Antifungal characterizations of a novel endo-ß-1,6-glucanase from Flavobacterium sp. NAU1659.

ß-1,6-Glucan plays a crucial role in fungal cell walls by linking the outer layer of mannoproteins and the inner layer of ß-1,3-glucan, contributing significantly to the maintenance of cell wall rigidity. Therefore, the hydrolysis of ß-1,6-glucan by ß-1,6-glucanase directly leads to the disintegration of the fungal cell wall. Here, a novel ß-1,6-glucanase FlGlu30 was identified from the endophytic Flavobacterium sp. NAU1659 and heterologously expressed in Escherichia coli BL21 (DE3). The optimal reaction conditions of purified FlGlu30 were 50℃ and pH 6.0, resulting in a specific activity of 173.1 U/mg using pustulan as the substrate. The hydrolyzed products of FlGlu30 to pustulan were mainly gentianose within 1 h of reaction. With the extension of reaction time, gentianose was gradually hydrolyzed to glucose, indicating that FlGlu30 is an endo-ß-1,6-glucanase. The germination of Magnaporthe oryzae Guy11 spores could not be inhibited by FlGlu30, but the appressorium formation of spores was completely inhibited under the concentration of 250.0 U/mL FlGlu30. The disruptions of cell wall and accumulation of intracellular reactive oxide species (ROS) were observed in FlGlu30-treated M. oryzae Guy11 cells, suggesting the significant importance of ß-1,6-glucan as a potential antifungal target and the potential application of FlGlu30. KEY POINTS: • ß-1,6-Glucan is a key component maintaining the rigid structure of fungal cell wall. • ß-1,6-Glucanase is an antifungal protein with significant potential applications. • FlGlu30 is the first reported ß-1, 6-glucanase derived from Flavobacterium.

Biochemical characterization and antifungal activity of a recombinant ß-1,3-glucanase FlGluA from Flavobacterium sp. NAU1659.

ß-1,3-glucanases can degrade ß-1,3-glucoside bonds in ß-glucan which is the main cell-wall component of most of fungi, and have the crucial application potential in plant protection and food processing. Herein, a ß-1,3-glucanase FlGluA from Flavobacterium sp. NAU1659 composed of 333 amino acids with a predicted molecular mass of 36.6 kDa was expressed in Escherichia coli BL21, purified and characterized. The deduced amino acid sequence of FlGluA showed the high identity with the ß-1,3-glucanase belonging to glycoside hydrolase (GH) family 16. Enzymological characterization indicated FlGluA had the highest activity on zymosan A, with a specific activity of 3.87 U/mg, followed by curdlan (1.16 U/mg) and pachymaran (0.88 U/mg). It exhibited optimal catalytic activity at the pH 5.0 and 40 °C, and was stable when placed at 4 °C for 12 h in the range of pH 3.0-8.0 or at a temperature below 50 °C for 3 h. Its catalytic activity was enhanced by approximately 36 % in the presence of 1 mM Cr3+. The detection of thin-layer chromatography and mass spectrometry showed FlGluA hydrolyzed zymosan A mainly to glucose and disaccharide, and trace amounts of tetrasaccharide and pentasaccharide, however, it had no action on laminaribiose, indicating its endo-ß-1,3-glucanase activity. The mycelium growth of F. oxysporum treated by FlGluA was inhibited, with approximately 37 % of inhibition rate, revealing the potential antifungal activity of the enzyme. These results revealed the hydrolytic properties and biocontrol activity of FlGluA, laying a crucial foundation for its potential application in agriculture and industry.

Functional analysis of a novel endo-ß-1,6-glucanase MoGlu16 and its application in detecting cell wall ß-1,6-glucan of Magnaporthe oryzae.

