Targeted metabolome and transcriptome analyses reveal changes in gibberellin and related cell wall-acting enzyme-encoding genes during stipe elongation in Flammulina filiformis.

Flammulina filiformis, a typical agaric fungus, is a widely cultivated and consumed edible mushroom. Elongation of its stipe (as the main edible part) is closely related to its yield and commercial traits; however, the endogenous hormones during stipe elongation and their regulatory mechanisms are not well understood. Gibberellin (GA) plays an important role in the regulation of plant growth, but little has been reported in macro fungi. In this study, we first treated F. filiformis stipes in the young stage with PBZ (an inhibitor of GA) and found that PBZ significantly inhibited elongation of the stipe. Then, we performed GA-targeted metabolome and transcriptome analyses of the stipe at both the young and elongation stages. A total of 13 types of GAs were detected in F. filiformis; the contents of ten of them, namely, GA3, GA4, GA8, GA14, GA19, GA20, GA24, GA34, GA44, and GA53, were significantly decreased, and the contents of three (GA5, GA9, and GA29) were significantly increased during stipe elongation. Transcriptome analysis showed that the genes in the terpenoid backbone biosynthesis pathway showed varying expression patterns: HMGS, HMGR, GPS, and FPPS were significantly upregulated, while CPS/KS had no significant difference in transcript level during stipe elongation. In total, 37 P450 genes were annotated to be involved in GA biosynthesis; eight of them were upregulated, twelve were downregulated, and the rest were not differentially expressed. In addition, four types of differentially expressed genes involved in stipe elongation were identified, including six signal transduction genes, five cell cycle-controlling genes, twelve cell wall-related enzymes and six transcription factors. The results identified the types and content of GAs and the expression patterns of their synthesis pathways during elongation in F. filiformis and revealed the molecular mechanisms by which GAs may affect the synthesis of cell wall components and the cell cycle of the stipe through the downstream action of cell wall-related enzymes, transcription factors, signal transduction and cell cycle control, thus regulating stipe elongation. This study is helpful for understanding the roles of GAs in stipe development in mushrooms and lays the foundation for the rational regulation of stipe length in agaric mushrooms during production.

Low temperature, mechanical wound, and exogenous salicylic acid (SA) can stimulate the SA signaling molecule as well as its downstream pathway and the formation of fruiting bodies in Flammulina filiformis.

Low temperature (LT) and mechanical wound (MW), as two common physics methods, have been empirically used in production to stimulate the primordia formation of Flammulina filiformis, which is typically produced using the industrial production mode. However, the detailed effect on the fruiting body formation and important endogenous hormones and signaling pathways in this process is poorly understood. In this study, LT, MW, their combination, i.e., MW + LT, and low concentration of SA (0.1 mM SA) treatments were applied to the physiologically mature mycelia of F. filiformis. The results showed that the primordia under the four treatments began to appear on the 5th-6th days compared with the 12th day in the control (no treatment). The MW + LT treatment produced the largest number of primordia (1,859 per bottle), followed by MW (757), SA (141), and LT (22), compared with 47 per bottle in the control. The HPLC results showed that the average contents of endogenous SA were significantly increased by 1.3 to 2.6 times under four treatments. A total of 11 SA signaling genes were identified in the F. filiformis genome, including 4 NPR genes (FfNpr1-4), 5 TGA genes (FfTga1-5), and 2 PR genes (FfPr1-2). FfNpr3 with complete conserved domains (ANK and BTB/POZ) showed significantly upregulated expression under all four above treatments, while FfNpr1/2/4 with one domain showed significantly upregulated response expression under the partial treatment of all four treatments. FfTga1-5 and FfPr1-2 showed 1.6-fold to 8.5-fold significant upregulation with varying degrees in response to four treatments. The results suggested that there was a correlation between "low temperature/mechanical wound-SA signal-fruiting body formation", and it will help researchers to understand the role of SA hormone and SA signaling pathway genes in the formation of fruiting bodies in fungi.

NAT10 promotes osteogenic differentiation of periodontal ligament stem cells by regulating VEGFA-mediated PI3K/AKT signaling pathway through ac4C modification.

