[Objective] To screen and identify the plant growth-promoting bacterial strain with saline-alkali tolerance and evaluate its functions. [Methods] The rhizosphere soil of wheat was collected from different saline-alkali regions, and the highly efficient saline-alkali-tolerant bacterial strain was isolated by enrichment under saline-alkali condition and dilution coating. The antifungal spectrum and plant growth-promoting effects of the isolate were evaluated in vitro, and the nitrogenase activity was measured by an ELISA kit. The strain was identified based on the morphological, physiological, biochemical characteristics and phylogenetic analysis. The effect of the strain on wheat growth under salt stress was investigated by a pot experiment. [Results] A highly abundant bacterium with saline-alkali tolerance was enriched from rhizosphere soil and designated as TaRb44, which could grow normally under 3% NaCl and pH 10.0. Strain TaRb44 showed strong antagonism against soil-borne pathogenic fungi such as Fusarium pseudograminearum causing wheat crown rot, Fusarium oxysporum f. sp. niveum causing watermelon Fusarium wilt, and Fusarium oxysporum f. sp. cubense causing banana wilt, as well as Botrytis cinerea causing gray mold of postharvest tomatoes. Further tests showed that strain TaRb44 produced a variety of plant growth-promoting substances such as siderophores, amylase, and cellulase, and it had nitrogen fixation activity with the nitrogenase level of 65.50 U/L. Finally, the strain TaRb44 was identified as Paenibacillus polymyxa, which significantly promoted the growth of wheat seedlings and increased the root biomass under both salt stress and non-salt stress conditions. [Conclusion] In this study, P. polymyxa TaRb44 was obtained with much excellent properties such as disease prevention, plant growth promotion, and saline-alkali tolerance. It serves as an elite strain for developing new microbial fertilizer and soil amendments in the future.

[Objective] To screen out a strain that can degrade lignocellulose for the application of lignocellulose deposited by delayed harvest of alfalfa under inappropriate climate and mechanical matching and provide strain resources for the efficient use of alfalfa. [Methods] The primary screening was carried out by culture with alkali lignin and sodium carboxymethyl cellulose. Based on color changes and fading circles, the strain with the ability to secrete ligninase and cellulase was screened out. The target strain was identified by 16S rRNA gene sequencing and whole genome sequencing, and then Kyoto encyclopedia of genes and genomes (KEGG) and carbohydrate-active enzymes (CAZy) were employed to annotate gene functions based on the whole genome sequence. The microstructure of the alfalfa stems degraded by the strain was observed by scanning electron microscopy. The nutrient and microbial community changes in alfalfa hay treated with the strain were evaluated. [Results] A strain S1 producing ligninase and cellulase was identified as Bacillus cereus by whole genome sequencing. A total of 305 genes involved in carbohydrate metabolism were annotated, including 139 genes encoding CAZy. After treatment with this strain, the microstructure of vascular bundles in alfalfa stems changed significantly. The crude protein content in alfalfa hay increased, while the lignin, neutral detergent fiber, and acid detergent fiber content and the alpha-diversity of microorganisms decreased over time. [Conclusion] The screened strain identified as B. cereus demonstrates a robust ability to degrade lignocellulose.

[Objective] To mine the macrofungal strains capable of efficiently degrading straw at low temperatures, thereby improving the utilization efficiency of straw resources, we measured the lignocellulose degradation abilities of 955 macrofungal strains. [Methods] First, we employed the plate method to screen the strains with carboxymethyl cellulase, xylanase, and laccase activities. Then, we carried out a filter paper degradation test to screen the cellulose-degrading strains. Finally, we performed liquid fermentation with the selected strains and measured their enzyme activities on days three, six, nine, and 12 to identify the dominant strains with strong lignocellulose-degrading abilities. [Results] We identified 11 macrofungal strains exhibiting strong lignocellulose degradation capabilities at a low temperature (15 ℃). The 11 strains were Trametes suaveolens, Irpex lacteus, Crucibulum laeve, Stereum hirsutum, Pleurotus ostreatus, Phlebia acerina, Agaricus xanthodermus, Neofomitella fumosipora, Pholiota multicingulata, Abortiporus biennis, and Armillaria cepistipes. Notably, C. laeve, A. xanthodermus, and P. multicingulata were newly reported for their high lignocellulose-degrading abilities. The maximum activities of carboxymethyl cellulase, xylanase, and laccase in the 11 strains reached 262.31, 91.03, and 196.50 U/mL, respectively. T. suaveolens exhibited carboxymethyl cellulase activity of 168.17 U/mL at 15 ℃, which was significantly higher than that (67.88 U/mL) observed at room temperature. P. ostreatus showed the carboxymethyl cellulase activity of 150.78 U/mL and the laccase activity of 154.32 U/mL. S. hirsutum achieved the laccase activity of 63.27 U/mL at 15 ℃, which was twice the level measured at room temperature. [Conclusion] We successfully identified 11 macrofungal strains with strong lignocellulose-degrading abilities at 15 ℃. The findings provide valuable microbial resources for the degradation of lignocellulose in cold regions and lay a theoretical basis for application of these strains in low-temperature industries.