As an essential component of the fungal cell wall, ß-1,6-glucan has an important role in the growth and development of fungi, but its distribution has not been investigated in Magnaporthe oryzae. Here, a novel ß-1,6-glucanase from M. oryzae, MoGlu16, was cloned and expressed in Pichia pastoris. The enzyme was highly active on pustulan, with a specific activity of 219.0 U/mg at pH 5.0 and 50°C, and showed great selectivity for continuous ß-1,6-glycosidic bonding polysaccharides. Based on this, ß-1,6-glucan was selectively visualized in the vegetative hyphae, conidia and bud tubes of M. oryzae using a hydrolytically inactive GFP-tagged MoGlu16 with point mutations at the catalytic position (His-MoGlu16E236A-Gfp). The spore germination and appressorium formation were significantly inhibited after incubation of 105/ml conidia with 0.03 µg/µl MoGlu16. Mycelia treated with MoGlu16 produced reactive oxygen species and triggered the cell wall integrity pathway, increasing the expression levels of genes involved in cell wall polysaccharide synthesis. These results revealed that MoGlu16 participated in the remodeling of cell wall in M. oryzae, laying a foundation for the analysis of cell wall structure.

A novel xylanase from a myxobacterium triggers a plant immune response in Nicotiana benthamiana.

Xylanases derived from fungi, including phytopathogenic and nonpathogenic fungi, are commonly known to trigger plant immune responses. However, there is limited research on the ability of bacterial-derived xylanases to trigger plant immunity. Here, a novel xylanase named CcXyn was identified from the myxobacterium Cystobacter sp. 0969, which displays broad-spectrum activity against both phytopathogenic fungi and bacteria. CcXyn belongs to the glycoside hydrolases (GH) 11 family and shares a sequence identity of approximately 32.0%-45.0% with fungal xylanases known to trigger plant immune responses. Treatment of Nicotiana benthamiana with purified CcXyn resulted in the induction of hypersensitive response (HR) and defence responses, such as the production of reactive oxygen species (ROS) and upregulation of defence gene expression, ultimately enhancing the resistance of N. benthamiana to Phytophthora nicotianae. These findings indicated that CcXyn functions as a microbe-associated molecular pattern (MAMP) elicitor for plant immune responses, independent of its enzymatic activity. Similar to fungal xylanases, CcXyn was recognized by the NbRXEGL1 receptor on the cell membrane of N. benthamiana. Downstream signalling was shown to be independent of the BAK1 and SOBIR1 co-receptors, indicating the involvement of other co-receptors in signal transduction following CcXyn recognition in N. benthamiana. Moreover, xylanases from other myxobacteria also demonstrated the capacity to trigger plant immune responses in N. benthamiana, indicating that xylanases in myxobacteria are ubiquitous in triggering plant immune functions. This study expands the understanding of xylanases with plant immune response-inducing properties and provides a theoretical basis for potential applications of myxobacteria in biocontrol strategies against phytopathogens.

Antifungal Activity and Mechanisms of 2-Ethylhexanol, a Volatile Organic Compound Produced by Stenotrophomonas sp. NAU1697, against Fusarium oxysporum f. sp. cucumerinum.

Researchers often consider microorganisms from Stenotrophomonas sp. to be beneficial for plants. In this study, the biocidal effects and action mechanisms of volatile organic compounds (VOCs) produced by Stenotrophomonas sp. NAU1697 were investigated. The mycelial growth and spore germination of Fusarium oxysporum f. sp. cucumerinum (FOC), which is a pathogen responsible for cucumber wilt disease, were significantly inhibited by VOCs emitted from NAU1697. Among the VOCs, 33 were identified, 11 of which were investigated for their antifungal properties. Among the tested compounds, 2-ethylhexanol exhibited the highest antifungal activity toward FOC, with a minimum inhibitory volume (MIV) of 3.0 µL/plate (equal to 35.7 mg/L). Damage to the hyphal cell wall and cell membrane integrity caused a decrease in the ergosterol content and a burst of reactive oxygen species (ROS) after 2-ethylhexanol treatment. DNA damage, which is indicative of apoptosis-like cell death, was monitored in 2-ethylhexanol-treated FOC cells by using micro-FTIR analysis. Furthermore, the activities of mitochondrial dehydrogenases and mitochondrial respiratory chain complex III in 2-ethylhexanol-treated FOC cells were significantly decreased. The transcription levels of genes associated with redox reactions and the cell wall integrity (CWI) pathway were significantly upregulated, thus indicating that stress was caused by 2-ethylhexanol. The findings of this research provide a new avenue for the sustainable management of soil-borne plant fungal diseases.

Characterization of a multi-domain exo-ß-1,3-galactanase from Paenibacillus xylanexedens.