Periodontal tissue regeneration engineering based on human periodontal ligament stem cells (hPDLSCs) provides a broad prospect for the treatment of periodontal disease. N-Acetyltransferase 10 (NAT10)-catalyzed non-histone acetylation is widely involved in physiological or pathophysiological processes. However, its function in hPDLSCs is still missing. hPDLSCs were isolated, purified, and cultured from extracted teeth. Surface markers were detected by flow cytometry. Osteogenic, adipogenic, and chondrogenic differentiation potential was detected by alizarin red staining (ARS), oil red O staining, and Alcian blue staining. Alkaline phosphatase (ALP) activity was assessed by ALP assay. Quantitative real-time PCR (qRT-PCR) and western blot were used to detect the expression of key molecules, such as NAT10, Vascular endothelial growth factor A (VEGFA), PI3K/AKT pathway, as well as bone markers (RUNX2, OCN, OPN). RNA-Binding Protein Immunoprecipitation PCR (RIP-PCR) was used to detect the N4-acetylcytidine (ac4C) mRNA level. Genes related to VEGFA were identified by bioinformatics analysis. NAT10 was highly expressed in the osteogenic differentiation process with enhanced ALP activity and osteogenic capability, and elevated expression of osteogenesis-related markers. The ac4C level and expression of VEGFA were obviously regulated by NAT10 and overexpression of VEGFA also had similar effects to NAT10. The phosphorylation level of PI3K and AKT was also elevated by overexpression of VEGFA. VEGFA could reverse the effects of NAT10 in hPDLSCs. NAT10 enhances the osteogenic development of hPDLSCs via regulation of the VEGFA-mediated PI3K/AKT signaling pathway by ac4C alteration.

Spermidine Synthase and Saccharopine Reductase Have Co-Expression Patterns Both in Basidiomycetes with Fusion Form and Ascomycetes with Separate Form.

Gene fusion is a process through which two or more distinct genes are fused into a single chimeric gene. Unlike most harmful fusion genes in cancer cells, in this study, we first found that spermidine synthetase- (SPDS, catalyst of spermidine biosynthesis) and saccharopine reductase- (SR, catalyst of the penultimate step of lysine biosynthesis) encoding genes form a natural chimeric gene, FfSpdsSr, in Flammulina filiformis. Through the cloning of full-length ORFs in different strains and the analysis of alternative splicing in developmental stages, FfSpdsSr has only one copy and unique transcript encoding chimeric SPDS-SR in F. filiformis. By an orthologous gene search of SpdsSr in more than 80 fungi, we found that the chimeric SpdsSr exists in basidiomycetes, while the two separate Spds and Sr independently exist in ascomycetes, chytridiomycetes, and oomycetes. Further, the transcript level of FfSpdsSr was investigated in different developmental stages and under some common environmental factors and stresses by RT-qPCR. The results showed that FfSpdsSr mainly up-regulated in the elongation stage and pileus development of F. filiformis, as well as under blue light, high temperature, H2O2, and MeJA treatments. Moreover, a total of 15 sets of RNA-Seq data, including 218 samples of Neurospora crassa, were downloaded from the GEO database and used to analyze the expression correlation of NcSpds and NcSr. The results showed that the separate NcSpds and NcSr shared highly similar co-expression patterns in the samples with different strains and different nutritional and environmental condition treatments. The chimeric SpdsSr in basidiomycetes and the co-expression pattern of the Spds and Sr in N. crassa indicate the special link of spermidine and lysine in fungi, which may play an important role in the growth and development of fruiting body and in response to the multiple environmental factors and abiotic stresses.

Flammutoxin, a Degradation Product of Transepithelial Electrical Resistance-Decreasing Protein, Induces Reactive Oxygen Species and Apoptosis in HepG2 Cells.

