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Root exudates play a crucial role in the symbiosis between leguminous plants and rhizobia. Our previous studies have shown that a fungal endophyte Phomopsis liquidambaris promotes peanut-rhizobia nodulation and nitrogen fixation, but the underlying mechanism are largely unknown. Here, we explore the role of peanut root exudates in Ph. liquidambaris-mediated nodulation enhancement. We first collected root exudates from Ph. liquidambaris-inoculated and un-inoculated peanuts and determined their effects on rhizobial growth, biofilm formation, chemotaxis, nodC gene expression, and peanut nodulation. Our results found a positive effect of Ph. liquidambaris-inoculated root exudates on these characteristics of rhizobia. Next, we compared the root exudates profile of Ph. liquidambaris-inoculated and un-inoculated plants and found that Ph. liquidambaris altered the concentrations of phenolic acids, flavonoids, organic acids and amino acids in root exudates. Furthermore, the rhizobial chemotaxis, growth and biofilm formation in response to the changed compounds at different concentrations showed that all of the test compounds induced rhizobial chemotactic behavior, and organic acids (citric acid and oxalic acid) and amino acid (glutamate, glycine and glutamine) at higher concentrations increased rhizobial growth and biofilm formation. Collectively, our results suggest that root exudates alterations contribute to Ph. liquidambaris-mediated peanut-rhizobia nodulation enhancement., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Abundant gene clusters of natural products are observed in the endophytic fungus Phomopsis liquidambaris ; however, most of them are silent. Herein, a plug-and-play DNA assembly tool has been applied for flavonoid synthesis in P. liquidambaris. A shuttle plasmid was constructed based on S. cerevisiae , E. coli , and P. liquidambaris with screening markers URA , Amp , and hygR , respectively. Each fragment or cassette was successively assembled by overlap extension PCR with at least 40-50 bp homologous arms in S. cerevisiae for generating a new vector. Seven native promoters were screened by the DNA assembly based on the fluorescence intensity of the mCherry reporter gene in P. liquidambaris , and two of them were new promoters. The key enzyme chalcone synthase was the limiting step of the pathway. The naringenin and kaempferol pathways were refactored and activated with the titers of naringenin and kaempferol of 121.53 mg/L and 75.38 mg/L in P. liquidambaris using fed-batch fermentation, respectively. This study will be efficient and helpful for the biosynthesis of secondary metabolites.

Background: Rice spikelet rot disease (RSRD) is an emerging disease that significantly reduces rice yield and quality. In this study, we evaluated the potential use of the broad-spectrum endophytic fungus Phomopsis liquidambaris B3 as a biocontrol agent against RSRD. We also compared the control effects of different treatments, including chemical fungicides and treatment with multiple strains and single strains in combination or individually, against RSRD. The objective of this study was to find an effective and environmentally friendly control strategy to reduce the occurrence of RSRD and improve the rice yield., Results: In pot experiments, the effect of B3 alone was better than that of fungicide or combined measures. The results showed that root colonization by B3 significantly reduced the incidence and disease index of RSRD by 41.0% and 53.8%, respectively. This was related to enhanced superoxide dismutase (SOD), peroxidase (POD), and polyphenol oxidase (PPO) activity, and to significantly upregulated expression levels of OsAOX, OsLOX, OsPAL, and OsPR10 in rice. Moreover, B3 improved the diversity of the bacterial community rather than the fungal community in the rice rhizosphere. It also led to a decrease in Fusarium proliferatum colonization and fumonisin content in the grain. Finally, root development was markedly promoted after B3 inoculation, and the yield improved by 48.60%. The result of field experiments showed that the incidence of RSRD and the fumonisin content were observably reduced in rice receiving B3, by 24.41% and 37.87%, respectively., Conclusion: The endophytic fungus Phomopsis liquidambaris B3 may become an effective tool to relieve rice spikelet rot disease. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Jasmonate restricts accumulation of constitutive and fungus-induced root soluble sugars at flowering stage, and thus reduces root beneficial fungal colonization, but little is known about how these are achieved. To determine whether jasmonate-mediated depletion of soluble sugars is the result of direct phytohormonal cross-talk or indirect induced defensive secondary metabolism, we first profiled soluble sugar and tryptophan (Trp)-derived defensive secondary metabolites in the roots of wild-type and jasmonate signaling-impaired Arabidopsis thaliana at flowering upon a beneficial fungus Phomopsis liquidambaris inoculation. Next, jasmonate and gibberellin signaling were manipulated to determine the relationship between jasmonate and gibberellin, and to quantify the effects of these phytohormones on fungal colonization degree, soluble sugar accumulation, Trp-derived secondary metabolites production, and sugar source-sink transport and metabolism. Gibberellin complementation increased Ph. liquidambaris colonization and rescued jasmonate-dependent root soluble sugar depletion and phloem sugar transport and root invertase activity without influencing jasmonate-induced Trp-derived secondary metabolites production at flowering. Furthermore, jasmonate signaling antagonized gibberellin biosynthesis in Ph. liquidambaris-inoculated roots. Our results suggest a phytohormonal antagonism model that jasmonate signaling restricts root soluble sugar accumulation through antagonizing gibberellin biosynthesis rather than through promoting Trp-derived secondary metabolites production and thus drives beneficial fungal colonization decline at flowering., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In agroecosystems, drought stress severely threatens crops development. Although potassium (K) is required in amounts by crops under drought stress, the mobilization and availablity of K are limited by the soil water status. Arbuscular mycorrhizal (AM) fungi can form mutualistic associations with most crops and play direct or indirect roles in the host drought resistance. Considering that the glomalin generated by living AM fungal hyphae can sequester multiple minerals, however, the function of mineral-sequestering glomalin in the crop drought resistance remains unclear. In this study, peanuts cultivated in the sterilized soil with a history of AM fungi inoculation showed significantly enhanced leaf K accumulation, drought resistance and pod yield under drought stress. Through the collection of different types of mineral-sequestering glomalin from living AM fungal hyphae, the peanut drought resistance was improved only when K-sequestering glomalin was added. Moreover, we found that peanut root exudates could prime the dissociation of glomalin-bound K and further satisfy the K requirement of crops. Our study is the first report that K-sequestering glomalin could improve drought performance and peanut pod yield, and it helps us to understand the ecological importance of improving AM symbiosis to face agricultural challenges., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Endophytic fungi can profoundly affect host productivity, but the underlying mechanisms of these effects are only partly understood. As the most important regulators of plant-soil feedback, root exudates can easily cause soil sickness in continuous monoculture systems by reducing certain microbes in the rhizosphere. In this study, exudates from roots colonized by the endophytic fungus Phomopsis liquidambaris significantly increased rhizosphere bacterial abundance, soil respiration, microbial biomass and enzyme activities in a long-term continuously cropped peanut soil. Further analysis revealed that P. liquidambaris-colonized root exudates clearly altered the carbon metabolism and rhizosphere bacterial diversity, which were closely correlated with changes in soil chemical properties caused by the exudates from the colonized roots. Finally, a synthetic root exudate experiment further confirmed that the root exudates derived from P. liquidambaris colonization can indeed play an important role in promoting peanut growth. Therefore, these results show that this endophytic fungus could improve the carbon metabolism and rhizosphere bacterial community in long-term monoculture soils via exudates from colonized roots, which contribute to the alleviation of soil sickness., (© 2020. Springer Science+Business Media, LLC, part of Springer Nature.)

Composting has become the most important way to recycle medicinal herbal residues (MHRs). The traditional composting method, adding a microbial agent at one time, has been greatly limited due to its low composting efficiency, mutual influence of microbial agents, and unstable compost products. This study was conducted to assess the effect of multi-phase inoculation on the lignocellulose degradation, enzyme activities, and fungal community during MHRs composting. The results showed that multi-phase inoculation treatment had the highest thermophilic temperature (68.2 °C) and germination index (102.68%), significantly improved available phosphorus content, humic acid, and humic substances concentration, accelerated the degradation of cellulose and lignin, and increased the activities of cellulase in the mature phase, xylanase, manganese peroxidase, and utilization of phenolic compounds. Furthermore, the non-metric multi-dimensional scaling showed that the composting process and inoculation significantly influenced fungal community composition. In multi-phase inoculation treatment, Thermomyces in mesophilic, thermophilic, and mature phase, unclassified_Sordariales, and Coprinopsis in mature phase were the dominant genus that might be the main functional groups to degrade lignocellulose and improve the MHRs composting process.