[Objective] This study isolated and identified an algicidal bacterium from the East China Sea near Wenzhou and investigated its algicidal characteristics and mechanisms, aiming to contribute a solid scientific basis to the microbial control against red tides. [Methods] The strain was identified by morphological observation, physiological and biochemical tests, and 16S rRNA gene sequence analysis. The algicidal characteristics such as algicidal activity, influences of environmental factors on the algicidal activity, and algicidal specificity were assessed. The algicidal mechanism was explored by electron microscopy, measurements of photosynthetic parameters, and determination of reactive oxygen species (ROS) and malondialdehyde levels and antioxidant enzyme activities. [Results] The algicidal strain J75 was identified as Pseudoalteromonas sp. The algicidal rate of strain J75 against Skeletonemacostatum reached 95.97% within 36 h. Strain J75 induced lysis of algal cells indirectly by secreting extracellular algicidal substances, maintaining the algicidal activity across broad ranges of temperatures (-20 °C to 80 °C) and pH levels (5.0 to 9.0). In addition, strain J75 demonstrated algicidal activities against other harmful microalgae, including Kareniamikimotoi, Prorocentrumminimum, and Phaeocystisglobosa. Under the stress of J75 cell-free supernatant, the algal cells showed morphological structure damage, a significant decrease in the photosynthetic activity, elevations in the levels of ROS, membrane lipid peroxidation, activities of superoxide dismutase, catalase, and peroxidase, and the level of glutathione. These results indicated that strain J75 caused oxidative damage to algal cells, ultimately leading to algal death. [Conclusion] The marine algicidal bacterium Pseudoalteromonas sp. J75, which holds significant potential for the control of red tides, demonstrates its efficacy by secreting algicidal compounds that inhibit the growth of diverse red tide microalgae. The exploration of the algicidal characteristics and mechanisms of this strain provides a theoretical basis for advancing red tide management strategies.

[Objective] The rhizosphere of plants hosts a diverse array of microorganisms that play a crucial role in plant growth and health. This study identified functional bacteria in the rhizosphere soil of an economic bamboo species Indosasa acutiligulata and assessed the effects of synthetic microbial communities (SynComs) on bamboo growth. The results are expected to provide implications for enhancing forest quality and utilizing beneficial microorganisms in bamboo cultivation. [Methods] Rhizosphere soil samples of In. acutiligulata were collected from the Jiuyi Mountain National Nature Reserve. Bacteria were isolated by the dilution culture method, and a phylogenetic tree was established by the maximum-likelihood algorithm based on 16S rRNA gene sequences. Specific media and colorimetric assays were employed to study the functions of strains. The strains with plant growth-promoting effects and no antagonistic effects between each other were combined. The effects of SynComs on the growth of Phyllostachys edulis seedlings were examined by re-inoculation experiments. [Results] Seventy strains of rhizosphere bacteria were isolated, representing 35 genera belonging to 21 families of four phyla. The dominant phylum was Pseudomonadota and the predominant family was Burkholderiaceae. Functional analyses revealed that 30 strains produced indole-3-acetic acid (IAA), while 16 strains produced siderophores. Among those with dual functions, there were four strains capable of solubilizing inorganic phosphorus, four strains capable of mineralizing organic phosphorus, and three strains capable of solubilizing potassium. Strains TR5, TN6, and TN26 exhibited capabilities to produce IAA and siderophores, as well as solubilize inorganic phosphorus and mineralize organic phosphorus. They were identified as Burkholderia pyrrocinia, Burkholderia paludis, and Paraburkholderia kirstenboschensis, respectively, based on physiological and biochemical properties and 16S rRNA gene sequences alignments. Re-inoculation experiments demonstrated that the SynCom FH, comprising strains TR5, TN6, and TN26, significantly enhanced the root, leaf, and rhizoma growth of Ph. edulis seedlings. [Conclusion] The rhizosphere of In. acutiligulata harbors diverse functional microorganisms capable of producing IAA and siderophores, solubilizing phosphorus, and releasing potassium. The re-inoculation experiments confirmed that the SynCom FH promotes the growth of Ph. edulis seedlings.