ß-1,3-Galactanases selectively degrade ß-1,3-galactan, thus it is an attractive enzyme technique to map high-galactan structure and prepare galactooligosaccharides. In this work, a gene encoding exo-ß-1,3-galactanase (PxGal43) was screened form Paenibacillus xylanexedens, consisting of a GH43 domain, a CBM32 domain and α-L-arabinofuranosidase B (AbfB) domain. Using ß-1,3-galactan (AG-II-P) as substrate, the recombined enzyme expressed in Escherichia coli BL21 (DE3) exhibited an optimal activity at pH 7.0 and 30 °C. The enzyme was thermostable, retaining >70 % activity after incubating at 50 °C for 2 h. In addition, it showed high tolerance to various metal ions, denaturants and detergents. Substrate specificity indicated that PxGal43 hydrolysis only ß-1,3-linked galactosyl oligosaccharides and polysaccharides, releasing galactose as an exo-acting manner. The function of the CBM32 and AbfB domain was revealed by their sequential deletion and suggested that their connection to the catalytic domain was crucial for the oligomerization, catalytic activity, substrate binding and thermal stability of PxGal43. The substrate docking and site-directed mutagenesis proposed that Glu191, Gln244, Asp138 and Glu81 served as the catalytic acid, catalytic base, pKa modulator, and substrate identifier in PxGal43, respectively. These results provide a better understanding and optimization of multi-domain bacterial GH43 ß-1,3-galactanase for the degradation of arabinogalactan.

Excellent Room Temperature Thermoelectric Performance in Mg3Sb2-Based Alloys via Multi-Functional Doping of Nb.

Mg3Sb2-based alloys are attracting increasing attention due to the excellent room temperature thermoelectric properties. However, due to the presence and easy segregation of charged Mg vacancies, the carrier mobility in Mg3Sb2-based alloys is always severely compromised that significantly restricts the room temperature performance. General vacancy compensation strategies cannot synergistically optimize the complicated Mg3Sb2 structures involving both interior and boundary scattering. Herein, due to the multi-functional doping effect of Nb, the electron scattering inside and across grains is significantly suppressed by inhibiting the accumulation of Mg vacancies, and leading to a smooth transmission channel of electrons. The increased Mg vacancies migration barrier and optimized interface potential are also confirmed theoretically and experimentally, respectively. As a result, a leading room temperature zT of 1.02 is achieved. This work reveals the multi-functional doping effect as an efficient approach in improving room temperature thermoelectric performance in complicated defect/interface associated Mg3Sb2-based alloys.

Heterologous Expression and Characterization of a Novel Mesophilic Maltogenic α-Amylase AmyFlA from Flavobacterium sp. NAU1659.

A novel amylase AmyFlA from Flavobacterium sp. NAU1659, AmyFlA, was cloned and expressed in Esherichia coli. Based on phylogenetic and functional analysis, it was identified as a novel member of the subfamily GH13_46, sharing high sequence identity. The protein was predicted to consist of 620 amino acids, with a putative signal peptide of 25 amino acids. The enzyme was able to hydrolyze soluble starch with a specific activity of 352.97 U/mg at 50 °C in 50 mM phosphate buffer (pH 6.0). The Km and Vmax values of AmyFlA were respectively 3.15 mg/ml and 566.36 µmol·ml-1·min-1 under optimal conditions. Its activity towards starch was enhanced by 63% in the presence of 1 mM Ca2+, indicating that AmyFlA was a Ca2+-dependent amylase. Compared to the reported maltogenic amylases, AmyFlA produced a lower variety of intermediate oligosaccharides at the start of the reaction so that the product mixture contained a higher proportion of maltose. These results indicate that AmyFlA may be potential application value in the production of high-maltose syrup.

Genome analysis of a plasmid-bearing myxobacterim Myxococcus sp. strain MxC21 with salt-tolerant property.

