiotics produced by B. amyloliquefaciens. Instead of density effects, this synergy was due to phage-driven increase in phage resistance that led to trade-off with the resistance to B. amyloliquefaciens antibiotics. While no evidence was found for pathogen resistance evolution to B. amyloliquefaciens antibiotics, the fitness cost of adaptation (reduced growth) was highest when the pathogen had evolved in the presence of both parasite and competitor. Qualitatively similar patterns were found between laboratory and greenhouse experiments even though the evolution of phage resistance was considerably attenuated in the tomato rhizosphere. These results suggest that evolutionary trade-offs can impose strong constraints on disease dynamics and that combining phages and antibiotic-producing bacteria could be an efficient way to control agricultural pathogens.

Altering Transplantation Time to Avoid Periods of High Temperature Can Efficiently Reduce Bacterial Wilt Disease Incidence with Tomato.

Tomato bacterial wilt caused by Ralstonia solanacearum bacterium is a severe problem in Southern China, where relatively high environmental temperatures commonly prevails during the crop seasons. Previous research has indicated that bacterial wilt disease incidence generally increases during the warm months of summer leading to reduced tomato yield. Moreover, the efficacy of bio-organic fertilizers (BOFs)-organic compost fortified with pathogen-suppressive bacteria-is often lost during the periods of high environmental temperatures. Here we studied if the disease incidence could be reduced and the BOF performance enhanced by simply preponing and postponing the traditional seedling transplantation times to avoid tomato plant development during periods of high environmental temperature. To this end, a continuous, two-year field experiment was conducted to evaluate the performance of BOF in two traditional (late-spring [LS] and early-autumn [EA]) and two alternative (early-spring [ES] and late-autumn [LA]) crop seasons. We found that changing the transplantation times reduced the mean disease incidence from 33.9% (LS) and 54.7% (EA) to 11.1% (ES) and 7.1% (LA), respectively. Reduction in disease incidence correlated with the reduction in R. Solanacearum pathogen density in the tomato plant rhizosphere and stem base. Applying BOF during alternative transplantation treatments improved biocontrol efficiency from 43.4% (LS) and 3.1% (EA) to 67.4% (ES) and 64.8% (LA). On average, the mean maximum air temperatures were positively correlated with the disease incidence, and negatively correlated with the BOF biocontrol efficacy over the crop seasons. Crucially, even though preponing the transplantation time reduced the tomato yield in general, it was still economically more profitable compared to LS season due to reduced crop losses and relatively higher market prices. Preponing and postponing traditional tomato transplantation times to cooler periods could thus offer simple but effective way to control R. solanacearum disease outbreaks.

Functional diversity and properties of multiple xylanases from Penicillium oxalicum GZ-2.

A multiple xylanase system with high levels of xylanase activity produced from Penicillium oxalicum GZ-2 using agricultural waste as a substrate has been previously reported. However, the eco-physiological properties and origin of the multiplicity of xylanases remain unclear. In the present study, eight active bands were detected using zymography, and all bands were identified as putative xylanases using MALDI-TOF-MS/MS. These putative xylanases are encoded by six different xylanase genes. To evaluate the functions and eco-physiological properties of xylanase genes, xyn10A, xyn11A, xyn10B and xyn11B were expressed in Pichia pastoris. The recombinant enzymes xyn10A and xyn10B belong to the glycoside hydrolase (GH) family 10 xylanases, while xyn11A and xyn11B belong to GH11 xylanases. Biochemical analysis of the recombinant proteins revealed that all enzymes exhibited xylanase activity against xylans but with different substrate specificities, properties and kinetic parameters. These results demonstrated that the production of multiple xylanases in P. oxalicum GZ-2 was attributed to the genetic redundancy of xylanases and the post-translational modifications, providing insight into a more diverse xylanase system for the efficient degradation of complex hemicelluloses.

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.

Suppression of bacterial wilt of tomato by bioorganic fertilizer made from the antibacterial compound producing strain Bacillus amyloliquefaciens HR62.

Ralstonia solanacearum (Smith) is an important soil-borne pathogen worldwide. We investigated the effects of a new bioorganic fertilizer, BIO62, which was made from organic fertilizer and antagonist Bacillus amyloliquefaciens HR62, on the control of bacterial wilt of tomato in greenhouse condition. The results showed that the application of BIO62 significantly decreased disease incidence by 65% and strongly reduced R. solanacearum populations both in the rhizosphere soil (8.04 log cfu g(-1) dry soil) and crown sections (5.63 log cfu g(-1) fresh plant section) at 28 days after pathogen challenge. Antibacterial compounds produced by HR62 were purified by silica gel, Sephadex LH-20, and HPLC and then identified using HPLC/electrospray ionization mass spectrometry analysis. Macrolactin A and 7-O-malonyl macrolactin A (molecular weights of 402 and 488 Da, respectively), along with surfactin B (molecular weights of 994, 1008, 1022, and 1036 Da), were observed to inhibit the growth of R. solanacearum.

A new acidophilic endo-ß-1,4-xylanase from Penicillium oxalicum: cloning, purification, and insights into the influence of metal ions on xylanase activity.

A new acidophilic xylanase (XYN11A) from Penicillium oxalicum GZ-2 has been purified, identified and characterized. Synchronized fluorescence spectroscopy was used for the first time to evaluate the influence of metal ions on xylanase activity. The purified enzyme was identified by MALDI TOF/TOF mass spectrometry, and its gene (xyn11A) was identified as an open reading frame of 706 bp with a 68 bp intron. This gene encodes a mature protein of 196 residues with a predicted molecular weight of 21.3 kDa that has the 100 % identity with the putative xylanase from the P. oxalicum 114-2. The enzyme shows a structure comprising a catalytic module family 10 (GH10) and no carbohydrate-binding module family. The specific activities were 150.2, 60.2, and 72.6 U/mg for beechwood xylan, birchwood xylan, and oat spelt xylan, respectively. XYN11A exhibited optimal activity at pH 4.0 and remarkable pH stability under extremely acidic condition (pH 3). The specific activity, K m and V max values were 150.2 U/mg, 30.7 mg/mL, and 403.9 µmol/min/mg for beechwood xylan, respectively. XYN11A is a endo-ß-1,4-xylanase since it release xylobiose and xylotriose as the main products by hydrolyzing xylans. The activity of XYN11A was enhanced 155 % by 1 mM Fe(2+) ions, but was inhibited strongly by Fe(3+). The reason of enhancing the xylanase activity of XYN11A with 1 mM Fe(2+) treatment may be responsible for the change of microenvironment of tryptophan residues studied by synchronous fluorescence spectrophotometry. Inhibition of the xylanase activity by Fe(3+) was first time demonstrated to associate tryptophan fluorescence quenching.