We recently reported that polyethylenimine (PEI; molecular weight of 600 Da) acted as a vaccine adjuvant for liposomal group A Streptococcus (GAS) vaccines, eliciting immune responses in vivo with IgG antibodies giving opsonic activity against five Australian GAS clinical isolates. However, to date, no investigation comparing the structure-activity relationship between the molecular weight of PEI and its adjuvanting activity in vaccine development has been performed. We hypothesized that the molecular weight and quantity of PEI in a liposomal vaccine will impact its adjuvanting properties. In this study, we successfully formulated liposomes containing different molecular weights of PEI (600, 1800, 10k and 25k Da) and equivalents of PEI (0.5, 1 and 2) of branched PEI. Outbred mice were administrated the vaccine formulations intranasally, and the mice that received a high ratio of PEI 600 reported a stronger immune response than the mice that received a lower ratio of PEI 600. Interestingly, mice that received the same quantity of PEI 600, PEI 10k and PEI 25k showed similar immune responses in vivo and in vitro. This comparative study highlights the ratio of PEI present in the liposome vaccines impacts adjuvanting activity, however, PEI molecular weight did not significantly enhance its adjuvanting properties. We also report that the stability of PEI liposomes is critical for vaccines to elicit the desired immune response., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A smartphone-combined dual-emission ratiometric fluorescence probe for specifically and visibly detecting cephalexin was first designed. In the probe, blue-emitting fluorescent carbon dots (CDs) was synthesized and covered with a layer of silica spacer. Red-emitting fluorescent CdTe QDs (r-QDs) was grafted onto the silica nanospheres as an analytical probe. Then, the cephalexin antibody was covalent grafted to the ratio sensor to increase the selectivity. The ratio of fluorescence intensity (FL) of r-QDs and CDs was quenched with the increasing concentration of cephalexin. The detection method has good linear response in the range of 1-500 μM and the detection limit was 0.7 μM. Then portable device based on smartphone detection was constructed according to the color change under UV lamp. The detection image was obtained through the smartphone camera, and the color picker APP installed in the smartphone captured the RGB value of the image. In addition, this method was also used to determine the amount of cephalexin in milk samples with recovery of 94.1%-102.2%. These results showed that it was a portable, simple and visible method to detect cephalexin in food analysis and environmental monitoring., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Identifying the immunogenic moieties and their precise structure of carbohydrates plays an important role for developing effective carbohydrate-based subunit vaccines. This study assessed the structure-immunogenicity relationship of carbohydrate moieties of a single repeating unit of group A carbohydrate (GAC) present on the cell wall of group A Streptococcus (GAS) using a rationally designed self-adjuvanted lipid-core peptide, instead of a carrier protein. Immunological evaluation of fully synthetic glyco-lipopeptides (particle size: 300-500 nm) revealed that construct consisting of higher rhamnose moieties (trirhamnosyl-lipopeptide) was able to induce enhanced immunogenic activity in mice, and GlcNAc moiety was not found to be an essential component of immunogenic GAC mimicked epitope. Trirhamnosyl-lipopeptide also showed 75-97% opsonic activity against four different clinical isolates of GAS and was comparable to a subunit peptide vaccine (J8-lipopeptide) which illustrated 65-96% opsonic activity.

Endophytic bacteria are always related to the host different traits, including the secondary metabolites production. However, the effect and mechanism of endophytic bacteria in the mushrooms fruit body on mycelia are still not clear. In this study, we investigated the effect of endophytic bacterial metabolites on the quality of Lyophyllum decastes mycelia. Soluble sugars, starch, protein, free amino acids, 5'-Nucleotides, EUC, and organic acids contents of mycelia were analyzed. We found that endophytic bacterial metabolites significantly increased the contents of soluble sugars, starch, protein, free amino acids, organic acids, and EUC. The present study thus suggests that endophytic bacteria could promote the quality of Lyophyllum decastes by improving non-volatile taste components of mycelia., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Flavonoids, which are mainly extracted from plants, are important antioxidants and play an important role in human diseases. However, the growing market demand is limited by low productivity and complex production processes. Herein, the flavonoids biosynthesis pathway of the endophytic fungus Phomopsis liquidambaris was revealed. The mitogen-activated protein kinase kinase (MAPKK) of the strain was disrupted using a newly constructed CRISPR-Cas9 system mediated by two gRNAs which was conducive to cause plasmid loss. The disruption of the MAPKK gene triggered the biosynthesis of flavonoids against stress and resulted in the precipitation of flavonoids from fermentation broth. Naringenin, kaempferol and quercetin were detected in fed-batch fermentation with yields of 5.65 mg/L, 1.96 mg/L and 2.37 mg/L from P. liquidambaris for dry cell weigh using the mixture of glucose and xylose and corn steep powder as carbon source and nitrogen source for 72 h, respectively. The biosynthesis of flavonoids was triggered by disruption of MAPKK gene in P. liquidambaris and the mutant could utilize xylose.

N6-methyladenosine (m6A) modification regulatory proteins are involved in the development of many types of cancer. KIAA1429 serves as a scaffold in bridging the catalytic core components of the m6A methyltransferase complex. The role of KIAA1429 in gastric cancer and its related mechanism has not been reported upon. The expression of KIAA1429 was detected in human gastric cancer tissues and cell lines by quantitative real-time polymerase chain reaction and western blot. The effects of KIAA1429 on gastric cancer proliferation were evaluated by cell counting kit assays, colony formation assays, flow cytometry assay, and in vivo experiments with nude mice. And messenger RNA (mRNA) high-throughput sequencing, RNA immunoprecipitation assay (RIP), luciferase assay, and a rescue experiment were used to identify the relationship between KIAA1429 and its specific targeted gene, c-Jun. We found that KIAA1429 was upregulated in gastric cancer tissues, and expressed lower in adjacent tissues. The upregulated KIAA1429 promoted proliferation and downregulated KIAA1429 was proved to inhibit proliferation of gastric cancer in vitro and in vivo. Then, we identified the potential KIAA1429 regulating gene as c-Jun by mRNAs high-throughput sequencing and RIP assay. By luciferase assay, we verified that KIAA1429 regulated the expression of c-Jun in an m6A-independent manner. Finally, the overexpression of c-Jun rescued the inhibition of proliferation caused by KIAA1429 knockdown in gastric cancer cells. KIAA1429 could act as an oncogene in gastric cancer by stabilizing c-Jun mRNA in an m6A-independent manner. This highlights the functional role for KIAA1429 as a potential prognostic biomarker and therapeutic target in gastric cancer., (© 2020 Wiley Periodicals, Inc.)

Group A Streptococcus (GAS) and GAS-related infections are a worldwide challenge, with no commercial GAS vaccine available. Polyethylenimine (PEI) attaches to the cells' surface and delivers cargo into endosomal and cytosolic compartments. We hypothesized that this will confer mucosal adjuvant properties for peptide antigens against group A Streptococcus (GAS). In this study, we successfully demonstrated the development of PEI incorporated liposomes for the delivery of a lipopeptide-based vaccine (LCP-1) against GAS. Outbred mice were administrated with the vaccine formulations intranasally, and immunological investigation showed that the PEI liposomes elicited significant mucosal and systemic immunity with the production of IgA and IgG antibodies. Antibodies were shown to effectively opsonize multiple isolates of clinically isolated GAS. This proof-of-concept study showed the capability for PEI liposomes to act as a safe vehicle for the delivery of GAS peptide antigens to elicit immune responses against GAS infection, making PEI a promising addition to liposomal mucosal vaccines.

The mechanisms underlying nitrogen (N)-regulated plant-fungi interactions are not well understood. N application modulates plant carbohydrate (C) sinks and is involved in the overall plant-fungal association. We hypothesized that N regulates plant-fungi interactions by influencing the carbohydrate metabolism. The mutualistic fungus Phomopsis liquidambaris was found to prioritize host hexose resources through in vitro culture assays and in planta inoculation. Rice-Ph. liquidambaris systems were exposed to N gradients ranging from N-deficient to N-abundant conditions to study whether and how the sugar composition was involved in the dynamics of N-mediated fungal colonization. We found that root soluble acid invertases were activated, resulting in increased hexose fluxes in inoculated roots. These fluxes positively influenced fungal colonization, especially under N-deficient conditions. Further experiments manipulating the carbohydrate composition and root invertase activity through sugar feeding, chemical treatments and the use of different soil types revealed that the external disturbance of root invertase could reduce endophytic colonization and eliminate endophyte-induced host benefits under N-deficient conditions. Collectively, these results suggest that the activation of root invertase is related to N deficiency-enhanced endophytic colonization via increased hexose generation. Certain combinations of farmland ecosystems with suitable N inputs could be implemented to maximize the benefits of plant-fungi associations., (© FEMS 2020.)