[Objective] To establish a theoretical foundation for the application and development of chitinases, this study isolated and screened chitin-degrading bacteria from the intestines of amphibians, optimized their fermentation conditions, characterized their enzymatic properties, and analyzed their whole genomes. [Methods] A strain capable of producing chitinase was isolated from the intestinal contents of Rana kukunoris and identified based on morphological characteristics and molecular biological evidence. The enzyme production conditions of the strain were optimized by single factor and response surface methodology (RSM) experiments, and the enzymatic properties were studied. Whole genome sequencing was carried out for identification of the chitinase gene family. [Results] The chitin-degrading strain JD-3 was identified as Carnobacteriummaltaromaticum. This strain achieved the highest enzyme activity of 12 mU/mL after fermentation with the inoculum amount of 4% at 31.4 ℃ and initial pH 4.9 for 2.47 d. The optimal reaction conditions of the enzyme were 20 ℃ and pH 3.0, and the enzyme maintained good stability at room temperature and under acidic conditions. The genome of JD-3 was 4 195 636 bp long, containing 6 circular contigs, 63 tRNA genes, 19 rRNA genes, and 3 864 protein coding sequences. Two chitinase genes belonging to the glycoside hydrolase family 18 (GH18) were identified and phylogenetically classified into two distinct categories. [Conclusion] We isolated an acid-tolerant chitin-degrading bacterium, C. maltaromaticum JD-3, from the intestines of plateau amphibians. The findings provide new insights into the development and utilization of microbial resources in the digestive systems of animals.

[Objective] Arbuscular mycorrhizal (AM) fungi are crucial components of the plant rhizosphere microbiota, capable of forming symbiotic relationships with 72% of terrestrial plants. However, AM fungi are plant-specific symbiotic fungi in soil, and they are difficult to be enriched for isolation and achieve artificial pure culture. This study aimed to develop a non-plant symbiotic culture system based on the addition of root exudates to solve the problem of difficult invitro culture of AM fungi. [Methods] The “multi-layer sandwich” culture system was used for the in vitro quasi-asymbiotic culture of AM fungal spores from soil. Molecular systematics methods were employed to identify the cultured AM fungi. [Results] A “multi-layer sandwich” culture system was used for the in vitro quasi-asymbiotic culture of AM fungi from soil. It was found that the root exudates of Astragalus sinicus effectively promoted the hyphal growth of AM fungi. A large number (951±45) of secondary spores were produced after 60 days of culture, exceeding those after 30 days and 45 days of culture. Further spore inoculation tests indicated that the secondary spores produced from this culture colonized the roots of A. sinicus seedlings. Two AM fungal species, Funneliformis mosseae and Paraglomus occultum, were identified by molecular characterization as suitable for the “multi-layer sandwich” culture system. Finally, a nutrient solution composed of simulated root exudate components from A. sinicus was used for the “multi-layer sandwich” culture of AM fungal spores. The results showed that the addition of root exudates significantly promoted the hyphal growth of AM fungi. [Conclusion] In the plant-assisted “multi-layer sandwich” culture system, the root exudates of A. sinicus can continually induce AM fungi to produce hyphae and secondary spores capable of colonizing host plants under non-symbiotic conditions. This study provides a new method for solving the problem related to the in vitro culture, isolation, and identification of AM fungi.