Proteins from Flammulina filiformis were prepared by sodium chloride extraction and fractionated by ammonium sulfate precipitation with increasing saturation degrees to obtain the protein fractions Ffsp-30, Ffsp-50, Ffsp-70, Ffsp-90, and Ffp-90. Among these protein fractions, Ffsp-50 possessed the most significant cytotoxic effect against three human gastrointestinal cancer cell lines, viz. HT-29, SGC-7901, and HepG2. SDS-PAGE and MALDI-TOF/TOF MS/MS analyses revealed that flammutoxin (FTX) was present as a dominating protein in Ffsp-50, which was further evidenced by HPLC-MS/MS determination. Furthermore, native FTX was purified from Ffsp-50 with a molecular weight of 26.78 kDa, exhibiting notable cytotoxicity against gastrointestinal cancer cell lines. Both Ffsp-50 and FTX exposure could enhance intercellular reactive oxygen species (ROS) generation and induce significant apoptosis in HepG2 cells. FTX was identified to be relatively conserved in basidiomycetes according to phylogenetic analysis, and its expression was highly upregulated in the primordium as well as the pileus of the fruiting body from the elongation and maturation stages, as compared with that in mycelium. Taken together, FTX could remarkably inhibit cell growth and induce ROS and apoptosis in HepG2 cells, potentially participating in the growth and development of the fruiting body. These findings from our investigation provided insight into the antigastrointestinal cancer activity of FTX, which could serve as a biological source of health-promoting and biomedical applications.

Flammulina filiformis Pkac Gene Complementing in Neurospora crassa Mutant Reveals Its Function in Mycelial Growth and Abiotic Stress Response

Flammulina filiformis is a popular edible mushroom that easily suffers from heat and oxidative stresses. The cyclic adenylate-dependent protein kinase A (cAMP/PKA) pathway is the main signaling pathway in response to environmental stress, and the PKAC is the terminal catalytic subunit of this pathway. In this study, the Pkac gene was identified in F. filiformis, which was highly conserved in basidiomycetes and ascomycetes. The transcription analysis showed that the Pkac gene was involved in the mycelial growth and the fruiting body development of fungi. In Neurospora crassa, the Pkac gene deletion (ΔPkac) resulted in the slower growth of the mycelia. We complemented the F. filiformis FfPkac to N. crassa ΔPkac mutant to obtain the CPkac strain. The mycelial growth in the CPkac strain was restored to the same level as the WT strain. In addition, the FfPkac gene showed significantly up-regulated expression under heat and oxidative stresses. By analyzing the differentially expressed genes of ΔPkac and Cpkac with WT, respectively, seven downstream genes regulated by Pkac were identified and may be related to mycelial growth. They were mainly focused on microbial metabolism in diverse environments, mitochondrial biogenesis, protein translation and nucleocytoplasmic transport. RT-qPCR results confirmed that the expression patterns of these seven genes were consistent with FfPkac under heat and oxidative stresses. The results revealed the conserved functions of PKAC in filamentous fungi and its regulatory mechanism in response to heat and oxidative stresses.

Genome-Wide Screening and Stability Verification of the Robust Internal Control Genes for RT-qPCR in Filamentous Fungi.

In real-time quantitative PCR (RT-qPCR), internal control genes (ICGs) are crucial for normalization. This study screened 6 novel ICGs: Pre-mRNA-splicing factor cwc15 (Cwf15); ER associated DnaJ chaperone (DnaJ); E3 ubiquitin-protein ligase NEDD4 (HUL4); ATP-binding cassette, subfamily B (MDR/TAP), member 1 (VAMP); Exosome complex exonuclease DIS3/RRP44 (RNB); V-type H+-transporting ATPase sub-unit A (V-ATP) from the 22-transcriptome data of 8 filamentous fungi. The six novel ICGs are all involved in the basic biological process of cells and share the different transcription levels from high to low. In order to further verify the stability of ICGs candidates, the six novel ICGs as well as three traditional housekeeping genes: ß-actin (ACTB); ß-tubulin (ß-TUB); glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) and the previously screened reference genes: SPRY-domain-containing protein (SPRYp); Ras-2 protein (Ras); Vacuolar protein sorting protein 26 (Vps26) were evaluated by geNorm and NormFinder statistical algorithms. RT-qPCR of 12 ICGs were performed at different developmental stages in Flammulina filiformis and under different treatment conditions in Neurospora crassa. The consistent results of the two algorithms suggested that the novel genes, RNB, V-ATP, and VAMP, showed the highest stability in F. filiformis and N. crassa. RNB, V-ATP, and VAMP have high expression stability and universal applicability and therefore have great potential as ICGs for standardized calculation in filamentous fungi. The results also provide a novel guidance for the screening stable reference genes in RT-qPCR and a wide application in gene expression analysis of filamentous fungi.

