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Abscisic acid (ABA) is a conserved and important “sesquiterpene signaling molecule” widely distributed in different organisms with unique biological functions. ABA coordinates reciprocity and competition between microorganisms and their hosts. In addition, ABA also regulates immune and stress responses in plants and animals. Therefore, ABA has a wide range of applications in agriculture, medicine and related fields. The plant pathogenic ascomycete B. cinerea has been extensively studied as a model strain for ABA production. Nevertheless, there is a relative dearth of research regarding the regulatory mechanism governing ABA biosynthesis in B. cinerea. Here, we discovered that H3K9 methyltransferase BcDIM5 is physically associated with the H3K14 deacetylase BcHda1. Deletion of Bcdim5 and Bchda1 in the high ABA-producing B. cinerea TB-31 led to severe impairment of ABA synthesis. The combined analysis of RNA-seq and ChIP-seq has revealed that the absence of BcDIM5 and BcHda1 has resulted in significant global deficiencies in the normal distribution and level of H3K9me3 modification. In addition, we found that the cause of the decreased ABA production in the ΔBcdim5 and ΔBchda1 mutants was due to cluster gene repression caused by the emergence of hyper-H3K9me3 in the ABA gene cluster. We concluded that the ABA gene cluster is co-regulated by BcDIM5 and BcHda1, which are essential for the normal distribution of the B. cinerea TB-31 ABA gene cluster H3K9me3. This work expands our understanding of the complex regulatory network of ABA biosynthesis and provides a theoretical basis for genetic improvement of high-yielding ABA strains.

In order to study the difference of cultivable seed-borne bacterial diversity between commercial varieties and wild species of Poa pratensis L., and their antibiotic resistance to sulfadiazine, tetracycline, oxytetracycline, ciprofloxacin, gentamicin, oxytetracycline and rifampin. In this study, 60 bacterium isolates were isolated by dilution-coated plate method. Through 16S rRNA sequence analysis, 40 representative isolates with different morphological characteristics were identified and phylogenetic tree was constructed. The results of diversity analysis showed that the seed-borne bacterial diversity of commercial varieties was richer than that of wild species. The antibiotic resistance of the isolated bacterial strains was studied by agar dilution method, and it was concluded that the antibiotic resistance of the seed-borne bacteria carried by commercial varieties was stronger than that of the wild species. Finally, the biofilm formation ability and swimming motility of the bacterial strain were measured, and the correlation between the two and the antibiotic resistance of the bacterial strain was analyzed. The analysis showed that the antibiotic resistance of bacterial strains in Poa pratensis L. was significantly correlated with their swimming motility. In addition, the swimming motility of the bacterial strains was significantly correlated with the biofilm formation ability. It is worth mentioning that this is the first time to study the drug-resistant bacteria distributed in the seed-borne bacteria of Poa pratensis L.

IntroductionThe primary objective of this study was to investigate the potential correlation between gut microbes and postoperative pulmonary infection in gastric cancer patients. Additionally, we aimed to deduce the mechanism of differential functional genes in disease progression to gain a better understanding of the underlying pathophysiology.MethodsA nested case–control study design was utilized to enroll patients with gastric cancer scheduled for surgery at West China Hospital of Sichuan University. Patients were categorized into two groups, namely, the pulmonary infection group and the control group, based on the development of postoperative pulmonary infection. Both groups were subjected to identical perioperative management protocols. Fecal samples were collected 24 h postoperatively and upon pulmonary infection diagnosis, along with matched controls. The collected samples were subjected to 16S rDNA and metagenomic analyses, and clinical data and blood samples were obtained for further analysis.ResultsA total of 180 fecal specimens were collected from 30 patients in both the pulmonary infection and control groups for 16S rDNA analysis, and 3 fecal samples from each group were selected for metagenomic analysis. The study revealed significant alterations in the functional genes of the intestinal microbiome in patients with postoperative pulmonary infection in gastric cancer, primarily involving Klebsiella, Enterobacter, Ruminococcus, and Collinsella. During postoperative pulmonary infection, gut flora and inflammatory factors were found to be associated with the lipopolysaccharide synthesis pathway and short-chain fatty acid (SCFA) synthesis pathway.DiscussionThe study identified enriched populations of Klebsiella, Escherella, and intestinal bacteria during pulmonary infection following gastric cancer surgery. These bacteria were found to regulate the lipopolysaccharide synthesis pathway, contributing to the initiation and progression of pulmonary infections. Inflammation modulation in patients with postoperative pulmonary infection may be mediated by short-chain fatty acids. The study also revealed that SCFA synthesis pathways were disrupted, affecting inflammation-related immunosuppression pathways. By controlling and maintaining intestinal barrier function, SCFAs may potentially reduce the occurrence of pulmonary infections after gastric cancer surgery. These findings suggest that targeting the gut microbiome and SCFA synthesis pathways may be a promising approach for preventing postoperative pulmonary infections in gastric cancer patients.