Myxobacteria are widely distributed in various habitats of soil and oceanic sediment. However, it is unclear whether soil-dwelling myxobacteria tolerate a saline environment. In this study, a salt-tolerant myxobacterium Myxococcus sp. strain MxC21 was isolated from forest soil with NaCl tolerance >2% concentration. Under 1% salt-contained condition, strain MxC21 could kill and consume bacteria prey and exhibited complex social behaviors such as S-motility, biofilm, and fruiting body formation but adopted an asocial living pattern with the presence of 1.5% NaCl. To investigate the genomic basis of stress tolerance, the complete genome of MxC21 was sequenced and analyzed. Strain MxC21 consists of a circular chromosome with a total length of 9.13 Mbp and a circular plasmid of 64.3 kb. Comparative genomic analysis revealed that the genomes of strain MxC21 and M. xanthus DK1622 share high genome synteny, while no endogenous plasmid was found in DK1622. Further analysis showed that approximately 21% of its coding genes from the genome of strain MxC21 are predominantly associated with signal transduction, transcriptional regulation, and protein folding involved in diverse niche adaptation such as salt tolerance, which enables social behavior such as gliding motility, sporulation, and predation. Meantime, a high number of genes are also found to be involved in defense against oxidative stress and production of antimicrobial compounds. All of these functional genes may be responsible for the potential salt-toleration. Otherwise, strain MxC21 is the second reported myxobacteria containing indigenous plasmid, while only a small proportion of genes was specific to the circular plasmid of strain MxC21, and most of them were annotated as hypothetical proteins, which may have a direct relationship with the habitat adaptation of strain MxC21 under saline environment. This study provides an inspiration of the adaptive evolution of salt-tolerant myxobacterium and facilitates a potential application in the improvement of saline soil in future.

Identification and Characterization of Novel Malto-Oligosaccharide-Forming Amylase AmyCf from Cystobacter sp. Strain CF23.

Malto-oligosaccharides (MOSs) from starch conversion is advantageous for food and pharmaceutical applications. In this study, an efficient malto-oligosaccharide-forming α-amylase AmyCf was identified from myxobacter Cystobacter sp. strain CF23. AmyCf is composed of 417 amino acids with N-terminal 41 amino acids as the signal peptide, and conserved glycoside hydrolase family 13 (GH13) catalytic module and predicted C-terminal domain with ß-sheet structure are also identified. Phylogenetic and functional analysis demonstrated that AmyCf is a novel member of GH13_6 subfamily. The special activity of AmyCf toward soluble starch and raw wheat starch is 9249 U/mg and 11 U/mg, respectively. AmyCf has broad substrate specificity toward different types of starches without requiring Ca2+. Under ideal circumstances of 60 °C and pH 7.0, AmyCf hydrolyzes gelatinized starch into maltose and maltotriose and maltotetraose as the main hydrolytic products with more than 80% purity, while maltose and maltotriose are mainly produced from the hydrolysis of raw wheat starch with more than 95% purity. The potential applicability of AmyCf in starch processing is highlighted by its capacity to convert gelatinized starch and raw starch granules into MOSs. This enzymatic conversion technique shows promise for the low-temperature enzymatic conversion of raw starch.

Myxobacteria restrain Phytophthora invasion by scavenging thiamine in soybean rhizosphere via outer membrane vesicle-secreted thiaminase I.

Public metabolites such as vitamins play critical roles in maintaining the ecological functions of microbial community. However, the biochemical and physiological bases for fine-tuning of public metabolites in the microbiome remain poorly understood. Here, we examine the interactions between myxobacteria and Phytophthora sojae, an oomycete pathogen of soybean. We find that host plant and soil microbes complement P. sojae's auxotrophy for thiamine. Whereas, myxobacteria inhibits Phytophthora growth by a thiaminase I CcThi1 secreted into extracellular environment via outer membrane vesicles (OMVs). CcThi1 scavenges the required thiamine and thus arrests the thiamine sharing behavior of P. sojae from the supplier, which interferes with amino acid metabolism and expression of pathogenic effectors, probably leading to impairment of P. sojae growth and pathogenicity. Moreover, myxobacteria and CcThi1 are highly effective in regulating the thiamine levels in soil, which is correlated with the incidence of soybean Phytophthora root rot. Our findings unravel a novel ecological tactic employed by myxobacteria to maintain the interspecific equilibrium in soil microbial community.

Structural and Functional Properties of Porous Corn Starch Obtained by Treating Raw Starch with AmyM.