Objective: To compare the short-term outcomes and long-term survivals of radical antegrade modular pancreatosplenectomy(RAMPS) and conventional distal pancreatectomy(CDP). Methods: A total of consecutive 304 patients including 176 male patients and 128 female patients who underwent RAMPS or CDP at Pancreas Center, the First Affiliated Hospital with Nanjing Medical University from May 2013 to June 2019 were retrospectively analyzed. The median age was 64.1 years old (range:39 to 85 years old). There were 101 patients underwent RAMPS and 203 patients underwent CDP. Measurement data with skewed distribution were presented as ( M ( Q(R) )) and comparison between groups was evaluated with the Wilcoxon rank sum test. Count data were analyzed using the χ(2) test or Fisher exact probability. Survival analyses were performed by the Kaplan-Meier method after a one to one propensity score matching(PSM) conducted to balance several variables. Results: An eighty-one to eighty-one patients were enrolled after PSM. The overall morbidity was 32.1%(26/81)and there were no in-hospital mortalities in RAMPS. The median operative time was 225(95)minutes in RAMPS, not significantly longer as compared with CDP(210(130)minutes, P= 0.916). The median greatest tumor diameter in RAMPS was 4.0(2.3)cm, not significantly larger as compared with CDP(4.5(2.2)cm, P= 0.520).There were 34.6%(28/81)patients who presented with T4 tumors by 8(th) AJCC TNM staging system in RAMPS, which was not significantly different as compared with CDP(39.5%, χ(2)=0.574, P= 0.902). The median number of examined lymph nodes was 9(9), not significantly greater in RAMPS as compared with CDP(10(11), P= 0.992). The rate of negative posterior margins using 1 mm rule in RAMPS was 70.3%(52/74), significantly higher as compared with CDP(53.6%(30/56), χ(2)=3.817, P= 0.044). The overall R0 resection rate was 44.6% (33/74) in RAMPS and 37.5% (21/56) in CDP, which was not significantly different(χ(2)=0.663, P= 0.474). The median overall survival was 16.5 months for RAMPS, 25.2 months for CDP, and there was no statistical difference between two groups( P= 0.981). The median overall survival was 16.0 months for patients with preoperative CA19-9≥300 U/ml who underwent RAMPS, 10.1 months for patients who underwent CDP, without significant difference( P= 0.082). Conclusions: RAMPS can improve the rate of negative posterior margins by 1 mm rule and probably increase R0 resection rate and the harvest of lymph nodes. RAMPS may be beneficial to some patients with preoperative CA19-9≥300 U/ml.

The access of rhizobia to legume host is a prerequisite for nodulation. Rhizobia are poorly motile in soil, while filamentous fungi are known to grow extensively across soil pores. Since root exudates-driven bacterial chemotaxis cannot explain rhizobial long-distance dispersal, mycelia could constitute ideal dispersal networks to help rhizobial enrichment in the legume rhizosphere from bulk soil. Thus, we hypothesized that mycelia networks act as vectors that enable contact between rhizobia and legume and influence subsequent nodulation. By developing a soil microcosm system, we found that a facultatively biotrophic fungus, Phomopsis liquidambaris, helps rhizobial migration from bulk soil to the peanut (Arachis hypogaea) rhizosphere and, hence, triggers peanut-rhizobium nodulation but not seen in the absence of mycelia. Assays of dispersal modes suggested that cell proliferation and motility mediated rhizobial dispersal along mycelia, and fungal exudates might contribute to this process. Furthermore, transcriptomic analysis indicated that genes associated with the cell division, chemosensory system, flagellum biosynthesis, and motility were regulated by Ph. liquidambaris, thus accounting for the detected rhizobial dispersal along hyphae. Our results indicate that rhizobia use mycelia as dispersal networks that migrate to legume rhizosphere and trigger nodulation. This work highlights the importance of mycelial network-based bacterial dispersal in legume-rhizobium symbiosis.

The endophytic fungus Phomopsis liquidambaris efficiently promotes the nitrogen metabolism and growth of host plants such as rice and peanut. However, a lack of genetic tools limits further research regarding the mechanisms of interaction between P. liquidambaris and its host plants. Herein, a CRISPR/Cas9 system for targeted gene disruption in this strain was first constructed and optimized. The knock-out efficiency increased to over 60% when the ku70 or ku80 gene (involved in nonhomologous end-joining, NHEJ) was disrupted. Furthermore, the CRISPR/Cas9 system was applied to disrupt the PmkkA gene, encoding a mitogen-activated protein kinase kinase (MAPKK) in the cell-wall integrity (CWI) MAPK pathway of the strain. The ΔPmkkA mutant strain induced higher reactive oxygen species (ROS) production, chitinase activity and glucanase activity in rice seedlings than wild-type P. liquidambaris (WT), resulting in growth inhibition and strong resistance on rice. These results suggested that the PmkkA gene is crucial during the interaction with rice and may play a role in inhibiting the immune system of host plants. The CRISPR-Cas9 system will be of great use for the study of the interaction between P. liquidambaris and its host plants., Competing Interests: Declaration of Competing Interest The authors declared that there is no conflict of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Phenanthrene can easily be absorbed into the plant from the soil and cannot be effectively degraded in it. Thus, it is greatly hazardous for food safety and human health. In our study, the biodegradability and remediation mechanism of endophytic fungus Phomopsis liquidambaris on phenanthrene in vivo of rice (Oryza sativa L.) was detected. The results showed that the fungus could successfully establish a symbiotic relationship with rice, thus had the potential to degrade phenanthrene absorbed into the plant. Changes of phenanthrene-degrading genes of fungus in the combined system were consistent with the trends of their corresponding enzymatic activities, and the phenanthrene-degrading enzyme activities and gene expression levels in roots of rice were higher than those in the shoot. Moreover, the combined system can enhance bioremediation by increasing root viability, chlorophyll content, and energy supply. The combined system had also significantly increased the PPO activity and SOD activity in shoot compared with the control treatment, while decreased the content of MDA when remediation in vivo. The study on the degradation mechanism of the combined system will help us to increase the practical application potential of endophyte to effectively repair contamination absorbed into plant seedlings., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: To explore the safety and effectiveness of left bundle branch area pacing (LBBAP) in children. Methods: Clinical data, pacing electrocardiogram and parameters of 6 patients (5 females and 1 male) who underwent permanent pacemaker implantation and LBBAP from January to June 2019 in the Department of Pediatric Cardiology of Anzhen Hospital were retrospectively analyzed. Results: The weight of the 6 patients aged between 9 and 14 years ranged from 26 to 48 kg. Five patients were diagnosed with third degree atrioventricular block, and 1 patient was diagnosed with cardiac dysfunction after right ventricular apical pacing. Cardiac function decreased in one patient and remained normal in the other five patients. Left ventricular end diastolic diameter (LVEDD) Z score was 1.85±0.65. V(1) lead showed changes like right bundle branch block in pacing electrocardiogram. No significant difference was found regarding QRS wave duration ((95±13) ms vs. (111±20) ms, t= -1.610, P> 0.05) between preoperation and postoperation. Pacing threshold was (0.85±0.26) V. The sensing threshold was (15.0±4.3) mV and the impedance was (717±72) Ω. P potential was recorded in 3 cases. The earliest left ventricular local activation time was (56±5) ms and remained stable at different output voltages. Postoperative echocardiography revealed that the electrodes were located near the endocardium of the left ventricular septum. No complications such as myocardial perforation and electrode dislocation occurred during follow-up. The pacing threshold, sensing threshold and impedance were (0.60±0.09)V, (16.1±3.9)mV, (662±78)Ω respectively at 3 months after operation. The patient with low left ventricular ejection fraction (LVEF) recovered to normal on the 3 rd day after LBBAP (45% vs. 57%). The LVEDD Z score decreased to (1.1±0.3) at 3 months after operation and was significantly lower than that before operation ( t= 2.38, P< 0.05). Conclusions: LBBAP in children can achieve narrow QRS pacing andphysiological pacing with stablepacing parameters. It can improve left ventricular enlargement caused by long-term bradycardia, and cardiac dysfunction and cardiac enlargement caused by long-term right ventricular apical pacing quickly and effectively.LBBAP is safe and feasible for older children in the near future. However, the long term potential risks of LBBAP need further observation and study.