The amount of chemical fertilizer for vegetables is on the high level in China. The use of organic fertilizers to meet the nutrient requirement of crops will be an inevitable practice in sustainable agriculture. In this study, we compared the effects of pig manure fertilizer, rabbit manure fertilizer and chemical fertilizer on yield, quality of Brassica rapa var. Chinensis, soil physico-chemical properties and microbial community by using two consecutive seasons of three fertilizers in a pot experiment. The results were as follows: (1) In the first season, the fresh yield of Brassica rapa var. Chinensis applying chemical fertilizer was significantly (p ≤ 5%) higher than those of applying the pig manure and rabbit manure fertilizer, and the results were the opposite in the second season. The total soluble sugar concentration of fresh Brassica rapa var. Chinensis applying rabbit manure fertilizer was significantly (p ≤ 5%) higher than those of applying pig manure fertilizer and chemical fertilizer in the first season, and the NO3-N content of fresh Brassica rapa var. Chinensis on the contrary. (2) The organic fertilizer increased the concentration of total nitrogen, total phosphorus and organic carbon in soil in both two seasons. Rabbit manure fertilizer increased the soil pH and EC and significantly (p ≤ 5%) reduced the soil NO3-N content. (3) The pig manure and rabbit manure fertilizer significantly (p ≤ 5%) increased the diversity and abundance of soil bacterial of Brassica rapa var. Chinensis, but had no significant effect on soil fungi. Pearson correlation analysis showed that soil TN, TP, organic carbon content and EC were significantly correlated with soil bacterial α - diversity. There were significant differences (p ≤ 5%) in the bacterial community structures between three treatments in two seasons, and significant differences (p ≤ 5%) in the fungal community structures between fertilizer treatments while not between two seasons. Pig manure and rabbit manure fertilizer decreased the relative abundance of soil Acidobacteria and Crenarchaeota, rabbit manure fertilizer significantly increased the abundance of Actinobacteria in the second season. Distance-based redundancy analysis (dbRDA) showed that soil EC, TN, and organic carbon content were key physico-chemical factors in determining bacterial community structure in Brassica rapa var. Chinensis soil, and soil NO3-N, EC, SOC concentration and soil pH in the fungal community structure.

The booming mudflat aquaculture poses an accumulation of organic matter and a certain environmental threat. Protease-producing bacteria are key players in regulating the nitrogen content in ecosystems. However, knowledge of the diversity of protease-producing bacteria in coastal mudflats is limited. This study investigated the bacterial diversity in the coastal mudflat, especially protease-producing bacteria and their extracellular proteases, by using culture-independent methods and culture-dependent methods. The clam aquaculture area exhibited a higher concentration of carbon, nitrogen, and phosphorus when compared with the non-clam area, and a lower richness and diversity of bacterial community when compared with the clam naturally growing area. The major classes in the coastal mud samples were Bacteroidia, Gammaproteobacteria, and Alphaproteobacteria. The Bacillus-like bacterial community was the dominant cultivated protease-producing group, accounting for 52.94% in the non-clam area, 30.77% in the clam naturally growing area, and 50% in the clam aquaculture area, respectively. Additionally, serine protease and metalloprotease were the principal extracellular protease of the isolated coastal bacteria. These findings shed light on the understanding of the microbes involved in organic nitrogen degradation in coastal mudflats and lays a foundation for the development of novel protease-producing bacterial agents for coastal mudflat purification.

Triclocarban (TCC), an emerging organic contaminant (EOC), has become a severe threat to soil microbial communities and ecological security. Here, the TCC-degrading strain Rhodococcus rhodochrous BX2 and DCA-degrading strain Pseudomonas sp. LY-1 (together referred to as TC1) were immobilized on biochar to remove TCC and its intermediates in TCC-contaminated soil. High-throughput sequencing was used to investigate the microbial community structure in TCC-contaminated soil. Analysis of co-occurrence networks was used to explore the mutual relationships among soil microbiome members. The results showed that the immobilized TC1 significantly increased the removal efficiency of TCC from 84.7 to 92.7% compared to CK (no TC1 cells on biochar) in 10 mg/L TCC liquid medium. The utilization of immobilized TC1 also significantly accelerated the removal of TCC from contaminated soil. Microbial community analysis revealed the crucial microorganisms and their functional enzymes participating in TCC degradation in soil. Moreover, the internal labor division patterns and connections of TCC-degrading microbes, with a focus on strains BX2 and LY-1, were unraveled by co-occurrence networks analysis. This work provides a promising strategy to facilitate the bioremediation of TCC in soil, which has potential application value for sustainable biobased economies.

IntroductionIdentifying spatial patterns of biodiversity along elevational gradients provides a unified framework for understanding these patterns and predicting ecological responses to climate change. Moreover, microorganisms and plants are closely interconnected (e.g., via the rhizosphere) and thus may share spatial patterns of diversity and show similar relationships with environments.MethodsThis study compared diversity patterns and relationships with environments in host plants and rhizosphere microorganisms (including various functional groups) along elevational gradients across three climatic zones.ResultsWe found that above-and belowground diversity decreased monotonically or showed a hump-shaped or U-shaped pattern along elevation gradients. However, the diversity patterns of plants, bacteria, and fungi varied depending on the taxon and climatic zone. Temperature and humidity strongly contribute to above-and belowground diversity patterns and community composition along elevational gradients. Nonetheless, soil factors might be important regulators of diversity patterns and the community composition of plants and microorganisms along these gradients. Structural equation modeling revealed that environmental factors had a stronger direct effect on rhizosphere microbial diversity than host plant diversity.DiscussionIn sum, spatial patterns of diversity and their relationships with environments in rhizosphere microorganisms and their host plants differed at the regional scale. Different functional groups (e.g., pathogen, mycorrhiza and nitrifier) of soil microorganisms may have divergent elevational patterns and environmental responses. These data improve our understanding of elevational diversity patterns, and provide new insights into the conservation of biodiversity and ecosystem management, especially under climate change.

