Physiological and transcriptome analysis of Dendrobium officinale under low nitrogen stress.

Nitrogen (N) is the main nutrient of plants, and low nitrogen usually affects plant growth and crop yield. The traditional Chinese herbal medicine Dendrobium officinale Kimura et. Migo is a typical low nitrogen-tolerant plant, and its mechanism in response to low nitrogen stress has not previously been reported. In this study, physiological measurements and RNA-Seq analysis were used to analyse the physiological changes and molecular responses of D. officinale under different nitrogen concentrations. The results showed that under low nitrogen levels, the growth, photosynthesis and superoxide dismutase activity were found to be significantly inhibited, while the activities of peroxidase and catalase, the content of polysaccharides and flavonoids significantly increased. Differentially expressed genes (DEGs) analysis showed that nitrogen and carbon metabolisms, transcriptional regulation, antioxidative stress, secondary metabolite synthesis and signal transduction all made a big difference in low nitrogen stress. Therefore, copious polysaccharide accumulation, efficient assimilation and recycling of nitrogen, as well as rich antioxidant components play critical roles. This study is helpful for understanding the response mechanism of D. officinale to low nitrogen levels, which might provide good guidance for practical production of high quality D. officinale .

Differential Expression of Genes Related to Growth and Aflatoxin Synthesis in Aspergillus flavus When Inhibited by Bacillus velezensis Strain B2.

Aspergillus flavus is a saprophytic soil fungus that infects and contaminates seed crops with the highly carcinogenic aflatoxin, which brings health hazards to animals and humans. In this study, bacterial strains B1 and B2 isolated from the rhizosphere soil of camellia sinensis had significant antagonistic activities against A. flavus. Based on the phylogenetic analysis of 16SrDNA gene sequence, bacterial strains B1 and B2 were identified as Bacillus tequilensis and Bacillus velezensis, respectively. In addition, the transcriptome analysis showed that some genes related to A. flavus growth and aflatoxin synthesis were differential expressed and 16 genes in the aflatoxin synthesis gene cluster showed down-regulation trends when inhibited by Bacillus velezensis strain B2. We guessed that the Bacillus velezensis strain B2 may secrete some secondary metabolites, which regulate the related gene transcription of A. flavus to inhibit growth and aflatoxin production. In summary, this work provided the foundation for the more effective biocontrol of A. flavus infection and aflatoxin contamination by the determination of differential expression of genes related to growth and aflatoxin synthesis in A. flavus when inhibited by B. velezensis strain B2.

Paracandidimonas lactea sp. nov., a urea-utilizing bacterium isolated from landfill.

A urea-utilizing bacterium, designated Q2-2 T, was isolated from landfill. Cells of strain Q2-2 T were Gram stain-negative, aerobic, short-rod bacteria. Strain Q2-2 T was observed to grow at a temperature range of 15-37℃ (optimum 30 â„ƒ), a pH range of 5.5-9.5 (optimum pH 8.0) and 0-4% (w/v) NaCl (optimum 1%). The major respiratory quinone was Q-8, and the major polar lipids were diphosphatidyl glycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, and phosphatidyl glycerol. Based on the 16S rRNA gene sequence, strain Q2-2 T had the highest similarity with Paracandidimonas caeni 24 T (98.0%), followed by Pusillimonas soli MJ07T (97.5%), Parapusillimonas granuli Ch07T (97.2%), Pusillimonas ginsengisoli DCY25T (97.1%) and Paracandidimonas soli IMT-305 T (96.4%). The ANI values between strain Q2-2 T and the above related type strains were 71.02%, 73.52%, 74.32%, 74.59% and 72.29%, respectively. The DNA G + C content of strain Q2-2 T was 61.1%. Therefore, strain Q2-2 T represents a novel species of the genus Paracandidimonas, for which the name Paracandidimonas lactea sp. nov. (type strain Q2-2 T = CGMCC 1.19179 T = JCM 34906 T) is proposed.

Infection of Nigrospora nonsegmented RNA Virus 1 Has Important Biological Impacts on a Fungal Host.