Phytoremediation uses plants and associated microbes to remove pollutants from the environment and is considered a promising bioremediation method. Compared with well-described single contaminant treatments, the number of studies reporting phytoremediation of soil mixed pollutants has increased recently. Endophytes, including bacteria and fungi, exhibit beneficial traits for the promotion of plant growth, stress alleviation, and biodegradation. Moreover, endophytes either directly or indirectly assist host plants to survive high concentrations of organic and inorganic pollutants in the soil. Endophytic microorganisms can also regulate the plant metabolism in different ways, exhibiting a variety of physiological characteristics. This review summarizes the taxa and physiological properties of endophytic microorganisms that may participate in the detoxification of contaminant mixtures. Furthermore, potential biomolecules that may enhance endophyte mediated phytoremediation are discussed. The practical applications of pollutant-degrading endophytes and current strategies for applying this valuable bio-resource to soil phytoremediation are summarized.

The role of flowering in root-fungal symbiosis is not well understood. Because flowering and fungal symbionts are supported by carbohydrates, we hypothesized that flowering modulates root-beneficial fungal associations through alterations in carbohydrate metabolism and transport. We monitored fungal colonization and soluble sugars in the roots of Arabidopsis thaliana following inoculation with a mutualistic fungus Phomopsis liquidambari across different plant developmental stages. Jasmonate signalling of wild-type plants, sugar transport, and root invertase of wild-type and jasmonate-insensitive plants were exploited to assess whether and how jasmonate-dependent sugar dynamics are involved in flowering-mediated fungal colonization alterations. We found that flowering restricts root-fungal colonization and activates root jasmonate signalling upon fungal inoculation. Jasmonates reduce the constitutive and fungus-induced accumulation of root glucose and fructose at the flowering stage. Further experiments with sugar transport and metabolism mutant lines revealed that root glucose and fructose positively influence fungal colonization. Diurnal, jasmonate-dependent inhibitions of sugar transport and soluble invertase activity were identified as likely mechanisms for flowering-mediated root sugar depletion upon fungal inoculation. Collectively, our results reveal that flowering drives root-fungus cooperation loss, which is related to jasmonate-dependent root soluble sugar depletion. Limiting the spread of root-fungal colonization may direct more resources to flower development., (© 2019 John Wiley & Sons Ltd.)

Background: Most allelochemicals are secondary products released from root excretions or plant residues that accumulate in continuous cropping systems and cause severe decline in peanut yield. Resveratrol is a plant-derived stilbene that is released from peanut residues and accumulates in the soil; however, its allelopathic effects on peanut production are overlooked. Effective management solutions need to be developed to relieve allelopathy caused by soil resveratrol. Here, the biodegradation of resveratrol by the fungal endophyte Phomopsis liquidambari was investigated in a mineral salt medium and a soil trial. Resveratrol and its metabolites (produced by degradation by P. liquidambari) were detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS)., Results: Resveratrol released from peanut residues reached a maximum concentration of 0.18 μg g -1 soil in litterbag experiments. Exogenous resveratrol inhibited peanut growth, nodule formation, and soil dehydrogenase activity, and reduced the soil microbial biomass carbon content and bacterial abundance, indicating an allelopathic role in peanut growth. More than 97% of the resveratrol was degraded within 72 and 168 h by P. liquidambari in pure culture and soil conditions, respectively. Resveratrol was first cleaved to 3,5-dihydroxybenzaldehyde and 4-hydroxybenzaldehyde, which were subsequently oxidized into 3,5-dihydroxybenzoic acid and 4-hydroxybenzoic acid, respectively. Fungal resveratrol cleavage oxygenase and the related gene expression were enhanced when P. liquidambari was induced by the resveratrol during the incubation., Conclusion: Our results indicate that the practical application of the fungal endophyte P. liquidambari has strong potential for biodegrading soil resveratrol, which can cause allelopathy in peanut continuous cropping systems. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

Watermelon Fusarium wilt is a common soil-borne disease that has significantly affected its yield. In this study, fusaric acid-deficient mutant designated as ΔFUBT (mutated from Fusarium oxysporum f. sp. niveum , FON) was obtained. The ΔFUBT mutant showed significant decrease in fusaric acid production but maintained wild-type characteristics, such as in vitro colony morphology, size, and conidiation. A field pot experiment demonstrated that ΔFUBT could successfully colonize the rhizosphere and the roots of watermelon, leading to significant reduction in FON colonization in the watermelon plant. In addition, ΔFUBT inoculation significantly improved the rhizosphere microenvironment and effectively increased the resistance in watermelon. This study demonstrated that a nonpathogenic Fusarium mutant (ΔFUBT) could be developed as an effective microbial control agent to alleviate Fusarium wilt disease in watermelon and increase its yield.

Filamentous fungi can produce many valuable secondary metabolites; among these fungi, endophytic fungi play an ecological role in mutualistic symbiosis with plants, including promoting plant growth, disease resistance, and stress resistance. However, the biosynthesis of most secondary metabolites remains unclear, and knowledge of the interaction mechanisms between endophytes and plants is still limited, especially for some novel fungi, due to the lack of genetic manipulation tools for novel species. Herein, we review the newly discovered strategies of gene disruption, such as the CRISPR-Cas9 system, the site-specific recombination Cre/loxP system, and the I-SceI endonuclease-mediated system in filamentous fungi. Gene expression systems contain using integration of target genes into the genome, host-dependent expression cassette construction depending on the host, a host-independent, universal expression system independent of the host, and reporter-guided gene expression for filamentous fungi. Furthermore, the Newly CRISPRi, CRISPRa, and the selection markers were also discussed for gene disruption and gene expression were also discussed. These studies lay the foundation for the biosynthesis of secondary metabolites in these organisms and aid in understanding the ecological function of filamentous fungi.

Endophytic fungi play important roles in the modification of ecosystem productivity; however, the underlying mechanisms are only partly understood. A 2-year field plot experiment verified that the endophytic fungus Phomopsis liquidambaris increased peanut ( Arachis hypogaea L.) yields and significantly increased nodulation and N 2 fixation regardless of whether N fertilizers were added. Root exudates collected from P. liquidambaris -colonized plants significantly improved nodulation and N 2 fixation. Rhizosphere stimulation experiments further showed that colonized root exudates had significantly decreased soil nitrate (NO 3 - ) concentrations, with decreased abundance and diversity of ammonia oxidizing archaea (AOA). In contrast, the abundance and diversity of diazotrophs significantly increased, and most diazotrophs identified were peanut nodulation-related strains ( Bradyrhizobium sp.). P. liquidambaris symbiosis increased the expression of phenolic and flavonoid synthesis-related genes, and the derived phenolics and flavonoids could effectively increase the chemotaxis, biofilm formation, and nodC gene expression (nodulation-related biological processes) of the Bradyrhizobium strain. Metabolic pattern analysis showed that phenolics and flavonoids are more likely to accumulate to higher levels in the rhizosphere soil of peanuts colonized with P. liquidambaris Finally, a synthetic root exudate experiment further confirmed the underlying mechanisms for the P. liquidambaris -induced improvement in nodulation and N 2 fixation, i.e., that the specific root exudates derived from P. liquidambaris colonization decrease nitrate concentration and increase the population and biological activities of peanut nodulation-related Bradyrhizobium species, which beneficially enhance peanut- Bradyrhizobium interactions. Therefore, this study is the first to provide new insight into a positive relationship between an exotic endophytic fungus, crop nodulation, and N 2 fixation increase. IMPORTANCE Endophytic fungi play an important role in balancing the ecosystem and boosting host growth; however, the underpinning mechanisms remain poorly understood. Here, we found that endophytic fungal colonization with P. liquidambaris significantly increased the productivity, nodulation, and N 2 fixation of peanuts through the secretion of specific root exudates. We provide a reasonable mechanism explaining how P. liquidambaris promotes peanut nodulation and N 2 fixation, whereby the specific root exudates produced by P. liquidambaris colonization decrease rhizosphere soil nitrate (NO3 - ) and increase the population and biological activities of peanut-nodulating-related Bradyrhizobium strains, which is beneficial to enhancing the peanut- Bradyrhizobium symbiotic interaction. Our study provides reliable empirical evidence to show the mechanism of how an exotic endophytic fungus drives an increase in nodulation and N 2 fixation, which will be helpful in erecting a resource-efficient and sustainable agricultural system., (Copyright © 2019 Xie et al.)