IntroductionAbscisic acid (ABA) is an important sesquiterpene compound that regulates the stress resistance of plants. Botrytis cinerea can synthesize ABA via the mevalonic acid pathway. To identify the functional genes that are involved in the biosynthesis of ABA, we performed insertion mutagenesis into B. cinerea TB-31.MethodsWe obtained the ABA-reduced mutant E154 by insertion mutagenesis, and we identified the insertion site was located upstream of the gene bcfrp1 by Thermal asymmetric interlaced PCR. We performed a detailed phenotypic characterization of the bcfrp1 knockout and complementation mutants in TB-31. Furthermore, transcriptome and proteome analyses were conducted to explore how bcfrp1 affects the level of the ABA biosynthesis.ResultsThe bcfrp1 gene encodes an F-box protein. The phenotypic results confirmed the positive contribution of bcfrp1 to the biosynthesis of ABA and growth. Between TB-31 and ΔBcfrp1, we obtained 4,128 and 1,073 differentially expressed genes and proteins, respectively. The impaired ABA biosynthesis in the ΔBcfrp1 mutants was primarily affected by the different levels of expression of the ABA biosynthetic gene cluster and the genes involved in the mevalonic acid pathway. In addition, we further characterized the differentially expressed genes and proteins that participated in the growth, secondary metabolism, and signal transduction in B. cinerea based on the transcriptome and proteome data.DiscussionThis research based on the transcriptome and proteome analyses to display the changes after the deletion of bcfrp1 in B. cinerea TB-31, will help us to explore the molecular mechanism of ABA biosynthesis in B. cinerea.

Porcine circovirus type 2 (PCV2) has been identified as the causal agent of postweaning multisystemic wasting syndrome (PMWS), an economically important multifactorial disease of the swine industry worldwide. This research designed a dual nested polymerase chain reaction (PCR) detection method to simultaneously monitor porcine circovirus type 2 (PCV2) and PCV3. The limit of detection (LoD) of sensitivity for PCV2 and PCV3 was ten copies/mL for both viruses. There was no cross-reaction with any other porcine pathogens tested and no non-specific amplification. The coincidence and repetition rates were both 100%. Through the systematic and clinical sampling, 15,130 samples collected from 30 large-scale pig farms in eight provinces in China (including Hubei, Hunan, Henan, Jiangxi, Shanxi, Guangdong, Hainan, and Heilongjiang) were subjected to early warning surveillance and/or clinical diagnosis. These results revealed that the overall positive rates of PCV3 and PCV2 were 0 and 28.29%, respectively, with the lowest level recorded in Jiangxi province. The highest carrying rate was observed in Hainan province. Pigs at different ages displayed varying carrying rates for PCV: fattening pigs and gilts had the highest and the lowest carrying rates for PCV, respectively. In addition, the excretion rates for PCV of colostrum, semen, and nasal, anal, and vulval swabs were tested. The colostrum, anal swabs, and semen had higher excretion rates for PCV; these were followed by the vulval and nasal swabs that had excretion rates for PCV. Furthermore, a high blood virus-carrying rate was detected in moribund pigs, especially in pigs with fever and red skin. As to the virus-carrying rate in the pig organs received from clinical necropsy, the highest rate was found in placental tissue, followed by the kidneys, and the virus also was detected in lymphoid organs, liver, stomach, and intestines. The PCV2-positive samples were sequenced to reveal the molecular epidemic dynamics of PCV2. The results indicated four major branches, namely, PCV2a, PCV2b, PCV2c, and PCV2d, concerning PCV2 molecular epidemiology in China, with PCV2a, PCV2b, and PCV2d dominating. In conclusion, the results obtained in this study elucidated the molecular epidemiology, transmission, and positive blood samples of PCV and provided new ideas for developing comprehensive PCV control technologies to begin eliminating the disease caused by PCV by cleaning pig farms.

Paris spp. are important medicinal plant and main raw material for many Chinese patent medicines, but viral diseases have became serious problems in cultivation of this group of important medicinal plants in China. In this study, eight viruses were identified in the diseased plants of Paris yunnanensis by high-throughput sequencing (HTS) and RT–PCR. These viruses include three novel viruses (two potyviruses and one nepovirus), Hippeastrum chlorotic ringspot virus (HCRV), Lychnis mottle virus (LycMoV), Paris mosaic necrosis virus (PMNV), Paris virus 1 and pepper mild mottle virus. The three new viruses were tentatively named Paris potyvirus 3 (ParPV-3), Paris potyvirus 4 (ParPV-4), Paris nepovirus 1 (ParNV-1) and their complete genome sequences were determined. Sequence analyses showed ParPV-3 and ParPV-4 shared the highest amino acid (aa) sequence identities of 54.3% to each other and 53.0–57.8% to other known potyviruses. ParNV-1 had aa sequence identities of 28.8–63.7% at protease-polymerase (Pro-Pol) with other nepoviruses. Phylogenetic analyses further support that the three viruses are new members of their corresponding genera. Analyses of the partial sequences of HCRV and LycMoV infecting P. yunnanensis revealed they diverged from existing isolates by aa sequence identities of 97.1% at glycoprotein precursor of HCRV and 93.3% at polyprotein of LycMoV. These two viruses are reported for the first time in Paris spp. A total of 123 field samples collected from P. yunnanensis in four counties of Yunnan, Southwest China were tested by RT–PCR for detecting each of the eight viruses. Results showed that nearly half of the samples were positive for at least one of the eight viruses. Two potyviruses, ParPV-3 (26.8%) and PMNV (24.4%), were predominant and widely distributed in the fields, while other viruses occurred in low rates and/or had limited distribution. This study insights into the virome infecting P. yunnanensis and provides valuable information for diagnosis and control of viral diseases in P. yunnanensis.