Ultrasonic treatment decreases Lyophyllum decastes fruiting body browning and affects energy metabolism.

Lyophyllum decastes is a common mushroom that is prone to browning during prolonged storage. In this study, the effects of ultrasonic treatment on metabolic gene expression, enzyme activity, and metabolic compounds related to L. decastes browning were investigated. Treatment of the fruiting body at 35 kHz and 300 W for 10 min reduced the browning index of L. decastes by 21.0 % and increased the L* value by 11.1 %. Ultrasonic treatment of the fruiting body resulted in higher levels of total phenols, flavonoids, and 9 kinds of amino acid with catalase (CAT) and peroxidase (POD) activities maintained at high levels. Higher cytochrome c oxidase (CCO), succinate dehydrogenase (SDH), phosphofructokinase (PFK), and pyruvate kinase (PK) activities may be ascribed to increased antioxidant capacity. Moreover, ultrasonication retained higher adenosine triphosphate (ATP) concentrations with an increased energy charge, while there were lower levels of adenosine diphosphate (ADP) and reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+), respectively. Meanwhile, lower lignin contents were observed, along with retarded polyphenol oxidase (PPO) and lipoxygenase (LOX) activities. Lower PPO activity reduced the fruiting body enzymatic browning rate through decreased expression of LdPpo1, LdPpo2, and LdPpo3 during storage at 4 °C for 16 days. This activity may be used to determine the effectiveness of ultrasonication.

Comprehensive Genetic Analysis of Monokaryon and Dikaryon Populations Provides Insight Into Cross-Breeding of Flammulina filiformis.

Flammulina filiformis, as one of the most popular edible fungi in East Asia, is produced in an industrialized and standardized way. However, its monotonous variety and product convergence have seriously restricted the development of the industry. In this study, 11 cultivated strains and 13 wild strains of F. filiformis were collected from multiple regions of China and Japan and were performed genome sequencing. Together with genome data of six strains previously released, in total 23 dikaryons (formed by two monokaryons mating, can making fruiting body), 35 monokaryons (formed by protoplast-regenerating of dikaryon and isolating) were used for genetic diversity and population structure analysis based on the high-throughput genotyping. Firstly, a set of SNP markers with intrapopulation polymorphism including 849,987 bi-allelic SNPs were developed and basically covered all of 11 chromosomes with a high distribution density of 24.16 SNP markers per kb. The cultivated dikaryotic strains were divided into three subgroups, and their breeding history was made inferences, which is consistent with the available pedigree records. The wild dikaryotic strains were divided into two subgroups and showed varied contributions of genetic components with high genetic diversity. All the investigated dikaryons have a symmetric distribution pattern with their two constituent monokaryons in principal component analysis. Finally, we summarized the pedigree relationship diagram of F. filiformis main strains including six modules, and the genotypes of hybrids can be directly phased by the known parental allele according to it. This study provides a method to distinguish two sets of monokaryon haplotypes, and several valuable genetic resources of wild F. filiformis, and an effective strategy for guiding F. filiformis breeding based on the population structure and pedigree relationship in future.

The Chromatin Modifier Protein FfJMHY Plays an Important Role in Regulating the Rate of Mycelial Growth and Stipe Elongation in Flammulina filiformis.