Nigrospora nonsegmented RNA virus 1 (NoNRV1) has been reported previously in the fungus Nigrospora oryzae, but its biological effects on its host are unknown. In this work, we isolated a strain 9-1 of N. oryzae from a chrysanthemum leaf and identified NoNRV1 infection in the isolated strain. The genome sequence of NoNRV1 identified here is highly homologous to that of the isolate HN-21 of NoNRV1 previously reported; thus, we tentatively designated the newly identified NoNRV1 as NoNRV1-ZJ. Drug treatment with Ribavirin successfully removed NoNRV1-ZJ from the strain 9-1, which provided us with an ideal control to determine the biological impacts of NoNRV1 infection on host fungi. By comparing the virus-carrying (9-1) and virus-cured (9-1C) strains, our results indicated that infection with NoNRV1 promoted the pigmentation of the host cells, while it had no discernable effects on host growth on potato dextrose agar plates when subjected to osmotic or oxidative stress. Interestingly, we observed inhibitory impacts of virus infection on the thermotolerance of N. oryzae and the pathogenicity of the host fungus in cotton leaves. Collectively, our work provides clear evidence of the biological relevance of NoNRV1 infection in N. oryzae, including pigmentation, hypovirulence, and thermotolerance.

Directional bioconversion and optimization of stevioside into rubusoside by Lelliottia sp. LST-1.

AIMS: The present study aimed to specifically transform stevioside (ST) into rubusoside (RS) through bioconversion with high efficiency, seeking to endow steviol glycosides (SGs) with subtle flavours for commercial acceptability. METHODS AND RESULTS: An endophytic bacterium named Lelliottia LST-1 was screened and confirmed to specifically convert ST into RS, reaching a conversion rate of 75.4% after response surface optimization. Phylogenetic analysis combined with complete genome sequencing demonstrated that LST-1 was also presumed to be a new species. To further explore the principle and process of biological transformation, the potential beta-glucosidases GH3-1, GH3-2, GH3-3 and GH3-4 were expressed, purified and reacted with SGs. High-performance liquid chromatography revealed that all enzymes hydrolysed ST and generated RS, but substrate specificity analysis indicated that GH3-2 had the highest substrate specificity towards STs and the highest enzyme activity. CONCLUSION: The potential ß-glucosidase GH3-2 in Lelliottia sp. LST-1 was found to specifically and efficiently convert ST to RS. SIGNIFICANCE AND IMPACT OF STUDY: The efficient biotransformation of ST into RS will be beneficial to its large-scale production and extensive application in the food and pharmaceutical industries.

Transcriptional Differences Guided Discovery and Genetic Identification of Coprogen and Dimerumic Acid Siderophores in Metarhizium robertsii.

Siderophores are small molecular iron chelators and participate in the multiple cellular processes in fungi. In this study, biosynthesis gene clusters of coprogens and dimerumic acids were identified by transcriptional level differences of genes related to iron deficiency conditions in Metarhizium robertsii. This leads to the characterization of new coprogen metachelin C (1) and five known siderophores metachelin A (2), metachelin A-CE (3), metachelin B (4), dimerumic acid 11-mannoside (5), and dimerumic acid (6). The structure of metachelin C (1) was elucidated by NMR spectroscopy and HR-ESI-MS analysis. Genetic deletions of mrsidA, and mrsidD abolished the production of compounds 1-6 that implied their involvement in the biosynthesis of coprogen and dimerumic acid. Interestingly, NRPS gene mrsidD is responsible for biosynthesis of both coprogen and dimerumic acid, thus we proposed plausible biosynthetic pathways for the synthesis of coprogen and dimerumic acid siderophores. Therefore, our study provides the genetic basis for understanding the biosynthetic pathway of coprogen and dimerumic acid in Metarhizium robertsii.

Analysis of saponins detoxification genes in Ilyonectria mors-panacis G3B inducing root rot of Panax notoginseng by RNA-Seq.