Aeromonas hydrophila is an important zoonotic pathogen responsible for septicemia, diarrhea and gastroenteritis, and has attracted considerable attention. The EnvZ/OmpR two-component system (TCS) mediates environmental stress responses in gram-negative bacteria. We investigated the role of the TCS in A. hydrophila by comparing the characteristics of the parental (23-C-23), EnvZ/OmpR knockout (23-C-23:ΔEnvZ/OmpR), and complemented strains (23-C-23:CΔEnvZ/OmpR). Under non-stress conditions, the 23-C-23:ΔEnvZ/OmpR strain showed a significant decrease in growth rate compared to that of 23-C-23. Transcriptome and metabonomic analysis indicated that many metabolic pathways were remarkably affected in the ΔEnvZ/OmpR strain, including the TCA cycle and arginine biosynthesis. In addition, the virulence of the ΔEnvZ/OmpR strain was attenuated in a Kunming mouse model. The ΔEnvZ/OmpR strain exhibited notably reduced tolerance to environmental stresses, including high temperature, different pH conditions, oxidative stress, and high osmotic stress. The downregulated expression of genes related to cell metabolism, motility, and virulence in the ΔEnvZ/OmpR mutant strain was further validated by real-time quantitative PCR. Consequently, our data suggest that the EnvZ/OmpR TCS is required for growth, motility, virulence, and stress response in A. hydrophila, which has significant implications in the development of novel antibacterial and vaccine therapies targeting EnvZ/OmpR against A. hydrophila.

The mobile colistin-resistance (mcr)-1 gene is primarily detected in Enterobacteriaceae species, such as Escherichia coli and Salmonella enterica, and represents a significant public health threat. Herein, we investigated the prevalence and characteristics of mcr-1-positive E. coli (MCRPEC) in hospitalized companion animals in a pet hospital in Shanghai, China, from May 2021 to July 2021. Seventy-nine non-duplicate samples were collected from the feces (n = 52) and wounds (n = 20) of cats and dogs and the surrounding hospital environment (n = 7). Seven MCRPEC strains, identified using screening assays and polymerase chain reaction, exhibited multidrug-resistant phenotypes in broth-microdilution and agar-dilution assays. Based in whole-genome sequencing and bioinformatics analyses, all seven isolates were determined to belong to sequence type (ST) 117. Moreover, the Incl2 plasmid was prevalent in these MCRPEC isolates, and the genetic environment of the seven E. coli strains was highly similar to that of E. coli SZ02 isolated from human blood. The isolates also harbored the β-lactamase gene blaCTX-M-65, and florfenicol resistance gene floR, among other resistance genes. Given that horizontal transfer occurred in all seven strains, E. coli plasmid transferability may accelerate the emergence of multidrug-resistant bacteria and may be transmitted from companion animals to humans. Therefore, the surveillance of MCRPEC isolates among companion animals should be strengthened.

Endophytic microbial communities of lichen are emerging as novel microbial resources and for exploration of potential biotechnological applications. Here, we focused on a medicinal lichen Usnea longissima, and investigated its bacterial and fungal endophytes. Using PacBio 16S rRNA and ITS amplicon sequencing, we explored the diversity and composition of endophytic bacteria and fungi in U. longissima collected from Tibet at five altitudes ranging from 2,989 to 4,048 m. A total of 6 phyla, 12 classes, 44 genera, and 13 species of the bacterial community have been identified in U. longissima. Most members belong to Alphaproteobacteria (42.59%), Betaproteobacteria (33.84%), Clostridia (13.59%), Acidobacteria (7%), and Bacilli (1.69%). As for the fungal community, excluding the obligate fungus sequences, we identified 2 phyla, 15 classes, 65 genera, and 19 species. Lichen-related fungi of U. longissima mainly came from Ascomycota (95%), Basidiomycota (2.69%), and unidentified phyla (2.5%). The presence of the sequences that have not been characterized before suggests the novelty of the microbiota. Of particular interest is the detection of sequences related to lactic acid bacteria and budding yeast. In addition, the possible existence of harmful bacteria was also discussed. To our best knowledge, this is the first relatively detailed study on the endophytic microbiota associated with U. longissima. The results here provide the basis for further exploration of the microbial diversity in lichen and promote biotechnological applications of lichen-associated microbial strains.

Abscisic acid (ABA), as a classic plant hormone, is a key factor in balancing the metabolism of endogenous plant hormones, and plays an important role in regulating the activation of mammalian innate immune cells and glucose homeostasis. Currently, Botrytis cinerea has been used for fermentation to produce ABA. However, the mechanism of the regulation of ABA biosynthesis in B. cinerea is still not fully understood. The putative methyltransferase LaeA/LAE1 is a global regulator involved in the biosynthesis of a variety of secondary metabolites in filamentous fungi. In this study, we demonstrated that BcLAE1 plays an important role in the regulation of ABA biosynthesis in B. cinerea TB-31 by knockout experiment. The deletion of Bclae1 caused a 95% reduction in ABA yields, accompanied by a decrease of the transcriptional level of the ABA synthesis gene cluster Bcaba1-4. Further RNA-seq analysis indicated that deletion of Bclae1 also affected the expression level of key enzymes of BOA and BOT in secondary metabolism, and accompanied by clustering regulatory features. Meanwhile, we found that BcLAE1 is involved in epigenetic regulation as a methyltransferase, with enhanced H3K9me3 modification and attenuated H3K4me2 modification in ΔBclae1 mutant, and this may be a strategy for BcLAE1 to regulate ABA synthesis.