Stipe elongation is an important process in the development of the fruiting body and is associated with the commodity quality of agaric fungi. In this study, F. filiformis was used as a model agaric fungus to reveal the function of the chromatin modifier gene containing the JmjC domain in stipe elongation. First, we identified a JmjC domain family gene (FfJmhy) with a 3684 bp length open reading frame (ORF) in F. filiformis. FfJmhy was predicted to have a histone H3K9 demethylation function, and was specifically upregulated during stipe rapid elongation. Further investigation revealed that the silencing of FfJmhy inhibited the mycelial growth, while overexpression of this gene had no effect on the mycelial growth. Comparative analysis revealed that the stipe elongation rate in FfJmhy overexpression strains was significantly increased, while it was largely reduced when FfJmhy was silenced. Taken together, these results suggest that FfJmhy positively regulates the mycelial growth and controls the elongation speed and the length of the stipe. Moreover, cell wall-related enzymes genes, including three exo-ß-1,3-glucanases, one ß-1,6-glucan synthase, four chitinases, and two expansin proteins, were found to be regulated by FfJmhy. Based on the putative functions of FfJmhy, we propose that this gene enhances the transcription of cell wall-related enzymes genes by demethylating histone H3K9 sites to regulate remodeling of the cell wall in rapid stipe elongation. This study provides new insight into the mechanism of rapid stipe elongation, and it is important to regulate the commodity quality of agaric fungi.

FFGA1 Protein Is Essential for Regulating Vegetative Growth, Cell Wall Integrity, and Protection against Stress in Flammunina filiformis.

Flammulina filiformis is a popular mushroom which has been regarded as a potential model fungus for mycelium growth, fruiting body development, and stress response studies. Based on a genome-wide search, four genes encoding heterotrimeric G protein α subunits were identified in F. filiformis. The data of conserved domain analysis showed that these genes contain only one subgroup I of Gα subunit (Gαi), similar to many other fungi. To explore the function of Gαi, FfGa1 over-expression (OE) and RNA interference (RNAi) strains were generated using the Agrobacterium tumefaciens-mediated transformation (ATMT) approach. RNAi transformant strains showed remarkably reduced growth on PDA medium and added sensitivity to cell wall-enforcing agents with maximum growth inhibition, but showed better growth in response to hypertonic stress-causing agents, while OE strains exhibited more resistance to thermal stress and mycoparasite Trichoderma as compared to the wild-type and RNAi strains. Taken together, our results indicated that FfGa1 positively regulates hyphal extension, and is crucial for the maintenance of cell wall integrity and protection against biotic and abiotic (hypertonic and thermal) stress.

Recent advances of the ionic chiral selectors for chiral resolution by chromatography, spectroscopy and electrochemistry.

Ionic chiral selectors have been received much attention in the field of asymmetric catalysis, chiral recognition, and preparative separation. It has been shown that the addition of ionic chiral selectors can enhance the recognition efficiency dramatically due to the presence of multiple intermolecular interactions, including hydrogen bond, π-π interaction, van der Waals force, electrostatic ion-pairing interaction, and ionic-hydrogen bond. In the initial research stage of the ionic chiral selectors, most of work center on the application in chromatographic separation (capillary electrophoresis, high-performance liquid chromatography, and gas chromatography). Differently, more and more attention has been paid on the spectroscopy (nuclear magnetic resonance, fluorescence, ultraviolet and visible absorption spectrum, and circular dichroism spectrum) and electrochemistry in recent years. In this tutorial review as regards the ionic chiral selectors, we discuss in detail the structural features, properties, and their application in chromatography, spectroscopy, and electrochemistry.

Strategies to Achieve a Ferrocene-Based Polymer with Reversible Redox Activity for Chiral Electroanalysis of Nonelectroactive Amino Acids.

In the past, various chiral isomers accompanied by electroactive units have been distinguished using electrochemical techniques, which can produce electrochemical signals by themselves. However, it is still difficult to use an electrochemical technique to detect nonelectroactive samples. To address this bottleneck, an electroactive chiral polymer (S,S)-p-CVB-Fc that contains one redox-active ferrocene unit was designed and synthesized in this study. The electroactive polymer can give electrochemical signals as an alternative to the tested chiral samples, regardless of whether the isomers have electroactive units. Then, it was fixed on the surface of a glassy carbon electrode as an electrochemical chiral sensor. When nonelectroactive amino acids including proline, threonine, and alanine were examined by the sensor, clear discrimination in the response of peak current could be observed toward l- and d-isomers at pH 6.5. The peak current ratios (IL/ID) for proline and alanine were 1.47 and 1.48, respectively. In contrast, for threonine, the d-isomer exhibited a higher peak current than the l -isomer with a ratio of 2.59. In summary, the results ensure that the current work can enlarge the testing scope of chiral samples in the field of chiral electroanalysis using an electroactive sensor.