The fungal endophyte Gilmaniella sp. AL12 can establish a beneficial association with the medicinal herb Atractylodes lancea , and improve plant growth and sesquiterpenoids accumulation, which is termed "double promotion." Our previous studies have uncovered the underling primary mechanism based on some physiological evidences. However, a global understanding of gene or protein expression regulation in primary and secondary metabolism and related regulatory processes is still lacking. In this study, we employed transcriptomics and proteomics of Gilmaniella sp. AL12-inoculated and Gilmaniella sp. AL12-free plants to study the impact of endophyte inoculation at the transcriptional and translational levels. The results showed that plant genes involved in plant immunity and signaling were suppressed, similar to the plant response caused by some endophytic fungi and biotroph pathogen. The downregulated plant immunity may contribute to plant-endophyte beneficial interaction. Additionally, genes and proteins related to primary metabolism (carbon fixation, carbohydrate metabolism, and energy metabolism) tended to be upregulated after Gilmaniella sp. AL12 inoculation, which was consistent with our previous physiological evidences. And, Gilmaniella sp. AL12 upregulated genes involved in terpene skeleton biosynthesis, and upregulated genes annotated as β-farnesene synthase and β-caryophyllene synthase. Based on the above results, we proposed that endophyte-plant associations may improve production (biomass and sesquiterpenoids accumulation) by increasing the source (photosynthesis), expanding the sink (glycolysis and tricarboxylic acid cycle), and enhancing the metabolic flux (sesquiterpenoids biosynthesis pathway) in A. lancea . And, this study will help to further clarify plant-endophyte interactions.

The endophytic fungus Phomopsis liquidambari play a key role in habitat adaptation of rice (Oryza sativa L.) with potential multiple beneficial. However, our previous published work on this subject remains incomplete. Here, we performed a soil nutrient (nitrogen and phosphorus) transformation with related functional genes and elucidated how rhizosphere microbiota vary their response to P. liquidambari interaction throughout the plant's life cycle under field conditions by Illumina Miseq sequencing platforms in a nutrient-limited paddy soil. Our results showed that P. liquidambari symbiosis decreased the nitrogen and phosphorus loss by 24.59% and 17.46% per pot, respectively. Additionally, we suggest that the application of P. liquidambari altered the activation of soil nitrogen and phosphorus functional genes to accelerate nutrient turnover in the rice rhizosphere. High-throughput sequencing with co-occurrence network and species-related network analysis further revealed that P. liquidambari colonization influenced the patterns of microbiota shift in the rhizosphere, especially during the heading stages. This led to an optimized microbial community through the promotion and inhibition of indigenous soil microbes with a higher level of available nutrient supplies. Our study strongly proposes rice-P. liquidambari symbiosis as a useful candidate for improving N and P acquisition and utilization., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Background: The continuous cropping of peanuts is a primary cause of yield and quality loss. Solutions to this problem should be therefore developed to ensure the sustainability of peanut production., Results: In this study, colonization by the endophytic fungus Phomopsis liquidambari was detected, which led to significantly improved rhizosphere soil microenvironment, enhanced N, P and K assimilation and suppressed incidence of peanut disease. Statistical analysis demonstrated that the yield enhancement was significantly correlated with improvement of the rhizosphere soil microenvironment and the peanut's physiological status by P. liquidambari colonization. In addition, P. liquidambari colonization also significantly improved peanut quality., Conclusion: Our results indicate that the practical application of the endophytic fungus P. liquidambari has a strong potential to alleviate the obstacles associated with continuous peanut cropping under field conditions. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Aims: To explore if and how symbiotic Phomopsis liquidambari-rice system influences below-ground straw decomposition and then nitrogen(N) transformation in response to environmental N levels., Methods and Results: Litter bag experiments were utilized to trace the decay process during rice growth phases (seedling (T1), tillering (T2), heading (T3) and maturing (T4) stage), with (E+) and without endophyte (E-), under low (LN), medium (MN) and high nitrogen (HN) supply. Litter, soil and plant samples were collected to evaluate the decay process, N transformations, plant quality and relative abundance of soil ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB) and P. liquidambari. The results showed that straw decomposition increased by 19·76% (LN, T2 stage), 14·05% (MN, T3 stage) and 16·88% (MN, T4 stage) in E+ pots when compared with E- pots. Further analysis revealed that no significant endophyte × N interaction was found for straw decay rate and that the decay rate was reduced by a higher N supply (LN, 37·16 ± 0·65%; MN, 32·27 ± 1·72%; HN, 29·44 ± 1·22%) at the T1 stage, whereas straw decay rate and N release increased by 9·38 and 11·16%, respectively, mainly by endophyte colonization at the T4 stage. The abundance of AOA and AOB were altered, corresponding with the decay rate. Soil mineral N, straw mineral N and plant quality were shown to increase in E+ pots, depending on environmental N conditions and growth phase. The yield increased by 2·98% for E+ plants under MN level., Conclusions: Symbiotic P. liquidambari-rice system promoted below-ground straw decomposition and N transformation, depending on environmental N levels and plant growth phase., Significance and Impact of the Study: This study provides evidence that fungal endophyte-plant systems are able to promote N transformation by increasing straw decomposition. A reasonable combination of N inputs could enhance its advantage in agriculture ecosystems., (© 2018 The Society for Applied Microbiology.)

Allelopathy is mediated by plant-derived secondary metabolites (allelochemicals) which are released by donor plants and affect the growth and development of receptor plants. The plant root is the first organ which senses soil allelochemicals this results in the production of a shorter primary root. However, the mechanisms underlying this process remain elusive. Here, we report that a model allelochemical benzoic acid (BA) inhibited primary root elongation of Arabidopsis seedlings by reducing the sizes of both the meristem and elongation zones, and that auxin signaling affected this process. An increase in auxin level in the root tips was associated with increased expression of auxin biosynthesis genes and auxin polar transporter AUX1 and PIN2 genes under BA stress. Mutant analyses demonstrated that AUX1 and PIN2 rather than PIN1 were required for the inhibition of primary root elongation during BA exposure. Furthermore, BA stimulated ethylene evolution, whereas blocking BA-induced ethylene signaling with an ethylene biosynthesis inhibitor (Co2+), an ethylene perception antagonist (1-methylcyclopropene) or ethylene signaling mutant lines etr1-3 and ein3eil1 compromised BA-mediated inhibition of root elongation and up-regulation of auxin biosynthesis-related genes together with AUX1 and PIN2, indicating that ethylene signal was involved in auxin-mediated inhibition of primary root elongation during BA stress. Further analysis revealed that the BA-induced reactive oxygen species (ROS) burst contributed to BA-mediated root growth inhibition without affecting auxin and ethylene signals. Taken together, our results reveal that the allelochemical BA inhibits root elongation by increasing auxin accumulation via stimulation of auxin biosynthesis and AUX1/PIN2-mediated auxin transport via stimulation of ethylene production and an auxin/ethylene-independent ROS burst., (� The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Beneficial fungal and rhizobial symbioses share commonalities in phytohormones responses, especially in auxin signalling. Mutualistic fungus Phomopsis liquidambari effectively increases symbiotic efficiency of legume peanut (Arachis hypogaea L.) with another microsymbiont, bradyrhizobium, but the underlying mechanisms are not well understood. We quantified and manipulated the IAA accumulation in ternary P. liquidambari-peanut-bradyrhizobial interactions to uncover its role between distinct symbioses. We found that auxin signalling is both locally and systemically induced by the colonization of P. liquidambari with peanut and further confirmed by Arabidopsis harbouring auxin-responsive reporter, DR5:GUS, and that auxin action, including auxin transport, is required to maintain fungal symbiotic behaviours and beneficial traits of plant during the symbiosis. Complementation and action inhibition experiments reveal that auxin signalling is involved in P. liquidambari-mediated nodule development and N 2 -fixation enhancement and symbiotic gene activation. Further analyses showed that blocking of auxin action compromised the P. liquidambari-induced nodule phenotype and physiology changes, including vascular bundle development, symbiosome and bacteroids density, and malate concentrations, while induced the accumulation of starch granules in P. liquidambari-inoculated nodules. Collectively, our study demonstrated that auxin signalling activated by P. liquidambari symbiosis is recruited by peanut for bradyrhizobial symbiosis via symbiotic signalling pathway activation and nodule carbon metabolism enhancement., (© 2018 John Wiley & Sons Ltd.)