Fungi secrete numerous effectors to modulate host defense systems. Understanding the molecular mechanisms by which fungal effectors regulate plant defense is of great importance for the development of novel strategies for disease control. In this study, we identified necrosis- and ethylene-inducing protein 1 (Nep1)-like protein (NLP) effector gene, CgNLP1, which contributed to conidial germination, appressorium formation, invasive growth, and virulence of Colletotrichum gloeosporioides to the rubber tree. Transient expression of CgNLP1 in the leaves of Nicotiana benthamiana induced ethylene production in plants. Ectopic expression of CgNLP1 in Arabidopsis significantly enhanced the resistance to Botrytis cinerea and Alternaria brassicicola. An R2R3 type transcription factor HbMYB8-like of rubber tree was identified as the target of CgNLP1.HbMYB8-like, localized on the nucleus, and induced cell death in N. benthamiana. CgNLP1 disrupted nuclear accumulation of HbMYB8-like and suppressed HbMYB8-like induced cell death, which is mediated by the salicylic acid (SA) signal pathway. This study suggested a new strategy whereby C. gloeosporioides exploited the CgNLP1 effector to affect invasion and suppress a host defense regulator HbMYB8-like to facilitate infection.

Colistin is one of the last-line drugs against difficult to treat and multidrug-resistant Gram-negative bacteria. The emergence of mobile colistin resistance gene mcr-1 increased worldwide attention on colistin resistance. mcr-1 is the dominant gene that caused resistance to colistin in chicken-derived Escherichia coli (E. coli) in China; it has a broad resistance spectrum and causes multiple drug resistance problems. There are only few studies on mcr-positive E. coli (MCRPEC) from laying ducks and quails in China. Here, the molecular and epidemiological characteristics of MCRPEC from three kinds of poultry farms (laying duck, quail, and broiler) were investigated in Changsha, China. A total of 17 mcr-positive E. coli (MCRPEC) strains were screened in 690 samples from the three kinds of poultry farms. This is the first report on MCRPEC, to our best knowledge, derived from quail. All the MCRPEC strains were resistant to colistin, sulfamethoxazole-trimethoprim, florfenicol, tetracycline, and ciprofloxacin, and mildly resistant to tigecycline, gentamicin, piperacillin/tazobactam, cefotaxime, and ceftiofur. All the strains were sensitive to meropenem and amikacin. By bioinformatics analysis, 17 MCRPEC strains belonging to 11 MLST types were distributed in phylogroups A (58.8%), B1 (23.5%), and phylogroup D (17.6%). mcr-1 was located in IncI2 plasmid with typical plasmid conjugation transfer part, type IV secretory system, and tellurium-resistant protein, increasing transmission capacity to other bacteria. Monitoring of colistin-resistant bacteria in poultry farms should be strengthened.

Pears (Pyrus sp.) are widely cultivated in China, and their yield accounts for more than 60% of global pear production. The fungal pathogen Valsa pyri is a major causal agent of pear canker disease, which results in enormous losses of pear production in northern China. In this study, we characterized a Zn2Cys6 transcription factor that contains one GAL4 domain and a fungal-trans domain, which are present in VpxlnR. The vpxlnR gene expression was upregulated in the invasion stage of V. pyri. To investigate its functions, we constructed gene deletion mutants and complementary strains. We observed that the growth of the vpxlnR mutants was reduced on potato dextrose agar (PDA), Czapek plus glucose or sucrose compared with that of the wild-type strain. Additionally, vpxlnR mutants exhibited loss of function in fruiting body formation. Moreover, vpxlnR mutants were more susceptible to hydrogen peroxide (H2O2) and salicylic acid (SA) and were reduced in their virulence at the early infection stage. According to a previous study, VpxlnR-interacting motifs containing NRHKGNCCGM were searched in the V. pyri genome, and we obtained 354 target genes, of which 148 genes had Clusters of Orthologous Groups (COG) terms. PHI-BLAST was used to identify virulence-related genes, and we found 28 hits. Furthermore, eight genes from the 28 PHI-BLAST hits were further assessed by yeast one-hybrid (Y1H) assays, and five target genes, salicylate hydroxylase (VP1G_09520), serine/threonine-protein kinase (VP1G_03128), alpha-xylosidase (VP1G_06369), G-protein beta subunit (VP1G_02856), and acid phosphatase (VP1G_03782), could interact with VpxlnR in vivo. Their transcript levels were reduced in one or two vpxlnR mutants. Taken together, these findings imply that VpxlnR is a key regulator of growth, development, stress, and virulence through controlling genes involved in signaling pathways and extracellular enzyme activities in V. pyri. The motifs interacting with VpxlnR also provide new insights into the molecular mechanism of xlnR proteins.

Fusarium is one of the most important phytopathogenic and mycotoxigenic fungi that caused huge losses worldwide due to the decline of crop yield and quality. To systematically investigate the infections of Fusarium species in ear rot of maize in the Guizhou Province of China and analyze its population structure, 175 samples of rotted maize ears from 76 counties were tested by combining immunoassays and molecular identification. Immunoassay based on single-chain variable fragment (scFv) and alkaline phosphatase (AP) fusion protein was first employed to analyze these samples. Fusarium pathogens were isolated and purified from Fusarium-infected samples. Molecular identification was performed using the partial internal transcribed spacer (ITS) and translation elongation factor 1α (TEF-1α) sequences. Specific primers were used to detect toxigenic chemotypes, and verification was performed by liquid chromatography tandem mass spectrometry (LC–MS/MS). One-hundred and sixty three samples were characterized to be positive, and the infection rate was 93.14%. Sixteen species of Fusarium belonging to six species complexes were detected and Fusarium meridionale belonging to the Fusarium graminearum species complex (FGSC) was the dominant species. Polymerase chain reaction (PCR) identification illustrated that 69 isolates (56.10%) were potential mycotoxin-producing Fusarium pathogens. The key synthetic genes of NIV, NIV + ZEN, DON + ZEN, and FBs were detected in 3, 35, 7, and 24 isolates, respectively. A total of 86.11% of F. meridionale isolates carried both NIV- and ZEN-specific segments, while Fusarium verticillioides isolates mainly represented FBs chemotype. All the isolates carrying DON-producing fragments were FGSC. These results showed that there are different degrees of Fusarium infections in Guizhou Province and their species and toxigenic genotypes display regional distribution patterns. Therefore, scFv-AP fusion-based immunoassays could be conducted to efficiently investigate Fusarium infections and more attention and measures should be taken for mycotoxin contamination in this region.