Heat stress promotes the conversion of putrescine to spermidine and plays an important role in regulating ganoderic acid biosynthesis in Ganoderma lucidum.

Heat stress (HS) is inescapable environmental stress that can induce the production of ganoderic acids (GAs) in Ganoderma lucidum. Our previous studies found that putrescine (Put) played an inhibitory role in GAs biosynthesis, which appeared to be inconsistent with the upregulated transcription of the Put biosynthetic gene GlOdc under HS. To uncover the mechanism underlying this phenomenon, two spermidine (Spd) biosynthetic genes, GlSpds1 and GlSpds2, were identified and upregulated under HS. Put and Spd increased by 94% and 160% under HS, respectively, suggesting that HS induces polyamine biosynthesis and promotes the conversion of Put to Spd. By using GlSpds knockdown mutants, it is confirmed that Spd played a stimulatory role in GAs biosynthesis. In GlOdc-kd mutants, Put decreased by 62-67%, Spd decreased by approximately 34%, and GAs increased by 15-22% but sharply increased by 75-89% after supplementation with Spd. In GlSpds-kd mutants, Put increased by 31-41%, Spd decreased by approximately 63%, and GAs decreased by 24-32% and were restored to slightly higher levels than a wild type after supplementation with Spd. This result suggested that Spd, rather than Put, is a crucial factor that leads to the accumulation of GAs under HS. Spd plays a more predominant and stimulative role than Put under HS, possibly because the absolute content of Spd is 10 times greater than that of Put. GABA and H2O2, two major catabolites of Spd, had little effect on GAs biosynthesis. This study provides new insight into the mechanism by which environmental stimuli regulate secondary metabolism via polyamines in fungi. KEY POINTS: • HS induces polyamine biosynthesis and promotes the conversion of Put to Spd in G. lucidum. • Put and Spd played the inhibitory and stimulatory roles in regulating GAs biosynthesis, respectively. • The stimulatory role of Spd was more predominant than the inhibitory role of Put in GAs biosynthesis.

Silver nanoparticle driven signal amplification for electrochemical chiral discrimination of amino acids.

ß-Cyclodextrin (ß-CD) modified silver nanoparticles (AgNPs), denoted as ß-CD/AgNPs, were prepared by a simple one-pot method. Due to the inherent chirality of ß-CD, the developed ß-CD/AgNPs exhibited higher affinity toward l-tyrosine (l-Tyr) than d-tyrosine (d-Tyr), leading to serious aggregation of AgNPs in the presence of l-Tyr. Consequently, the l-Tyr induced aggregation of AgNPs can result in signal amplification in the differential pulse voltammograms (DPVs) of l-Tyr, which can be applied for the electrochemical chiral discrimination of the Tyr enantiomers. Other chiral amino acids including tryptophan and phenylalanine can also be successfully discriminated with the ß-CD/AgNPs, suggesting high universality of the developed chiral sensor.

Enantioselective Limiting Transport into a Fixed Cavity via Supramolecular Interaction for the Chiral Electroanalysis of Amino Acids Regardless of Electroactive Units.