The fungal endophyte Phomopsis liquidambari can improve nitrification rates and alter the abundance and composition of ammonia-oxidizers in the soil rhizosphere of rice. Aerenchyma is related to oxygen transport efficiency and contributes to the enhanced rhizospheric nitrification under flooding conditions. However, whether and how P. liquidambari affects aerenchyma formation is largely unknown. We therefore conducted pot and hydroponic experiments to investigate the changes of aerenchyma area, ethylene and indole-3-acetic acid (IAA) levels in rice with or without P. liquidambari infection. Our results showed that the larger aerenchyma area in rice roots with P. liquidambari inoculation was associated with markedly up-regulated expression of genes related to aerenchyma formation. Meanwhile, P. liquidambari inoculation substantially elevated root porosity (POR) and radial oxygen loss (ROL), leading to the enhancement of oxidation-reduction potential (ORP) under pot condition. Besides, P. liquidambari significantly increased IAA and ethylene levels in rice by stimulating the expression of genes involved in auxin and ethylene biosyntheses. Furthermore, auxin that partly acting upstream of ethylene signalling played an essential role in P. liquidambari-promoted aerenchyma formation. These results verified the direct contribution of P. liquidambari in promoting aerenchyma formation via the accumulation of IAA and ethylene in rice roots, which provides a constructive suggestion for improving hypoxia tolerance through plant-endophyte interactions., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

The bacterial endophyte Pseudomonas fluorescens ALEB7B significantly enhances photosynthate accumulations in Atractylodes lancea. These carbohydrates are preferentially used by the host plant to synthesize secondary metabolites, rather than to increase plant biomass accumulation. Mechanisms underlying the allocation of endophyte-increased carbohydrate in different plant metabolic processes are largely unknown. We have studied how P. fluorescens ALEB7B enhances photosynthate accumulation and how bacterial elicitors regulate metabolic flux and increase medicinal sesquiterpenoid formation in A. lancea using the sterile tissue culture plantlets. P. fluorescens ALEB7B enhances plant photosynthate accumulation by synthesizing and secreting indole-3-acetic acid, which has been demonstrated using high-performance liquid chromatography analysis. The increased endogenous indole-3-acetic acid promotes plant root development and then assimilation. Increased carbohydrates provide the material basis for the formations of terpenoid hydrocarbon scaffolds, which has been proved using gas chromatography analysis. Further, protein and polysaccharide elicitors secreted by P. fluorescens ALEB7B have been separated and purified from the bacterial fermentation broth, which have been applied to A. lancea plantlets. Both elicitors can stimulate the conversions of terpenoid hydrocarbon scaffolds to oxygenous sesquiterpenoids, the active medicinal ingredients in A. lancea, by triggering the oxidative burst in planta. Moreover, this study separates an ABC transporter substrate-binding protein from protein elicitors secreted by P. fluorescens ALEB7B with an ÄKTA Prime Plus Purifier System and firstly shows that this protein is essential to induce oxygenous sesquiterpenoid accumulation in A. lancea. This study provides new perspectives for mechanisms of medicinal oxygenous terpenoid synthesis, which has important reference values to the cultivation of medicinal plants that have terpenoids as their active ingredients, such as Artemisia annua and Taxus chinensis., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Heat stable antifungal factor (HSAF) is considered to be a potential biological pesticide due to its broad antifungal activity and novel mode of action. However, few studies have reported on HSAF production during fermentation. Thus, this work was executed to optimize the medium composition to maximize HSAF production by Lysobacter enzymogenes OH11, with soybean flour, glucose and CaCl 2 identified as suitable nutrients with concentrations of 8·00, 7·89 and 0·72 g l -1 respectively. Simultaneously, the quantitative analysis of HSAF production was established by eliminating the emulsification problem, and the highest HSAF production was determined to be 356·34 ± 13·86 mg l -1 using the optimized medium, 12-fold higher than when using the 10% TSB medium (29·34 ± 2·57 mg l -1 ). Furthermore, the cost of this medium was assessed and nearly 31-fold lower than that of 10% TSB. This study suggests that the optimized medium is not only effective but also economical for HSAF production., Significance and Impact of the Study: Significance and Impact of the Study: Heat stable antifungal factor (HSAF) exhibits a potent and broad antifungal activity with a novel mode of action. Increased production and reduced cost of raw materials are particularly important for the future production of HSAF, however, no report was involved in these studies. This study aimed to improve the production of HSAF with cheap raw materials through the medium optimization, which would lay the foundation for the application of HSAF in biological control., (© 2018 The Society for Applied Microbiology.)

Genetically engineered bacterial whole-cell bioreporters were deployed to investigate bioavailable mercury (b-Hg) and phenanthrene (b-PHE). Characterized by high sensitivity and specificity in aqueous solutions, the bioreporter system could detect in amended soils the concentrations of b-Hg and b-PHE in the ranges of 19.6 to 111.6 and 21.5 to 110.9 μg kg, respectively. The sensitivity of the system allowed for the combined analysis of b-Hg and b-PHE from real environmental samples. Therefore, soil samples from three large refinery facilities were tested, and the results from the instrumental analysis strongly correlated with the ones obtained with the bioreporter method. Large-scale and fast screening of soil contamination across the Yangtze River Delta in Eastern China was conducted. More than 36% of the samples contained b-Hg, whereas the fractions of b-PHE were below the detection limit for all the samples. These results indicated a higher toxicity and more hazardous condition for Hg contamination than for PHE. Population densities and airborne 10-μm particulate matter (PM10) concentrations were used as parameters for comparison with the spatial distribution of the b-Hg and b-PHE fractions. The results revealed that the bioreporters could offer a rapid and cost-efficient method to test soil samples from contaminated areas and provide a screening tool for environmental risk assessment., (Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.)

Filamentous ascomycete Phomopsis sp. are common inhabitants of natural ecosystems and, as saprophytes, are largely responsible for the destructive decay of litterfall, promoting the carbon and nitrogen cycles. Phomopsis liquidambari B3 can establish mutualistic symbiosis with a broad spectrum of crop plants. Colonizing dynamics observations and a growth promotion assay of rice and Arabidopsis thaliana revealed that the B3 colonization strategy is host-adapted and resulted in different growth promotions influenced by N availability. However, the biochemical mechanisms and underlying genetics of the saprophyte transition to an endophyte are poorly understood. Here, the transcriptome features of generalist P. liquidambari and highlighted gene sets involved in the lifestyle transition from saprophytism to endophytism were reported. Most notable were genes for translation, ribosome biogenesis and MAPK signaling, several of which were only up-regulated in endophytic B3. Coordinated up-regulation of genes encoding enzymes involved in phenylalanine, tyrosine and tryptophan biosynthesis were preceded by secondary metabolite induction, which was encountered with host defense. Quantitative PCR validates the reliability of RNA-seq. Dissection at the molecular level facilitated a deeper understanding of P. liquidambari adaptation to hosts and the complex natural environment to play a role in sustainable agriculture and carbon and nitrogen cycles., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The continuous cropping obstacles in monoculture fields are a major production constraint for peanuts. Application of the endophytic fungus Phomopsis liquidambari has increased peanut yields, and nodulation and N 2 fixation increases have been considered as important factors for P. liquidambari infection-improved peanut yield. However, the mechanisms involved in this process remain unknown. This work showed that compared with only Bradyrhizobium inoculation, co-inoculation with P. liquidambari significantly elevated endogenous H 2 O 2 and NO levels in peanut roots. Pre-treatment of seedlings with specific scavengers of H 2 O 2 (CAT) and NO (cPTIO) blocked P. liquidambari-induced nodulation and N 2 fixation. CAT not only suppressed the P. liquidambari-induced nodulation and N 2 fixation, but also suppressed the enhanced H 2 O 2 and NO generation. Nevertheless, the cPTIO did not significantly inhibit the induced H 2 O 2 biosynthesis, implying that H 2 O 2 acted upstream of NO production. These results were confirmed by observations that exogenous H 2 O 2 and sodium nitroprusside (SNP) reversed the inhibition of P. liquidambari-increased nodulation and N 2 fixation by the specific scavengers. The transcriptional activities of the symbiosis-related genes SymRK and CCaMK of peanut-Bradyrhizobium interactions also increased significantly in response to P. liquidambari, H 2 O 2 and SNP treatments. The pot experiment further confirmed that the P. liquidambari infection-enhanced H 2 O 2 and NO signalling pathways were significantly related to the increase in peanut nodulation and N 2 fixation. This is the first report that endophytic fungus P. liquidambari can increase peanut-Bradyrhizobium interactions via enhanced H 2 O 2 /NO-dependent signalling crosstalk, which is conducive to the alleviation of continuous cropping obstacles via an increase in nodulation and N 2 fixation.