Introduction: Diabetic foot infections (DFIs) pose a huge challenge for clinicians. Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is one of the most significant pathogens of DFI. Early pathogen identification will greatly benefit the diagnosis and treatment of the disease. However, existing diagnostic methods are not effective in early detection.Methods: We developed an assay that coupled loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) techniques to enable quick and specific detection of Staphylococcus aureus and differentiate MRSA in samples from patients with DFI. Furthermore, the results were compared using a reference culture, quantitative real-time polymerase chain reaction (qRT-PCR), and metagenomics next generation sequencing (mNGS).Results: The CRISPR-LAMP assay targeting nuc and mecA successfully detected S. aureus strains and differentiated MRSA. The limit of detection (LoD) of the real-time LAMP for nuc and mecA was 20 copies per microliter reaction in comparison to two copies per μL reaction for the qRT-PCR assay. The specificity of the LAMP-CRISPR assay for nuc was 100%, without cross-reactions with non-S. aureus strains. Evaluating assay performance with 18 samples from DFI patients showed that the assay had 94.4% agreement (17/18 samples) with clinical culture results. The results of mNGS for 8/18 samples were consistent with those of the reference culture and LAMP-CRISPR assay.Conclusion: The findings suggest that the LAMP-CRISPR assay could be promising for the point-of-care detection of S. aureus and the differentiation of MRSA in clinical samples. Furthermore, combining the LAMP-CRISPR assay and mNGS provides an advanced platform for molecular pathogen diagnosis of DFI.

This study was aimed to investigate the effects of Clostridium butyricum (C. butyricum) immunity and intestinal epithelial barrier function at the intestinal mucosal level, by using Salmonella enteritidis (S. enteritidis) to infect specific-pathogen-free (SPF) chickens and intestinal epithelial cells (IEC). We found that C. butyricum could decrease cytokine levels (IFN-γ, IL-1β, IL-8, and TNF-α) via the TLR4-, MyD88-, and NF-κB-dependent pathways in intestinal tissues and intestinal epithelial cells. Additionally, C. butyricum could attenuate bacteria-induced intestinal damage and increase the expression level of muc-2 and ZO-1 in the intestine and intestinal epithelial cells. Furthermore, C. butyricum altered the intestinal microbial composition, increased the diversity of the bacterial communities in the cecum of Salmonella-infected chickens. In conclusion, C. butyricum effectively attenuated inflammation and epithelial barrier damage, altered the intestinal microbial composition, increased the diversity of the bacterial communities in the intestine of Salmonella-infected chickens. The result suggests that C. butyricum might be an effective and safe therapy for the treatment of Salmonella infection.

Greater feed efficiency (FE) is critical in increasing profitability while reducing the environmental impact of pig production. Previous studies that identified swine FE-associated bacterial taxa were limited in either sampling sites or sequencing methods. This study characterized the microbiomes within the intestine of FE contrasting Duroc × (Landrace × Yorkshire) (DLY) pigs with a comprehensive representation of diverse sampling sites (ileum, cecum, and colon) and a metagenomic sequencing approach. A total of 226 pigs were ranked according to their FE between weaning to 140 day old, and six with extreme phenotypes were selected, three for each of the high and low groups. The results revealed that the cecum and colon had similar microbial taxonomic composition and function, and had higher capacity in polysaccharide metabolism than the ileum. We found in cecum that the high FE pigs had slightly higher richness and evenness in their micriobiota than the low FE pigs. We identified 12 phyla, 17 genera, and 39 species (e.g., Treponema porcinum, Treponema bryantii, and Firmicutes bacterium CAG:110) that were potentially associated with swine FE variation in cecum microbiota through LEfSe analysis. Species enriched in the cecum of the high FE pigs had a greater ability to utilize dietary polysaccharides and dietary protein according to the KEGG annotation. Analysis of antibiotic resistance based on the CARD database annotation indicated that the macB resistant gene might play an important role in shaping the microbial community in the cecum of pigs with contrasting FE. The bacteria from the genus Prevotella was highly enriched in the cecum of low FE pigs, which may impair the establishment of a more effective nutrient harvesting microbiota because of the interaction between Prevotella and other benefical microbes. These findings improved our understanding of the microbial compositions in the different gut locations of DLY pigs and identified many biomarkers associated with FE variation wich may be used to develop strategies to improve FE in pigs.