Saponins are kinds of antifungal compounds produced by Panax notoginseng to resist invasion by pathogens. Ilyonectria mors-panacis G3B was the dominant pathogen inducing root rot of P. notoginseng, and the abilities to detoxify saponins were the key to infect P. notoginseng successfully. To research the molecular mechanisms of detoxifying saponins in I. mors-panacis G3B, we used high-throughput RNA-Seq to identify 557 and 1519 differential expression genes (DEGs) in I. mors-panacis G3B with saponins treatments for 4H (Hours) and 12H (Hours) compared with no saponins treatments, respectively. Among these DEGs, we found 93 genes which were simultaneously highly expressed in I. mors-panacis G3B with saponins treatments for 4H and 12H, they mainly belong to genes encoding transporters, glycoside hydrolases, oxidation-reduction enzymes, transcription factors and so on. In addition, there were 21 putative PHI (Pathogen-Host Interaction) genes out of those 93 up-regulated genes. In this report, we analyzed virulence-associated genes in I. mors-panacis G3B which may be related to detoxifying saponins to infect P. notoginseng successfully. They provided an excellent starting point for in-depth study on pathogenicity of I. mors-panacis G3B and developed appropriate root rot disease management strategies in the future.

Profiles of Bacillus spp. Isolated from the Rhizosphere of Suaeda glauca and Their Potential to Promote Plant Growth and Suppress Fungal Phytopathogens.

Members of the genus Bacillus are known to play an important role in promoting plant growth and protecting plants against phytopathogenic microorganisms. In this study, 21 isolates of Bacillus spp. were obtained from the root micro-ecosystem of Suaeda glauca. Analysis of the 16S rRNA genes indicated that the isolates belong to the species Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus subtilis, Bacillus pumilus, Bacillus aryabhattai and Brevibacterium frigoritolerans. One of the interesting findings of this study is that the four strains B1, B5, B16 and B21 are dominant in rhizosphere soil. Based on gyrA, gyrB, and rpoB gene analyses, B1, B5, and B21 were identified as B. amyloliquefaciens and B16 was identified as B. velezensis. Estimation of antifungal activity showed that the isolate B1 had a significant inhibitory effect on Fusarium verticillioides, B5 and B16 on Colletotrichum capsici (syd.) Butl, and B21 on Rhizoctonia cerealis van der Hoeven. The four strains grew well in medium with 1-10% NaCl, a pH value of 5-8, and promoted the growth of Arabidopsis thaliana. Our results indicate that these strains may be promising agents for the biocontrol and promotion of plant growth and further study of the relevant bacteria will provide a useful reference for the development of microbial resources.

Foetidibacter luteolus gen. nov., sp. nov., isolated from the rhizosphere of Salvia miltiorrhiza.

A polyphosphate-producing bacterium, YG09T, was isolated from the rhizosphere of Salvia miltiorrhiza. Its colonies were 2.0-3.0 mm in diameter, smooth, circular, convex and yellow after growth on R2A at 28 °C for 72 h, with aerobic, Gram-stain-negative, non-motile and rod-shaped bacteria. The strain was found to grow at 10-40 °C (optimum 37 °C), pH 5.5-8.0 (optimum 6.0), with 0-0.6% (w/v) NaCl (optimum 0). Chemotaxonomic analysis showed menaquinone-7 as the only quinone present; C15: 1 iso G, C15: 1 iso, C16: 0, C16: 0 3OH, C17: 0 iso 3OH, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) as the major fatty acids (> 5%), and phosphatidylethanolamine, three unidentified phospholipids, four unidentified polar lipids, three unidentified aminolipids, and one unidentified amino phospholipid as the polar lipids. The DNA G + C content was 44.6 mol%. The 16S rRNA gene sequences of the isolate showed highest similarities to Panacibacter ginsenosidivorans Gsoil 1550T (93.6%), Filimonas endophytica SR2-06T (93.4%), Parasegetibacter terrae SGM2-10T (92.8%), and Arvibacter flaviflagrans C-1-16T (92.7%), within the family Chitinophagaceae of the phylum Bacteroidetes. The ANI values between strain YG09T and Panacibacter ginsenosidivoran Gsoil 1550T, Filimonas endophytica SR2-06T and Filimonas lacunae YT21T were 69.4, 68.3 and 68.7%, respectively. Based on phenotypic, genotypic and phylogenetic analyses, strain YG09T represents a novel genus in the family Chitinophagaceae, for which the name Foetidibacter luteolus gen. sp. nov. is proposed. The type strain is Foetidibacter luteolus YG09T (= MCCC 1K04042T = KCTC 72595T).