Although an increasing number of researchers are developing electroanalytical protocols for the chiral recognition of amino acids, the electroactive units of the tested isomers still need to provide corresponding electrical signals. In this study, a supramolecular system was developed for the chiral electroanalysis of amino acids regardless of electroactive units. As a model system, an enantiopure electroactive molecule Fc-(S,S)-1 that includes a ferrocenyl group was synthesized and acted as a guest. Moreover, hydrophobic cyclobis-(paraquat-p-phenylene) (CBPQT4+-2) was used as the host. In the presence of π-π stacking and the attraction of π-electrons, CBPQT4+-2 can encapsulate Fc-(S,S)-1 into its cavity. Next, a screen-printed electrode was utilized for electrochemical chiral recognition. The host was fixed on the surface of the working electrode, and the guest was used as the electroactive chiral selector to support electron transfer. Once different configurations of amino acids (threonine, histidine, glutamine, and leucine) were mixed with the guest, regardless of whether they contained electroactive units, differences in the cyclic voltammetry results of the probe enantiomers could be observed, namely, in the peak currents or peak potentials. However, glutamine was an exception because the L-isomer had a stronger binding affinity with Fc-(S,S)-1 + Cu(II), which would limit the transport of the complex into the cavity of CBPQT4+-2, thereby resulting in a low peak current. Thus, an inverse phenomenon was observed with glutamine. In summary, we believe that this work can increase the testing scope for the chiral recognition of different kinds of isomers using electrochemical tools.

Enantioselective Recognition of Chiral Tryptophan with Achiral Glycine through the Strategy of Chirality Transfer.

Glycine (Gly), an achiral amino acid, has never been reported for enantioselective recognition owing to the absence of chiral sites. Herein, a facile strategy of chirality transfer is proposed to endow Gly with chirality. Optically active CuO, L-CuO, is first prepared, which can be used for the decoration of Gly through the formation of the Cu(Gly)2 complex. Successful chirality transfer from L-CuO to Gly is confirmed by circular dichroism (CD) spectra. The formation of the Cu(Gly)2 complex is further confirmed by Fourier transform infrared spectra and X-ray photoelectron spectroscopy. Next, the resultant L-CuO-Gly is used for chiral analysis of the isomers of tryptophan (Trp). Because of the higher affinity of L-CuO-Gly toward L-Trp than its isomer, the Trp isomers exhibit significant differences in their oxidation peak currents at the L-CuO-Gly-modified glassy carbon electrode (GCE) (IL-Trp/ID-Trp = 5.24). Finally, the practicability of the developed L-CuO-Gly/GCE is assessed, and the results indicate that it could be a reliable chiral sensor for the quantitative analysis of Trp isomers in nonracemic mixtures.

First Report of Pseudopestalotiopsis theae Causing Leaf Spot of Date Palm (Phoenix dactylifera) in China.

Date palm (Phoenix dactylifera L.) is a popular landscape tree in Fujian province, in South China. In November 2018 and June 2019, a leaf spot disease was observed on date palm in Fuzhou city. A survey of date palm plants grown in four different locations revealed that the disease incidence was almost 20%. The spots were brown with a yellow margin, 1 to 20 mm in diameter, and oval to irregular. In later stages, the spots gradually expanded and coalesced, became dry and died. For isolation, small pieces (0.5 cm2) were cut from leaf spots obtained from seven trees and disinfested with 70% alcohol. Leaf pieces were then placed onto 2% potato dextrose agar (PDA) and incubated at 25±2°C for 3 to 4 days. One fungus was consistently isolated from fifteen leaves. Fungal colonies were white with undulating margins and a light cream on the reverse side. Black globose to oblate conidiomata were irregularly distributed throughout ten-day-old colonies. The conidiogenous cells were septate, colorless, smooth-walled, straight to slightly curved, ampulliform or subcylindrical, and 6.0 to 13.5 × 1.3 to 3.0 µm [(n=50); x̄ ± SD = 9.5 ± 2× 2 ± 0.5µm]. Conidia were fusiform and five-celled with constrictions at the septa, measuring 18.5 to 31.5 × 5.0 to 7.5 µm [(n=50); x̄ ± SD = 25.5 ± 2 × 6.5± 0.2µm]. The three median cells were light to dark brown and the two end cells were colorless. Apical cells had 2 to 4 appendages ranging from 10.2 to 22.5 µm long. Basal cells had one appendage ranging from 3.5 to 5.5 µm long. The internal transcribed spacer (ITS) region of the ribosomal DNA and translation elongation factor 1-alpha (TEF1-α) gene of fungus were amplified using primers ITS1/ITS4 and EF1728F/EF1986R, respectively. Amplified products (ITS: MN294700 and TEF1-α: MN970514) showed 99% sequence identity to Pestalotiopsis sp., and Pseudoestalotiopsis theae sequences in GenBank. A comparison of MRC12 sequences with the type culture sequences (ITS: JQ683727 and TEF1-a: JQ683743) also showed high similarity, where ITS sequences exhibited only a three-nucleotide difference at the start of the sequences. No differences, however, were found between the TEF1-α sequences. On the basis of morphology and molecular characteristics, the fungus was identified as Ps. theae (Sawada) Maharachch., K.D. Hyde & Crous Steyaert (Maharachchikumbura et al. 2014). To confirm pathogenicity, five disinfested leaves on three healthy five-year-old date palm plants in a nursery (average temperature 26°C), were punctured 3 to 5 times with a sterilized needle, and then 10 to 15 mL conidial suspension (105 conidia/mL in sterilized distilled water) was sprayed over punctured areas of the leaves. For the control treatment, punctured leaves were sprayed with sterilized distilled water. All inoculated leaves plus the control were covered with plastic bags. After 10 days, brown leaf spots similar in appearance to those observed in the field appeared on all wounded leaves, and Ps. theae was successfully re-isolated; the control leaves remained asymptomatic. Previously, Ps. theae was reported on oil palm (Elaeis guineensis Jacq.) from Sierra Leone and Thailand (Turner, 1971; Suwannarach et al. 2013). To our knowledge, this is the first report of Ps. theae on date palm in China. This report expands the host range Ps. theae to date palm and underscores the potential threat of an emerging leaf spot pathogen on Phoenix species. References Maharachchikumbura, K.D., et al. 2014. Stud. Mycol. 79: 121-186. Suwannarach, N., et al. 2013. J. Gen. Plant Pathol. 79: 277-279. Turner, P.D. 1971. Phytopathol. 14: 1-58.