D-amino acids (DAAs) are an important component of the refractory dissolved organic matter pool in the ocean. Microbes play a vital role in promoting the recycling of DAAs in the ocean. However, the diversity of marine DAA-utilizing bacteria and how they metabolize DAAs are seldom studied. Here, by enrichment culture with DAAs as the sole nitrogen source, bacteria of 12 families from three phyla were recovered from surface seawater and sediment from Kongsfjorden, Arctic, and seven DAA-utilizing bacterial strains were isolated. These strains have different DAA-utilizing abilities. Of the seven DAAs used, Halomonas titanicae SM1922 and Pseudoalteromonas neustonica SM1927 were able to utilize seven and five of them, respectively, while the other strains were able to utilize only one or two. Based on genomic, transcriptional and biochemical analyses, the key genes involved in DAA metabolism in each strain were identified and the metabolic pathways for the seven DAAs in these marine bacteria were identified. Conversion of DAAs into α-keto acids is generally the main pathway in marine DAA-utilizing bacteria, which is performed by several key enzymes, including DAA oxidoreductases/dehydrogenases, D-serine ammonia-lyases, D-serine ammonia-lyase DSD1s and DAA transaminases. In addition, conversion of DAAs into LAAs is another pathway, which is performed by amino acid racemases. Among the identified key enzymes, D-serine ammonia-lyase DSD1 and Asp racemase are first found to be employed by bacteria for DAA utilization. These results shed light on marine DAA-utilizing bacteria and the involved DAA metabolism pathways, offering a better understanding of the DAA recycling in the ocean.

Gut microbiota has indispensable roles in nutrient digestion and energy harvesting, especially in processing the indigestible components of dietary polysaccharides. Searching for the microbial taxa and functional capacity of the gut microbiome associated with feed efficiency (FE) can provide important knowledge to increase profitability and sustainability of the swine industry. In the current study, we performed a comparative analysis of the fecal microbiota in 50 commercial Duroc × (Landrace × Yorkshire) (DLY) pigs with polarizing FE using 16S rRNA gene sequencing and shotgun metagenomic sequencing. There was a different microbial community structure in the fecal microbiota of pigs with different FE. Random forest analysis identified 24 operational taxonomic units (OTUs) as potential biomarkers to improve swine FE. Multiple comparison analysis detected 8 OTUs with a significant difference or tendency toward a difference between high- and low-FE pigs (P < 0.01, q < 0.1). The high-FE pigs had a greater abundance of OTUs that were from the Lachnospiraceae and Prevotellaceae families and the Escherichia-Shigella and Streptococcus genera than low-FE pigs. A sub-species Streptococcus gallolyticus subsp. gallolyticus could be an important candidate for improving FE. The functional capacity analysis found 18 KEGG pathways and CAZy EC activities that were different between high- and low-FE pigs. The fecal microbiota in high FE pigs have greater functional capacity to degrade dietary cellulose, polysaccharides, and protein and may have a greater abundance of microbes that can promote intestinal health. These results provided insights for improving porcine FE through modulating the gut microbiome.

The co-culture of Trametes versicolor and Ganoderma applanatum is a model of intense basidiomycete interaction, which induces many newly synthesized or highly produced features. Currently, one of the major challenges is an identification of the origin of induced features during the co-culture. Herein, we report a 13C-dynamic labeling analysis used to determine an association of induced features and corresponding fungus even if the identities of metabolites were not available or almost nothing was known of biochemical aspects. After the co-culture of T. versicolor and G. applanatum for 10 days, the mycelium pellets of T. versicolor and G. applanatum were sterilely harvested and then mono-cultured in the liquid medium containing half fresh medium with 13C-labeled glucose as carbon source and half co-cultured supernatants collected on day 10. 13C-labeled metabolome analyzed by LC-MS revealed that 31 induced features including 3-phenyllactic acid and orsellinic acid were isotopically labeled in the mono-culture after the co-culture stimulation. Twenty features were derived from T. versicolor, 6 from G. applanatum, and 5 features were synthesized by both T. versicolor and G. applanatum. 13C-labeling further suggested that 12 features such as previously identified novel xyloside [N-(4-methoxyphenyl)formamide 2-O-beta-D-xyloside] were likely induced through the direct physical interaction of mycelia. Use of molecular network analysis combined with 13C-labeling provided an insight into the link between the generation of structural analogs and producing fungus. Compound 1 with m/z 309.0757, increased 15.4-fold in the co-culture and observed 13C incorporation in the mono-culture of both T. versicolor and G. applanatum, was purified and identified as a phenyl polyketide, 2,5,6-trihydroxy-4, 6-diphenylcyclohex-4-ene-1,3-dione. The biological activity study indicated that this compound has a potential to inhibit cell viability of leukemic cell line U937. The current work sets an important basis for further investigations including novel metabolites discovery and biosynthetic capacity improvement.

This study was designed to evaluate the protection mechanism of oral administration of Clostridium butyricum against Salmonella enteritidis (SE) colonization in broilers. In the current study, 180 one-day-old healthy Arbor Acres (AA) broilers were meanly grouped into three, with three replicates of 20 birds each. An negative control group was fed basal diet without SE challenge and a positive control (PC) group was fed the basal diet and challenged with SE [106 colony forming unit (CFU)/0.2 mL]. An experimental (EXP) group was fed the basal diet, orally administered with C. butyricum (106 CFU/mL) and challenged with SE (106 CFU/0.2 mL). The results showed that compared to the PC group, the SE loads in livers, spleens, and cecal contents of chickens in EXP group were significantly reduced (P < 0.05) except in spleens at the 2-day post-infection; the production of interferon-γ, interleukin (IL)-1β, IL-8, and tumor necrosis factor-α in the livers, spleens, and cecal tissues of chickens in EXP group were decreased to different extents. The results of quantitative real-time polymerase chain reaction further revealed that the inflammation of chickens in EXP group was alleviated by C. butyricum via down-regulating TLR4, MyD88, and NF-κB-dependent pathways. Collectively, these findings indicated that oral administration of C. butyricum could be a suitable alternative for preventing SE infection in broilers.