Porous starch is attracting considerable attention for its high surface area and shielding ability, properties which are useful in many food applications. In this study, native corn starch with 15, 25, and 45% degrees of hydrolysis (DH-15, DH-25, and DH-45) were prepared using a special raw starch-digesting amylase, AmyM, and their structural and functional properties were evaluated. DH-15, DH-25, and DH-45 exhibited porous surface morphologies, diverse pore size distributions and pore areas, and their adsorptive capacities were significantly enhanced by improved molecular interactions. Structural measures showed that the relative crystallinity decreased as the DH increased, while the depolymerization of starch double helix chains promoted interactions involving disordered chains, followed by chain rearrangement and the formation of sub-microcrystalline structures. In addition, DH-15, DH-25, and DH-45 displayed lower hydrolysis rates, and DH-45 showed a decreased C∞ value of 18.9% with higher resistant starch (RS) content and lower glucose release. Our results indicate that AmyM-mediated hydrolysis is an efficient pathway for the preparation of porous starches with different functionalities which can be used for a range of applications.

Toward Energy-Efficient Routing of Multiple AGVs with Multi-Agent Reinforcement Learning.

This paper presents a multi-agent reinforcement learning (MARL) algorithm to address the scheduling and routing problems of multiple automated guided vehicles (AGVs), with the goal of minimizing overall energy consumption. The proposed algorithm is developed based on the multi-agent deep deterministic policy gradient (MADDPG) algorithm, with modifications made to the action and state space to fit the setting of AGV activities. While previous studies overlooked the energy efficiency of AGVs, this paper develops a well-designed reward function that helps to optimize the overall energy consumption required to fulfill all tasks. Moreover, we incorporate the e-greedy exploration strategy into the proposed algorithm to balance exploration and exploitation during training, which helps it converge faster and achieve better performance. The proposed MARL algorithm is equipped with carefully selected parameters that aid in avoiding obstacles, speeding up path planning, and achieving minimal energy consumption. To demonstrate the effectiveness of the proposed algorithm, three types of numerical experiments including the ϵ-greedy MADDPG, MADDPG, and Q-Learning methods were conducted. The results show that the proposed algorithm can effectively solve the multi-AGV task assignment and path planning problems, and the energy consumption results show that the planned routes can effectively improve energy efficiency.

Quality Characteristics of Reduced-Fat Emulsified Sausages Made with Yeast Mannoprotein Enzymatically Prepared with a ß-1,6-glucanase.

Mannoproteins, as yeast polysaccharides, have been utilized in food the industry as dietary fibers, emulsifying agents or fat replacers. Mannoprotein MP112, produced from yeast by enzymatic hydrolysis of myxobacterial ß-1,6-glucanase GluM, exhibits excellent emulsifying properties in emulsion preparation. In this study, we aimed to examine the application of stable emulsion with the addition of mannoprotein MP112 (MP112 emulsion) to reduce the fat content of sausages. The addition of MP112 emulsion in emulsified sausages significantly reduced the fat content and increased the moisture and protein contents of emulsified sausages without the expense of their good sensory quality. Moreover, the textural properties of sausages were markedly improved with the higher hardness, chewiness and cohesiveness, especially in the 50-75% replacement ratio of MP112 emulsion. On the other hand, MP112 emulsion replacement of animal fat markedly improved the nutritional composition of emulsified sausages; they displayed a higher PUFA/SFA ratio and lower n-6/n-3 ratio due to their saturated fatty acids being replaced by poly-unsaturated fatty acids. Meanwhile, the oxidative stability of sausages was improved linearly, corresponding to the increased replacement ratio of MP112 emulsion. Our results show that mannoprotein-based emulsions could be used as potential fat alternatives in developing reduced-fat meat products.

Insights into the Antifungal Properties of Myxobacteria Outer Membrane ß-1,6-Glucanase.

Fungal cell wall decomposition enzymes exhibit great potential for the development of efficient antifungal agents. However, their practical application is restricted due to incomplete understanding of the action mode. In our previous study, we identified that a novel outer membrane (OM) ß-1,6-glucanase GluM is deployed by predatory myxobacteria to feed on fungi. In this work, we provide deep insights into the antifungal mechanism of ß-1,6-glucanase and its potential in improving plant disease resistance. The fungal cell wall decomposition ability of GluM resulted in irregular hyphae morphology, changed chitin distribution, increased membrane permeability, and leakage of cell constituents in Magnaporthe oryzae Guy11. Under the attack pattern, the cell wall integrity pathway was activated by strain Guy11 for self-protection. GluM exhibited a distinct endo-model toward fungal cell wall; the favorite substrate of GluM toward fungal ß-1,6-glucan may give reason for its efficient antifungal activity compared with Trichoderma ß-1,6-glucanase. Moreover, released glucans from GluM hydrolysis of fungal cell wall functioned as an elicitor and induced rice immunity by means of jasmonic acid pathway. Based on the dual roles of antifungal properties, gluM transgenic plants conferred enhanced resistance against fungal infection.