Horizontal gene transfer allowed the emergence of broad host range entomopathogens.

The emergence of new pathogenic fungi has profoundly impacted global biota, but the underlying mechanisms behind host shifts remain largely unknown. The endophytic insect pathogen Metarhizium robertsii evolved from fungi that were plant associates, and entomopathogenicity is a more recently acquired adaptation. Here we report that the broad host-range entomopathogen M. robertsii has 18 genes that are derived via horizontal gene transfer (HGT). The necessity of degrading insect cuticle served as a major selective pressure to retain these genes, as 12 are up-regulated during penetration; 6 were confirmed to have a role in penetration, and their collective actions are indispensable for infection. Two lipid-carrier genes are involved in utilizing epicuticular lipids, and a third (MrNPC2a) facilitates hemocoel colonization. Three proteases degraded the procuticular protein matrix, which facilitated up-regulation of other cuticle-degrading enzymes. The three lipid carriers and one of the proteases are present in all analyzed Metarhizium species and are essential for entomopathogenicity. Acquisition of another protease (MAA_01413) in an ancestor of broad host-range lineages contributed to their host-range expansion, as heterologous expression in the locust specialist Metarhizium acridum enabled it to kill caterpillars. Our work reveals that HGT was a key mechanism in the emergence of entomopathogenicity in Metarhizium from a plant-associated ancestor and in subsequent host-range expansion by some Metarhizium lineages.

Duplication of a Pks gene cluster and subsequent functional diversification facilitate environmental adaptation in Metarhizium species.

The ecological importance of the duplication and diversification of gene clusters that synthesize secondary metabolites in fungi remains poorly understood. Here, we demonstrated that the duplication and subsequent diversification of a gene cluster produced two polyketide synthase gene clusters in the cosmopolitan fungal genus Metarhizium. Diversification occurred in the promoter regions and the exon-intron structures of the two Pks paralogs (Pks1 and Pks2). These two Pks genes have distinct expression patterns, with Pks1 highly expressed during conidiation and Pks2 highly expressed during infection. Different upstream signaling pathways were found to regulate the two Pks genes. Pks1 is positively regulated by Hog1-MAPK, Slt2-MAPK and Mr-OPY2, while Pks2 is positively regulated by Fus3-MAPK and negatively regulated by Mr-OPY2. Pks1 and Pks2 have been subjected to positive selection and synthesize different secondary metabolites. PKS1 is involved in synthesis of an anthraquinone derivative, and contributes to conidial pigmentation, which plays an important role in fungal tolerance to UV radiation and extreme temperatures. Disruption of the Pks2 gene delayed formation of infectious structures and increased the time taken to kill insects, indicating that Pks2 contributes to pathogenesis. Thus, the duplication of a Pks gene cluster and its subsequent functional diversification has increased the adaptive flexibility of Metarhizium species.

Alternative transcription start site selection in Mr-OPY2 controls lifestyle transitions in the fungus Metarhizium robertsii.

Metarhizium robertsii is a versatile fungus with saprophytic, plant symbiotic and insect pathogenic lifestyle options. Here we show that M. robertsii mediates the saprophyte-to-insect pathogen transition through modulation of the expression of a membrane protein, Mr-OPY2. Abundant Mr-OPY2 protein initiates appressorium formation, a prerequisite for infection, whereas reduced production of Mr-OPY2 elicits saprophytic growth and conidiation. The precise regulation of Mr-OPY2 protein production is achieved via alternative transcription start sites. During saprophytic growth, a single long transcript is produced with small upstream open reading frames in its 5' untranslated region. Increased production of Mr-OPY2 protein on host cuticle is achieved by expression of a transcript variant lacking a small upstream open reading frame that would otherwise inhibit translation of Mr-OPY2. RNA-seq and qRT-PCR analyses show that Mr-OPY2 is a negative regulator of a transcription factor that we demonstrate is necessary for appressorial formation. These findings provide insights into the mechanisms regulating fungal lifestyle transitions.

Genome-wide identification of pathogenicity, conidiation and colony sectorization genes in Metarhizium robertsii.