A total of 154 non-duplicate Salmonella isolates were recovered from 1,105 rectal swabs collected from three large-scale chicken farms (78/325, 24.0%), three large-scale duck farms (56/600, 9.3%) and three large-scale pig farms (20/180, 11.1%) between April and July 2016. Seven serotypes were identified among the 154 isolates, with the most common serotype in chickens and ducks being Salmonella enteritidis and in pigs Salmonella typhimurium. Antimicrobial susceptibility testing revealed that high antimicrobial resistance rates were observed for tetracycline (72.0%) and ampicillin (69.4%) in all sources. Class 1 integrons were detected in 16.9% (26/154) of these isolates and contained gene cassettes aadA2, aadA1, drfA1-aadA1, drfA12-aadA2, and drfA17-aadA5. Three β-lactamase genes were detected among the 154 isolates, and most of the isolates carried blaTEM−1(55/154), followed by blaPSE−1(14/154) and blaCTX−M−55 (11/154). Three plasmid-mediated quinolone resistance genes were detected among the 154 isolates, and most of the isolates carried qnrA (113/154), followed by qnrB (99/154) and qnrS (10/154). Fifty-four isolates carried floR among the 154 isolates. Multilocus sequence typing (MLST) analysis showed that nine sequence types (STs) were identified; ST11 was the most frequent genotype in chickens and ducks, and ST19 was identified in pigs. Our findings indicated that Salmonella was widespread, and the overuse of antibiotics in animals should be reduced considerably in developing countries.

Laccases are a family of copper-containing oxidases with important applications in bioremediation and other various industrial and biotechnological areas. There have been over two dozen reviews on laccases since 2010 covering various aspects of this group of versatile enzymes, from their occurrence, biochemical properties, and expression to immobilization and applications. This review is not intended to be all-encompassing; instead, we highlighted some of the latest developments in basic and applied laccase research with an emphasis on laccase-mediated bioremediation of pharmaceuticals, especially antibiotics. Pharmaceuticals are a broad class of emerging organic contaminants that are recalcitrant and prevalent. The recent surge in the relevant literature justifies a short review on the topic. Since low laccase yields in natural and genetically modified hosts constitute a bottleneck to industrial-scale applications, we also accentuated a genus of laccase-producing white-rot fungi, Cerrena, and included a discussion with regards to regulation of laccase expression.

Pseudomonas aeruginosa is one of the most important foodborne pathogens that can persist in leafy green vegetables and subsequently produce biofilms. In this study, the synergistic effect of thymoquinone and nisin in reducing biofilm formation of P. aeruginosa on lettuce was evaluated, and their anti-virulence and anti-biofilm mechanisms were also investigated. At concentrations ranging from 0.5 to 2 mg/ml, thymoquinone inhibited the production of autoinducers and virulence factors, and enhanced the susceptibility of P. aeruginosa biofilms to nisin as evidenced by the scanning electron microscopy and confocal laser scanning microscopy. Integrated transcriptomics, metabolomics, and docking analyses indicated that thymoquinone treatment disrupted the quorum sensing (QS) system, altered cell membrane component, and down-regulated the expressions of genes related to virulence, efflux pump, and antioxidation. The changed membrane component and repressed efflux pump system enhanced membrane permeability and facilitated the entrance of nisin into cells, thus improving the susceptibility of biofilms to nisin. The dysfunctional QS and repressed antioxidant enzymes lead to the enhancement of oxidative stress. The enhanced oxidative stress disrupted energy metabolism and protein metabolism and ultimately attenuated the virulence and pathogenicity of P. aeruginosa PAO1. Our study indicated that thymoquinone has the potential to function as a QS-based agent to defend against foodborne pathogens in combination with nisin. [ABSTRACT FROM AUTHOR]

Laccases can oxidize a wide range of aromatic compounds and are industrially valuable. Laccases often exist in gene families and may differ from each other in expression and function. Quantitative real-time polymerase chain reaction (qPCR) was used for transcription profiling of eight laccase genes in Cerrena sp. strain HYB07 with validated reference genes. A high laccase activity of 280.0 U/mL was obtained after submerged fermentation for 5 days. Laccase production and laccase gene transcription at different fermentation stages and in response to various environmental cues were revealed. HYB07 laccase activity correlated with transcription levels of its predominantly expressed laccase gene, Lac7. Cu2+ ions were indispensable for efficient laccase production by HYB07, mainly through Lac7 transcription induction, and no aromatic compounds were needed. HYB07 laccase synthesis and biomass accumulation were highest with non-limiting carbon and nitrogen. Glycerol and inorganic nitrogen sources adversely impacted Lac7 transcription, laccase yields, and fungal growth. The present study would further our understanding of transcription regulation of laccase genes, which may in turn facilitate laccase production as well as elucidation of their physiological roles. [ABSTRACT FROM AUTHOR]

Natural attenuation is an effective and feasible technology for controlling groundwater contamination. This study investigated the potential effectiveness and mechanisms of natural attenuation of 1,1,1-trichloroethane (TCA) contaminants in shallow groundwater in Shanghai by using a column simulation experiment, reactive transport model, and 16S rRNA gene clone library. The results indicated that the majority of the contaminant mass was present at 2-6 m in depth, the contaminated area was approximately 1000 m x 1000 m, and natural attenuation processes were occurring at the site. The effluent breakthrough curves from the column experiments demonstrated that the effectiveness of TCA natural attenuation in the groundwater accorded with the advection-dispersion-reaction equation. The kinetic parameter of adsorption and biotic dehydrochlorination of TCA was 0.068 m3/kg and 0.0045 d-1. The contamination plume was predicted to diminish and the maximum concentration of TCA decreased to 280 μg/L. The bacterial community during TCA degradation in groundwater belonged to Trichococcus, Geobacteraceae, Geobacter, Mucilaginibacter, and Arthrobacter. [ABSTRACT FROM AUTHOR]