Predation of oomycetes by myxobacteria via a specialized CAZyme system arising from adaptive evolution.

As social micropredators, myxobacteria are studied for their abilities to prey on bacteria and fungi. However, their predation of oomycetes has received little attention. Here, we show that Archangium sp. AC19 secretes a carbohydrate-active enzyme (CAZyme) cocktail during predation on oomycetes Phytophthora. These enzymes include three specialized ß-1,3-glucanases (AcGlu13.1, -13.2 and -13.3) that act as a cooperative consortium to target ß-1,3-glucans of Phytophthora. However, the CAZymes showed no hydrolytic effects on fungal cells, even though fungi contain ß-1,3-glucans. Heterologous expression of AcGlu13.1, -13.2 or -13.3 enzymes in Myxococcus xanthus DK1622, a model myxobacterium that antagonizes but does not predate on P. sojae, conferred a cooperative and mycophagous ability that stably maintains myxobacteria populations as a mixture of engineered strains. Comparative genomic analyses suggest that these CAZymes arose from adaptive evolution among Cystobacteriaceae myxobacteria for a specific prey killing behavior, whereby the presence of Phytophthora promotes growth of myxobacterial taxa by nutrient release and consumption. Our findings demonstrate that this lethal combination of CAZymes transforms a non-predatory myxobacterium into a predator with the ability to feed on Phytophthora, and provides new insights for understanding predator-prey interactions. In summary, our work extends the repertoire of myxobacteria predatory strategies and their evolution, and suggests that these CAZymes can be engineered as a functional consortium into strains for biocontrol of Phytophothora diseases and hence crop protection.

Realizing Excellent Structural and Thermoelectric Performance in Mg3Sb2-Based Alloys by Manipulating Mg Intrinsic Migration Kinetics with Interstitial Ni Doping.

N-type Mg3Sb2 is attracting increasing focus for its outstanding room-temperature (RT) thermoelectric (TE) performance; however, achieving reliable n-type conduction remains challenging due to negatively charged Mg vacancies. Doping with compensation charges is generally used but does not fundamentally resolve the high intrinsic activity and easy formation of Mg vacancies. Herein, a robust structural and thermoelectric performance is obtained by manipulating Mg intrinsic migration activity by precisely incorporating Ni at the interstitial site. Density functional theory (DFT) indicates that a strong performance originates from a significant thermodynamic preference for Ni occupying the interstitial site across the complete Mg-poor to -rich window, which dramatically promotes the Mg migration barrier and kinetically immobilizes Mg. As a result, the detrimental vacancy-associated ionized scattering is eliminated with a leading room-temperature ZT up to 0.85. This work reveals that interstitial occupation in Mg3Sb2-based materials is a novel approach promoting both structural and thermoelectric performance.

Myxobacterial Outer Membrane ß-1,6-Glucanase Induced the Cell Death of Fusarium oxysporum by Destroying the Cell Wall Integrity.