A mutant strain G₉-72 with a high yield of ectoine was obtained from wild type Halomonas campaniensis after nine rounds of ultraviolet mutagenesis. The differentially expressed genes/proteins (DEGs/DEPs) and the molecular mechanism underlying the excessive increase in the ectoine yield remain to be explored for the mutant strain. [Objective] To explore the DEGs/DEPs between the wild type strain XH26 and G₉-72 and decipher the molecular mechanism of efficient ectoine production by conjoint analysis. [Methods] A non-salt (NS, 0 mol/L NaCl) group and a high-salt (HS, 1.5 mol/L NaCl) group were designed for the culture of XH26 and G₉-72. Illumina HiSeq and quantitative mass spectrometry were employed to identify the DEGs/DEPs between the two strains by transcriptomics-proteomics conjoint analysis. Furthermore, RT-qPCR was carried out to verify the expression of significant DEGs. [Results] The transcriptomics analysis revealed 11 amino acid metabolic pathways (44 DEGs) associated with ectoine anabolism, and the proteomics analysis revealed ten amino acid metabolic pathways (50 DEPs) associated with ectoine anabolism. The transcriptomics-proteomics conjoint analysis identified 15 significant DEGs, including seven genes (ectB, betB, betA, asd, doeD, doeC, and gabD) with up-regulated mRNA and protein level, four genes (ItaE, gdhA, gabT, and acnB) with down-regulated mRNA and protein levels, three genes (gltD, atoB, and narG) with down-regulated mRNA levels and up-regulated protein levels, and one gene narK with up-regulated mRNA level and no protein level. Additionally, the RT-qPCR results were consistent with the transcriptomics analysis. [Conclusion] The excessive increase in the ectoine yield of the mutant strain was associated with key genes in the ectoine metabolic pathway (including the synthesis genes asd and ectB and the catabolism genes doeD and doeC) and indirectly associated with several genes (betB, betA, ItaE, gltD, gadA, and acnB) in the upstream metabolic pathway. Notably, ectoine biosynthesis was highly associated with the Ala/Asp/Glu/His metabolic pathway (gabD, gdhA, gabT, and atoB) and nitrogen source metabolism (narK and narG).

[Objective] To reveal the phylogenetic diversity of 12 marine siderophore-producing bacteria isolated from intertidal sediment samples of Naozhou Island in Leizhou Bay of South China Sea, and to unravel the components, functions, genetic diversity and genetic evolution of siderophore biosynthetic gene clusters (BGCs) of the isolates as well as representatives of Microbulbifer. [Methods] The phenotypic characteristics as well as the siderophore-producing activity of the strains were observed by conventional methods. The phylogenetic diversity (including taxon, species, and genetic diversity) of the strains was analyzed based on 16S rRNA gene sequences. Then, the exact phylogenetic status of the representative strain JSM ZJ756 was investigated comprehensively by means of comparative genomics analysis based on whole-genome sequences, including comparisons of G+C content, average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH) estimated values. We then used multiple bioinformatic stools including antiSMASH 7.0, BLASTn, BLASTp, and MEGA 11 for rapid identification, annotation, and sequence alignment of BGCs, thus exploring the components, functions, genetic diversity, and genetic evolution of siderophore BGCs. [Results] All the 12 isolates were Gram-negative, aerobic, non-sporulating and slightly halophilic rods with siderophore-producing activity. The results of phylogenetic analyses based on 16S rRNA gene sequences as well as whole-genome sequences showed that all the 12 strains belonged to Microbulbifer, representing 6 to 8 species and forming 4 clades with Microbulbifer type strains in the phylogenetic tree. Among them, strain JSM ZJ756 should be a new member of Microbulbifer zhoushanensis. JSM ZJ756 and 9 representative strains of Microbulbifer each carried one copy of NI-siderophore BGCs, and 8 out of the 10 NI-siderophore BGCs shared the similarities ≤40% with known BGCs. According to the types and the similarities of known BGCs with the NI-siderophore BGCs identified in this study, the 10 NI-siderophore BGCs could be categorized into 5 functional subtypes: Ochrobactin (JSM ZJ756 and 2 type strains, JYFX01000060.1, similarity of 28%), Vibrioferrin (M. mangrove DD-13^T, AB082123.1, 100%; M. epialgicus DSM 18651^T, CP005094.1, 85%), Putrebactin (M. agarilyticus GP101, NIBS01000001.1, 40%), Aerobactin (M. echini JCM 30400^T, AB199785.1, 22%), and unknown functional subtype (3 type strains including M. variabilis ATCC 700307^T). The results of BLASTn and BLASTp analyses showed that the core biosynthetic gene sequences of the NI-siderophore BGCs identified were unique, and encoded novel proteins. The results of genetic evolution analysis showed that the core biosynthetic genes of the NI-siderophore BGCs found exhibited high genetic diversity. In the phylogenetic tree based on core biosynthetic gene sequences, 9 out of the 10 NI-siderophore BGCs were grouped to 3 clades, but M. agarilyticus GP101 exhibited an independent evolution path. The comparative analysis revealed the genetic evolution of NI-siderophore BGCs being consistent with that predicted based on the 16S rRNA sequences. [Conclusion] All the 12 siderophore-producing strains isolated from intertidal sediment samples of Naozhou Island in Leizhou Bay of South China Sea belong to Microbulbifer, demonstrating high phylogenetic diversity, and JSM ZJ756 is a new member of M. zhoushanensis. The NI-siderophore BGCs of JSM ZJ756 and representative strains of Microbulbifer are novel and showcase high diversity, which indicates that those strains possess high potential of producing a variety of novel siderophores. Moreover, strong positive correlations exist between the biological functions and genetic evolution of the NI-siderophore BGCs and the phylogeny of JSM ZJ756 and 9 representative strains of Microbulbifer. Therefore, we hypothesize that the 12 marine siderophore-producing bacteria and the representatives of Microbulbifer are typical new-resource microbes, and the taxonomy of Microbulbifer, and the metabolic mechanism and genetic evolution as well as the biotechnological potential of the NI-siderophore BGCs are worth being further explored.