The mechanisms that trigger the switch from endophytic fungi to saprophytic fungi are largely unexplored. Broad host range Phomopsis liquidambari is established in endophytic and saprophytic systems with rice ( Oryza sativa L.). Endophytic P. liquidambari promotes rice growth, increasing rice yield and improving the efficiency of nitrogen fertilizer. This species's saprophytic counterpart can decompose rice litterfall, promoting litter organic matter cycling and the release of nutrients and improving the soil microbial environment. Fluorescence microscopy, confocal laser scanning microscopy and quantitative PCR investigated the colonization dynamics and biomass of P. liquidambari in rice in vivo . P. liquidambari formed infection structures similar to phytopathogens with infected vascular tissues that systematically spread to acrial parts. However, different from pathogenic infection, P. liquidambari colonization exhibits space restriction and quantity restriction. Direct comparison of a fungal transcriptome under three different habitats provided a better understanding of lifestyle conversion during plant-fungi interactions. The isolated total RNA of Ck (pure culture), EP (endophytic culture) and FP (saprophytic culture) was subjected to Illumina transcriptome sequencing. To the best of our knowledge, this study is the first to investigate Phomopsis sp. using RNA-seq technology to obtain whole transcriptome information. A total of 27,401,258 raw reads were generated and 22,700 unigenes were annotated. Functional annotation indicated that carbohydrate metabolism and biosynthesis of secondary metabolites played important roles. There were 2522 differentially expressed genes (DEGs) between the saprophytic and endophytic lifestyles. Quantitative PCR analysis validated the DEGs of RNA-seq. Analysis of DEGs between saprophytic and endophytic lifestyles revealed that most genes from amino acids metabolism, carbohydrate metabolism, fatty acid biosynthesis, secondary metabolism and terpenoid and steroid biosynthesis were up-regulated in EP. Secondary metabolites of these pathways may affect fungal growth and development and contribute to signaling communication with the host. Most pathways of xenobiotic biodegradation and metabolism were upregulated in FP. Cytochrome P450s play diverse vital roles in endophytism and saprophytism, as their highly specialized functions are evolutionarily adapted to various ecological niches. These results help to characterize the relationship between fungi and plants, the diversity of fungi for ecological adaptations and the application prospects for fungi in sustainable agriculture.

Endophytes and plants can establish specific long-term symbiosis through the accumulation of secondary metabolites. Previous studies have shown that the endophytic fungus Gilmaniella sp. AL12 can stimulate Atractylodes lancea to produce volatile oils. The purpose of this report is to investigate key factors involved in the stimulation of A. lancea by AL12 and reveal the mechanism. We identified the active component from AL12 as an extracellular mannan with a polymerization degree of 26-42. Differential membrane proteomics of A. lancea was performed by 2D electrophoresis. The results showed that there were significant differences in the expression of 83 proteins. Based on these results, we conclude that AL12 secreted mannan contributes to the antagonistic balance seen in interactions between AL12 and A. lancea. One portion of the mannan was degraded to mannose for hexokinase activation, promoting photosynthesis and energy metabolism, with a potential metabolic fluxes flowing towards terpenoid biosynthesis. The other portion of the mannan directly enhanced autoimmunity of A. lancea through G protein-mediated signal transduction and the mannan-binding lectin pathway. Volatile oil accumulation was ultimately promoted in subsequent defense reactions. This study provides a new perspective on the regulation of secondary metabolites by endophytic fungal elicitors in medicinal plants.

Microbial community structure and functions of rhizosphere soil of rice were investigated after applying low and high doses of nitrogenous fertilizer and Phomopsis liquidambari. Average well color development, substrate richness, catabolic diversity and soil enzymes activities varied after applying N-fertilizer and P. liquidambari and were greater in P. liquidambari treated soil than only N-fertilization. Multivariate analysis distinctly separated the catabolic and enzymes activity profile which statistically proved alteration of microbial functional diversity. Nitrogen fertilizer altered microbial community structure revealed by the increased content of total PLFAs, specific subgroup marker PLFAs except fungal PLFAs and by the decreased ratio of G(+)/G(-), sat/monunsat, iso/anteiso, F/B except trans/cis while P. liquidambari inoculation enhanced N-fertilization effect except increased fungal PLFA and decreased trans/cis. PCA using identified marker PLFAs revealed definite discrimination among the treatments which further statistically confirmed structural changed of microbial community. Nitrogenase activity representative of N-fixing community decreased in N-fertilizer treatment while P. liquidambari inoculation increased. In short, application of P. liquidambari with low doses of N-fertilizer improved rice growth and reduced N-fertilizer requirement by increasing enzymes activities involved in C, N and P cycling, structural and functional diversity of microbes, nitrogenase activity involved in N2 fixation and accumulation of total-N.

Main Conclusion: Pseudomonas fluorescens induces gibberellin and ethylene signaling via hydrogen peroxide in planta . Ethylene activates abscisic acid signaling. Hormones increase sesquiterpenoid biosynthesis gene expression and enzyme activity, inducing essential oil accumulation. Atractylodes lancea is a famous Chinese medicinal plant, whose main active components are essential oils. Wild A. lancea has become endangered due to habitat destruction and over-exploitation. Although cultivation can ensure production of the medicinal material, the essential oil content in cultivated A. lancea is significantly lower than that in the wild herb. The application of microbes as elicitors has become an effective strategy to increase essential oil accumulation in cultivated A. lancea. Our previous study identified an endophytic bacterium, Pseudomonas fluorescens ALEB7B, which can increase essential oil accumulation in A. lancea more efficiently than other endophytes; however, the underlying mechanisms remain unknown (Physiol Plantarum 153:30-42, 2015; Appl Environ Microb 82:1577-1585, 2016). This study demonstrates that P. fluorescens ALEB7B firstly induces hydrogen peroxide (H2O2) signaling in A. lancea, which then simultaneously activates gibberellin (GA) and ethylene (ET) signaling. Subsequently, ET activates abscisic acid (ABA) signaling. GA and ABA signaling increase expression of HMGR and DXR, which encode key enzymes involved in sesquiterpenoid biosynthesis, leading to increased levels of the corresponding enzymes and then an accumulation of essential oils. Specific reactive oxygen species and hormone signaling cascades induced by P. fluorescens ALEB7B may contribute to high-efficiency essential oil accumulation in A. lancea. Illustrating the regulation mechanisms underlying P. fluorescens ALEB7B-induced essential oil accumulation not only provides the theoretical basis for the inducible synthesis of terpenoids in many medicinal plants, but also further reveals the complex and diverse interactions among different plants and their endophytes.