The ß-1,6-glucan is the key linker between mannoproteins in the outermost part of the cell wall and ß-1,3-glucan/chitin polysaccharide to maintain the rigid structure of the cell wall. The ß-1,6-glucanase GluM, which was purified from the fermentation supernatant of Corallococcus sp. EGB, was able to inhibit the germination of Fusarium oxysporum f. sp. cucumerinum conidia at a minimum concentration of 2.0 U/mL (0.08 µg/mL). The survival rates of GluM-treated conidia and monohyphae were 10.4% and 30.7%, respectively, which were significantly lower than that of ß-1,3-glucanase treatment (Zymolyase, 20.0 U/mL; equate to 1.0 mg/mL) (72.9% and 73.9%). In contrast to ß-1,3-glucanase treatment, the high-osmolarity glycerol (HOG) pathway of F. oxysporum f. sp. cucumerinum cells was activated after GluM treatment, and the intracellular glycerol content was increased by 2.6-fold. Moreover, the accumulation of reactive oxygen species (ROS) in F. oxysporum f. sp. cucumerinum cells after GluM treatment induced apoptosis, but it was not associated with the increased intracellular glycerol content. Together, the results indicate that ß-1,6-glucan is a promising target for the development of novel broad-spectrum antifungal agents. IMPORTANCE Phytopathogenic fungi are the most devastating plant pathogens in agriculture, causing enormous economic losses to global crop production. Biocontrol agents have been promoted as replacements to synthetic chemical pesticides for sustainable agriculture development. Cell wall-degrading enzymes (CWDEs), including chitinases and ß-1,3-glucanases, have been considered as important armaments to damage the cell wall. Here, we found that F. oxysporum f. sp. cucumerinum is more sensitive to ß-1,6-glucanase GluM treatment (0.08 µg/mL) than ß-1,3-glucanase Zymolyase (1.0 mg/mL). The HOG pathway was activated in F. oxysporum f. sp. cucumerinum cells after GluM treatment, and the intracellular glycerol content was significantly increased. Moreover, the decomposition of F. oxysporum f. sp. cucumerinum cell wall by GluM induced the burst of intracellular ROS and apoptosis, which eventually leads to cell death. Therefore, we suggest that the ß-1,6-glucan of the fungal cell wall may be a better antifungal target compared to the ß-1,3-glucan.

Phylogenetic diversity of stochasticity-dominated predatory myxobacterial community drives multi-nutrient cycling in typical farmland soils.

Predatory myxobacteria are keystone taxa in the soil microbial food web that potentially regulate soil microbial community structure and ecosystem functions. However, little is known about the community assembly processes of myxobacteria in typical farmland soils over large geographic scales, in addition to their relationship with soil multi-nutrient cycling. Here, we used high-throughput sequencing techniques and phylogenetic null modeling analysis to investigate the distribution patterns and assembly processes of myxobacteria communities, in addition to interactions between myxobacteria communities and soil multi-nutrient cycling. Anaeromyxobacter (28.5 %) and Haliangium (19.6 %) were the dominant myxobacteria genera in all samples, and myxobacteria community similarities exhibited distinct distance-decay relationships. Stochastic processes (~77.8 %) were the dominant ecological processes driving the assembly of predatory myxobacteria communities over large geographical scales and under three fertilization regimes. Myxobacteria community structure was influenced by geographic factors (location and climate), soil factors (soil pH, soil organic carbon, total nitrogen, and total potassium), and fertilization, with myxobacteria community assembly being more sensitive to geographic factors. Organic-inorganic combined fertilization (NPKM) increased the proportions of deterministic processes in myxobacteria community assembly. Moreover, myxobacteria community assembly and diversity were closely associated with soil multi-nutrient cycling. Hence, myxobacteria phylogenetic α-diversity represented by NTI index is a potential bioindicators for soil multi-nutrient cycling. Overall, our findings comprehensively reveal the mechanisms of assembly of myxobacteria communities in soils over large geographic scales, and provide a theoretical basis for further research on the role of predatory bacteria on soil nutrient cycling in agro-ecosystems.

Preparation of chitooligosaccharides with a low degree of polymerization and anti-microbial properties using the novel chitosanase AqCsn1.

Chitosanases hydrolyze chitosan into chitooligosaccharides (COSs) with various biological activities, which are widely employed in many areas including plant disease management. In this study, the novel chitosanase AqCsn1 belonging to the glycoside hydrolase family 46 (GH46) was cloned from Aquabacterium sp. A7-Y and heterologously expressed in Escherichia coli BL21 (DE3). AqCsn1 displayed the highest hydrolytic activity towards chitosan with 95% degree of deacetylation at 40 °C and pH 5.0, with a specific activity of 13.18 U/mg. Product analysis showed that AqCsn1 hydrolyzed chitosan into (GlcN)2 and (GlcN)3 as the main products, demonstrating an endo-type cleavage pattern. Evaluation of antagonistic activity showed that the hydrolysis products of AqCsn1 suppress the mycelial growth of Magnaporthe oryzae and Phytophthora sojae in a concentration-dependent manner, and the inhibition rate of P. sojae reached 39.82% at a concentration of 8 g/L. Our study demonstrates that AqCsn1 and hydrolysis products with a low degree of polymerization might have potential applications in the biological control of agricultural diseases.