An ionic-based carbon dot for enantioselective discrimination of nonaromatic amino alcohols.

Here, ionized chiral carbon dots, (S,S)-C-dots-1 (λex = 430 nm, λem = 480 nm), were synthesized via a facile route with relatively high quantum yield (∼24.4%) and used as a fluorescent chiral sensor. One of the advantages of the synthetic process is that it avoids the loss of the chiral center. That is, the chiral bromo compound can directly form an ionic pair with the pyridyl group, which is derived from the amine precursor in the first step. Furthermore, (S,S)-C-dots-1 shows clear discrimination toward different configurations of nonaromatic amino alcohols in the presence of Cu(ii). When the (R)-isomer is added to a solution of (S,S)-C-dots-1 + Cu(ii), it shows much higher fluorescent intensity than the (S)-isomer. The values of IR/IS are 2.9 and 2.3 for 2-aminobutan-1-ol and 2-aminopropan-1-ol, respectively. In summary, we believe that this work can expand the synthetic routes and potential applications of functional carbon dots in the field of enantioselective sensing.

A facile synthesis of two ionized fluorescent carbon dots and selective detection toward Fe2+ and Cu2.

In this study, a facile synthesis of two ionized carbon dots (CDs-2 and CDs-3) is reported, in which different ionic pairs are formed at the surface of the carbon core. In contrast to CDs-3, the accumulation of carbon core can be clearly observed in the TEM image of CDs-2. This is due to the linkage of the dibromine alkyl group. Compared with naked CDs in the absence of the ionic pair, the maximum emission wavelength undergoes a red-shift of nearly 60 nm. Moreover, protic solvents (water, ethanol and N,N'-dimethyl formamide) have an apparent effect on the emission intensities of CDs-2 and CDs-3. The time-resolved average lifetimes of CDs-2 and CDs-3 are calculated as 56.34 ns and 54.50 ns, respectively. Furthermore, they both have much better fluorescence stability in the solution with pH ranging from 2 to 11 due to the presence of the imidazolium cation. It is interesting to see that CDs-2 and CDs-3 have much different responses towards Cu2+ and Fe2+. The CDs-3 solution generates clear fluorescence quenching when treated with Fe2+. In brief, we believe that these findings can inspire more research developments in the synthesis and further application of functional CDs.