Atractylodes lancea is a well-known, but endangered, Chinese medicinal plant whose volatile oils are its main active components. As the volatile oil content in cultivated A. lancea is much lower than that in the wild herb, the application of microbes or related elicitors to promote growth and volatile oil accumulation in the cultivated herb is an important area of research. This study demonstrates that the endophytic bacterium Pseudomonas fluorescens ALEB7B isolated from the geo-authentic A. lancea can release several nitrogenous volatiles, such as formamide and N,N-dimethyl-formamide, which significantly promote the growth of non-infected A. lancea. Moreover, the main bacterial volatile benzaldehyde significantly promotes volatile oil accumulation in non-infected A. lancea via activating plant defense responses. Notably, the bacterial nitrogenous volatiles cannot be detected in the A. lancea - Pseudomonas fluorescens symbiont while the benzaldehyde can be detected, indicating the nitrogenous volatiles or their precursors may have been consumed by the host plant. This study firstly demonstrates that the interaction between plant and endophytic bacterium is not limited to the commonly known physical contact, extending the ecological functions of endophyte in the phytosphere and deepening the understandings about the symbiotic interaction., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Lactic acid bacteria (LAB) are suitable for rice straw silage fermentation, but have been studied rarely, and rice straw as raw material for ensiling is difficult because of its disadvantages, such as low nutrition for microbial activities and low abundances of natural populations of LAB. So we investigated the effect of application of LAB and chemical additives on the fermentation quality and microbial community of wilted rice straw silage. Treatment with chemical additives increased the concentrations of crude protein (CP), water soluble carbohydrate (WSC), acetic acid and lactic acid, reduced the concentrations of acid detergent fiber (ADF) and neutral detergent fiber (NDF), but did not effectively inhibit the growth of spoilage organisms. Inoculation with LABs did not improve the nutritional value of the silage because of poor growth of LABs in wilted rice straw. Inoculation with LAB and addition of chemical materials improved the quality of silage similar to the effects of addition of chemical materials alone. Growth of aerobic and facultatively anaerobic bacteria was inhibited by this mixed treatment and the LAB gradually dominated the microbial community. In summary, the fermentation quality of wilted rice straw silage had improved by addition of LAB and chemical materials., (© 2015 Japanese Society of Animal Science.)

Ethylene, the first known gaseous phytohormone, is involved in plant growth, development as well as responses to environmental signals. However, limited information is available on the role of ethylene in endophytic fungi induced secondary metabolites biosynthesis. Atractylodes lancea is a traditional Chinese herb, and its quality depends on the main active compounds sesquiterpenoids. This work showed that the endophytic fungus Gilmaniella sp. AL12 induced ethylene production in Atractylodes lancea. Pre-treatment of plantlets with ethylene inhibiter aminooxyacetic acid (AOA) suppressed endophytic fungi induced accumulation of ethylene and sesquiterpenoids. Plantlets were further treated with AOA, salicylic acid (SA) biosynthesis inhibitor paclobutrazol (PAC), jasmonic acid inhibitor ibuprofen (IBU), hydrogen peroxide (H2O2) scavenger catalase (CAT), nitric oxide (NO)-specific scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO). With endophytic fungi inoculation, IBU or PAC did not inhibit ethylene production, and JA and SA generation were suppressed by AOA, showing that ethylene may act as an upstream signal of JA and SA pathway. With endophytic fungi inoculation, CAT or cPTIO suppressed ethylene production, and H2O2 or NO generation was not affected by 1-aminocyclopropane-1-carboxylic acid (ACC), showing that ethylene may act as a downstream signal of H2O2 and NO pathway. Then, plantlets were treated with ethylene donor ACC, JA, SA, H2O2, NO donor sodium nitroprusside (SNP). Exogenous ACC could trigger JA and SA generation, whereas exogenous JA or SA did not affect ethylene production, and the induced sesquiterpenoids accumulation triggered by ACC was partly suppressed by IBU and PAC, showing that ethylene acted as an upstream signal of JA and SA pathway. Exogenous ACC did not affect H2O2 or NO generation, whereas exogenous H2O2 and SNP induced ethylene production, and the induced sesquiterpenoids accumulation triggered by SNP or H2O2 was partly suppressed by ACC, showing that ethylene acted as a downstream signal of NO and H2O2 pathway. Taken together, this study demonstrated that ethylene is an upstream signal of JA and SA, and a downstream signal of NO and H2O2 signaling pathways, and acts as an important signal mediating sesquiterpenoids biosynthesis of Atractylodes lancea induced by the endophytic fungus.

The biodegradation potential of sinapic acid, one of the most representative methoxy phenolic pollutants presented in industrial wastewater, was first studied using an endophytic fungus called Phomopsis liquidambari. This strain can effectively degrade sinapic acid in flasks and in soil and the possible biodegradation pathway was first systematically proposed on the basis of the metabolite production patterns and the identification of the metabolites by GC-MS and HPLC-MS. Sinapic acid was first transformed to 2,6-dimethoxy-4-vinylphenol that was further degraded via 4-hydroxy-3,5-dimethoxybenzaldehyde, syringic acid, gallic acid, and citric acid which involved in the continuous catalysis by phenolic acid decarboxylase, laccase, and gallic acid dioxygenase. Moreover, their activities and gene expression levels exhibited a 'cascade induction' response with the changes in metabolic product concentrations and the generation of fungal laccase significantly improved the degradation process. This study is the first report of an endophytic fungus that has great potential to degrade xenobiotic sinapic acid, and also provide a basis for practical application of endophytic fungus in the bioremediation of sinapic acid-contaminated industrial wastewater and soils., (Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

The progress of genetically engineered microbial whole-cell biosensors for chemosensing and monitoring has been developed in the last 20 years. Those biosensors respond to target chemicals and produce output signals, which offer a simple and alternative way of assessment approaches. As actual pollution caused by human activities usually contains a combination of different chemical substances, how to employ those biosensors to accurately detect real contaminant samples and evaluate biological effects of the combined chemicals has become a realistic object of environmental researches. In this review, we outlined different types of the recent method of genetically engineered microbial whole-cell biosensors for combined chemical evaluation, epitomized their detection performance, threshold, specificity, and application progress that have been achieved up to now. We also discussed the applicability and limitations of this biosensor technology and analyzed the optimum conditions for their environmental assessment in a combined way.

Background: Continuous cropping practices cause a severe decline in peanut yield. The aim of this study was to investigate the remediation effect of Serratia marcescens on continuously cropped peanut soil. A pot experiment was conducted under natural conditions to determine peanut agronomic indices, soil microorganism characteristics, soil enzyme activities and antagonism ability to typical pathogens at different growth stages. Four treatments were applied to red soil as follows: an active fermentation liquor of S. marcescens (RZ-21), an equivalent sterilized fermentation liquor (M), an equivalent fermentation medium (P) and distilled water (CK)., Results: S. marcescens significantly inhibited the two typical plant pathogens Fusarium oxysporum A1 and Ralstonia solanacearum B1 and reduced their populations in rhizosphere soil. The RZ-21 treatment significantly increased peanut yield, vine dry weight, root nodules and taproot length by 62.3, 33, 72 and 61.4% respectively, followed by the M treatment. The P treatment also increased root nodules and root length slightly. RZ-21 also enhanced the activities of soil urease, sucrase and hydrogen peroxidase at various stages. In addition, RZ-21 and M treatments increased the average population of soil bacteria and decreased the average population of fungi in the three critical peanut growth stages, except for M in the case of the fungal population at flowering, thus balancing the structure of the soil microorganism community., Conclusion: This is the first report of S. marcescens being applied to continuously cropped peanut soil. The results suggest that S. marcescens RZ-21 has the potential to improve the soil environment and agricultural products and thus allow the development of sustainable management practices., (© 2015 Society of Chemical Industry.)

This investigation examined the reduction of pesticide residues on straw inoculated with lactic acid bacteria (LAB) during ensiling. Lactobacillus casei WYS3 was isolated from rice straw that contained pesticide residues. Non-sterilized rice straw, which was inoculated with L. casei WYS3, showed increased removal of chlorpyrifos after ensiling, compared with rice straw that was not inoculated with L. casei WYS3 or sterilized rice straw. In pure culture, these strains can bind chlorpyrifos as indicated by high-performance liquid chromatography analysis. Viable L. casei WYS3 was shown to bind 33.3-42% of exogenously added chlorpyrifos. These results are similar to those of acid-treated cells but less than those of heat-treated cells, which were found to bind 32.0% and 77.2% of the added chlorpyrifos respectively. Furthermore, gas chromatography-mass spectrometry analysis determined that L. casei WYS3 detoxified chlorpyrifos via P-O-C cleavage. Real-time polymerized chain reaction analysis determined that organophosphorus hydrolase gene expression tripled after the addition of chlorpyrifos to LAB cultures, compared with the control group (without chlorpyrifos). This paper highlights the potential use of LAB starter cultures for the detoxification and removal of chlorpyrifos residues in the environment.