Metarhizium robertsii occupies a wide array of ecological niches and has diverse lifestyle options (saprophyte, insect pathogen and plant symbiont), that renders it an unusually effective model for studying genetic mechanisms for fungal adaptation. Here over 20,000 M. robertsii T-DNA mutants were screened in order to elucidate genetic mechanism by which M. robertsii replicates and persists in diverse niches. About 287 conidiation, colony sectorization or pathogenicity loci, many of which have not been reported in other fungi were identified. By analysing a series of conidial pigmentation mutants, a new fungal pigmentation gene cluster, which contains Mr-Pks1, Mr-EthD and Mlac1 was identified. A conserved conidiation regulatory pathway containing Mr-BrlA, Mr-AbaA and Mr-WetA regulates expression of these pigmentation genes. During conidiation Mr-BlrA up-regulates Mr-AbaA, which in turn controls Mr-WetA. It was found that Hog1-MAPK regulates fungal conidiation by controlling the conidiation regulatory pathway, and that all three pigmentation genes exercise feedback regulation of conidiation. This work provided the foundation for deeper understanding of the genetic processes behind M. robertsii adaptive phenotypes, and advances our insights into conidiation and pigmentation in this fungus.

Deletion of a Histone Acetyltransferase Leads to the Pleiotropic Activation of Natural Products in Metarhizium robertsii.

Histone deacetylation normally decreases the gene expression in organisms. By genome-wide deletions of epigenetic regulators in entomopathogenic fungus Metarhizium robertsii, unexpected activations of orphan secondary metabolite genes have been found upon the disruption of a histone acetyltransferase (HAT) gene Hat1. This led to the characterization of 11 new natural products, including eight isocoumarin derivatives meromusides A-H and two nonribosomal peptides meromutides A and B. Therefore, disruption of HAT represents a new approach to mine chemical diversity from fungi.

MAPK cascade-mediated regulation of pathogenicity, conidiation and tolerance to abiotic stresses in the entomopathogenic fungus Metarhizium robertsii.

Metarhizium robertsii has been used as a model to study fungal pathogenesis in insects, and its pathogenicity has many parallels with plant and mammal pathogenic fungi. MAPK (Mitogen-activated protein kinase) cascades play pivotal roles in cellular regulation in fungi, but their functions have not been characterized in M. robertsii. In this study, we identified the full complement of MAPK cascade components in M. robertsii and dissected their regulatory roles in pathogenesis, conidiation and stress tolerance. The nine components of the Fus3, Hog1 and Slt2-MAPK cascades are all involved in conidiation. The Fus3- and Hog1-MAPK cascades are necessary for tolerance to hyperosmotic stress, and the Slt2- and Fus3-MAPK cascades both mediate cell wall integrity. The Hog1 and Slt2-MAPK cascades contribute to pathogenicity; the Fus3-MAPK cascade is indispensable for fungal pathogenesis. During its life cycle, M. robertsii experiences multiple microenvironments as it transverses the cuticle into the haemocoel. RNA-seq analysis revealed that MAPK cascades collectively play a major role in regulating the adaptation of M. robertsii to the microenvironmental change from the cuticle to the haemolymph. The three MAPKs each regulate their own distinctive subset of genes during penetration of the cuticle and haemocoel colonization, but they function redundantly to regulate adaptation to microenvironmental change.

Toxicity of nickel ions and comprehensive analysis of nickel ion-associated gene expression profiles in THP-1 cells.

The aim of the present study was to explore the toxic effects and underlying mechanisms of nickel ions during therapeutic nickel­based alloy­treatment in congenital heart disease by investigating the metal­induced cytotoxicity to the human monocyte­derived macrophage cell line THP­1. THP­1 cells were treated with NiCl2·6H2O (25, 50, 100, 200, 400 and 800 µM) for 24, 48 and 72 h, respectively. MTT was applied to detect THP­1 cell proliferation following NiCl2 treatment. Apoptosis of THP­1 cells was quantified using flow cytometry. Illumina sequencing was used for screening the associated genes, whose mRNA expression levels were further confirmed by quantitative real­time polymerase chain reaction. High concentrations of nickel ions had a significant suppressive effect on cell proliferation at the three concentrations investigated (200, 400 and 800 µM). Treatment with nickel ions (25­400 µM) for 48 h reduced cell viability in a dose­dependent manner. The mRNA expression levels of RELB, FIGF, SPI­1, CXCL16 and CRLF2 were significantly increased following nickel treatment. The results of the present study suggested that nickel ions exert toxic effects on THP­1 cell growth, which may indicate toxicity of the nickel ion during treatment of congenital heart disease. The identification of genes modified by the toxic effects of nickel on THP­1 cells (EPOR, RELB, FIGF, SPI­1, TGF­ß1, CXCL16 and CRLF2) may aid in the development of interventional measures for the treatment/prevention of nickel ion­associated toxic effects during the treatment of congenital heart disease.