[Objective] To screen out a yeast strain that can efficiently assimilate ammonia nitrogen, optimize the solid-state fermentation conditions based on the nutrient composition, antioxidant activity, and amino acid content of the feed, and provide a scientific basis for the production of single-cell protein feed with this yeast strain. [Methods] Five strains of Candida utilis, four strains of Pichia anomala, five strains of Saccharomyces cerevisiae, and three strains of Issatchenkia orientalis were cultured with (NH₄)₂SO₄ as the sole nitrogen source. The yeast strain with the highest ammonia utilization rate and the highest glutamine synthetase (GS) activity was selected as the test strain, and then the fermentation parameters and fermentation substrates were optimized for this strain. The routine nutrient composition, content of phytate phosphorusand amino acids, and scavenging rates against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline- 6-sulfonicacid) (ABTS) free radicals of the fermented feed were determined. [Results] C. utilis CJ121 showed an ammonia utilization rate of 55.39% and a GS activity of 0.29 μmol/(h·g), which were higher than those of other yeast strains. The optimal fermentation process for C. utilis CJ12 was fermentation with a (NH₄)₂SO₄ addition amount of 2% and an inoculation amount of 8% for 36 h. The optimal fermentation substrate was composed of 94.8% wheat bran, 5% soybean meal, 0.1% protease, and 0.1% cellulase. After fermentation with C. utilis CJ12, the content of crude protein and organic nitrogen increased by 15.95% and 28.46%, respectively (P<0.05), while that of dry matter, crude fiber, crude fat, and phytate phosphorus decreased by 4.19%, 19.41%, 12.29%, and 13.51%, respectively (P<0.05). The total amino acid content increased after fermentation (P<0.05), with glutamine and glutamic acid levels being 111.38 times and 3.02 times those of the control group, respectively. However, the control group exhibited higher levels of tryptophan, asparagine, and 4-aminobutyric acid, which were 13.41, 8.27, and 6.41 times those of the experimental group, respectively. In addition, the scavenging abilities against DPPH and ABTS free radicals increased after fermentation (P<0.05), with the IC₅₀ values decreasing by 73.51% and 6.01%, respectively (P<0.05). [Conclusion] C. utilis CJ12 has high capacities of utilizing ammonia nitrogen and synthesizing glutamine. This strain improves the nutritional value and antioxidant performance of feed after solid-state fermentation, thus demonstrating the potential for producing functional single-cell protein feed.