Endoscopic closure of acquired oesophagorespiratory fistulas with cardiac septal defect occluders or vascular plugs.

OBJECTIVES: To report an endoscopic treatment for inoperable oesophagorespiratory fistulas (ORFs). PATIENTS AND METHODS: Six patients with inoperable acquired tracheobronchial-oesophageal fistulas (four males and two females; mean age, 70.2 ± 10.28 years) were included. Cardiac septal defect occluders or vascular plugs were implanted through a flexible bronchoscope to close the ORFs. Monthly follow-ups were done for 16 months. RESULTS: All fistulas were successfully closed immediately after the procedure. The severe aspirated pneumonia was controlled in 7-10 days. The two mechanically ventilated patients were weaned successfully from the ventilator, and the general condition of patients improved rapidly. However, the fistulas recanalised in four patients because of the cutting effect of the edge of the occluders 1-12 months after the procedure. Other reasons, such as compression of the tracheal intubation balloon and repeated inflammation of the oesophageal diverticulum, may also have contributed to the recanalisation. CONCLUSIONS: Endoscopic closure of acquired ORFs with cardiac septal defect occluders or vascular plugs improve patients' general condition immediately after the procedure, but may result in recanalisation longterm. The occlusion might be considered an abridgement to surgery.

[Efficacy and experience in right ventricular pacing-percutaneous balloon aortic valvuloplasty].

OBJECTIVE: To evaluate the efficacy and experience in right ventricular pacing-percutaneous balloon aortic valvuloplasty (RVP-PBAV) for congenital aortic stenosis (AS). METHOD: A total of sixteen children with AS accepted the treatment with RRVP-PBAV. The patients were at ages 6 months to 15 years, their median age was 5.4 years. Their body weight was between 8.5 and 59.0 kg, average (22.3 ± 16.5) kg. The gradient pressure across the aortic valve was measured for all the patients and aortic regurgitation was observed. The follow-up time ranged from 1 month to 5.5 years. RESULT: All patients underwent RVP-PBAV successfully. The ratios of balloon/valve were 0.86 to 1.12. The gradient pressure varied from preoperative Δp = (96 ± 32) mmHg (1 mmHg = 0.133 kPa) to the immediate postoperative ΔP = (41 ± 26) mmHg, (P < 0.05). One case had postoperative restenosis, and 3 cases were complicated with bicuspid aortic valve deformity. CONCLUSION: The treatment with RVP-PBAV for congenital aortic stenosis is safe and reliable. Rapid ventricular pacing is a safe procedure to stabilize the balloon during balloon aortic valvuloplasty and may decrease the incidence of aortic insufficiency.

[Genetics and pedigree analysis of primary carnitine deficiency cardiomyopathy in 6 cases].

OBJECTIVE: To investigate the mutation and background of SLC22A5 in 6 patients with primary carnitine deficiency (PCD) who only presented as cardiomyopathy. METHOD: Genomic DNA were abstracted from the blood of the patients and their parents. Using high-throughput sequencing to determine the mutation site.Using Sanger method to confirm the mutated alleles in PCD patients and detect the corresponding sequences in their patients. Using SIFT and PolyPhen to predict the function of protein for detected missense mutations. RESULT: Three different mutations were identified, including 2 nonsense mutations (R254X and R289X), 1 missense mutation (C113Y), R254X was the most frequently seen mutation. Four patients had compound heterozygous mutations and 2 patients had homozygous mutations. Their parents were found to have heterozygous mutations in corresponding alleles. CONCLUSION: R254X, R289X and C113Y might be associated with primary carnitine deficiency.