Leaf spot on Alocasia macrorrhizos caused by Fusarium asiaticum in Sichuan, China.

Alocasia macrorrhizos (Giant elephant's ear), a perennial herb in the Araceae family, is native to South Asia and the Asia-Pacific (Takano, et al. 2012). It is cultivated as a medicinal and ornamental plant, and has a considerable economic importance in China. In September 2020, a severe infection of unknown leaf spot disease was observed on these plants at the Sichuan Agricultural University, Sichuan, China. The leaf spots first appeared as yellow dots. As these lesions expanded, they became circular to oval and light brown with darker brown edges. Around the lesions, the leaf tissue was chlorotic, thereby creating a yellow halo. When the infection became severe, spots merged into larger irregular lesions. Eventually, the diseased leaves senesced and dried. To identify the pathogen, five leaf samples of diseased plants were collected, and symptomatic tissues were surface-disinfected with 75% ethanol for 30 s followed by 3% NaCl solution for 30 s. Samples were rinsed three times in sterilized water, placed on potato dextrose agar (PDA), and incubated at 25°C ± 1°C in the dark. The colony grown on PDA was white (3 days), the center was brown (5 days), turned pink to dark red (8 days) with fluffy aerial mycelium and pigmentation with age. Ten pure cultures were inoculated into carnation leaf agar (CLA) medium and incubated at 25°C in an incubator (12 h for one light-dark cycle). In CLA medium, pathogen produced hyaline, sickle-shaped, macroconidia with 3 to 5 septa, and an average size of 30 to 50 × 4 to 5 µm (n = 30) macroconidia but no microconidia in 10 days. Chlamydospores were spherical to subspherical (5.4 to 13.8 µm). Morphological characteristics of the all isolates were consistent with the description of the Fusarium asiaticum (Leslie and Summerell 2006). To validate this identification, RNA polymerase II (RPB2) (Liu et al. 1999), translation elongation factor (EF-1) (Geiser et al. 2004), and ß-tubulin (TUB2) gene region of five isolates were amplified and sequenced (O' Donnell et al. 2015; White et al. 1990). The sequence of one representative isolate (ZL10) sequence was submitted to GenBank (ON215729, ON215730, and ON215731). The NCBI BLAST identified the top hits, 100%, 100%, and 99.87% for RPB2, EF, and TUB gene sequences, respectively, all indicating to Fusarium asiaticum. Pairwise matched of RPB2 and EF genes by MycoBank Fusarium MSIL showed the top hit rate of 100% for F. asiaticum (MH582120 and MH582249). For Koch's postulate and pathogenicity test, spore suspensions (1 × 10^7 conidia/ml) collected from PDA and CLA cultures with 0.05% Tween 80 buffer were used to inoculate with a spray bottle on leaves of a one year old A. macrorrhizos plants. Two leaves of each plant (20 pots in total) were inoculated with the spore suspension (approximately 2000 µl per leaf). An equal number of control leaves were applied with water and 0.05% Tween 80 buffer. Twenty days later, the inoculated plants showed similar symptoms to those of the original diseased plants while the controls remained asymptomatic. Fusarium asiaticum was reisolated from the infected leaves and confirmed using morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results. This first report raises awareness of a new leaf spot disease infecting a commercial A. macrorrhizos in China. It provides an insight for a need of systematic survey identifying current spread, disease origin, and ultimately developing disease management strategies. Funding: Funding was provided by Sichuan Agricultural University Subject Dual Support Program (Grant No. 2121993055). Funding was provided by Deyang Science and Technology Bureau (Sichuan Province) for key R&D projects in agriculture and rural areas (Grant No. 2021NZ048). Funding was provided by the Sichuan Provincial Department of science and technology for the Sichuan Provincial Science and technology project for connecting and Promoting Rural Revitalization (Grant No, 2022ZHXC0007) References： Geiser, D. M., et al. 2004. Eur. J. Plant Pathol. 110:473. https://doi.org/10.1023/B:EJPP.0000032386.75915.a0 Crossref, ISI, Google Scholar Leslie, J. F., and Summerall, B. A., eds. 2006. Page 176 in The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA. https://doi.org/10.1002/9780470278376 Liu, Y. J., et al. 1999. Mol. Biol. Evol. 16:1799. https://doi.org/10.1093/oxfordjournals.molbev.a026092 O'Donnell, K., and Cigelnik, E. 1997. Mol. Phylogenet. Evol. 7:103. https://doi.org/10.1006/mpev.1996.0376 Takano K T, et al. 2012, Plant Bio., 14(4). https://doi.org/10.1111/j.1438-8677.2011.00541.x.

Smartphone-assisted visual ratio-fluorescence detection of hypochlorite based on copper nanoclusters.

A sensitive naked eye and ratio-fluorescence sensor for Curcumin (CCM) and hypochlorite (ClO-) determination based on copper nanoclusters (Cu NCs) was developed. The fluorescence of the Cu NCs can be quenched due to inner filter effect (IFE) between CCM and Cu NCs, and the ratio fluorescence probe was formed. After adding ClO- to Cu NCs-CCM system, the phenolic and methoxy groups of CCM were oxidized to quinones, then the fluorescence of CCM was quenched and the fluorescence of Cu NC was restored. Moreover, the continuous detection of CCM and ClO- is accompanied by the change of solution color. Therefore, CCM and ClO- semiquantitative visual and fluorescence dual channel detection were realized. The detection results show that the detection based on Cu NCs-CCM probe has a wide detection range (0-412 µM) and low detection limit (24 µM), and a good recovery rate is obtained in adulterated milk and tap water detection. Furthermore, smartphone was introduced for image digital colorimetric analysis through the acquisition, recognition and RGB data processing of solution colors, providing an effective scheme for the field rapid detection of hypochlorite.

Comparing of backfat microRNAomes of Landrace and Neijiang pig by high-throughput sequencing.

BACKGROUND: MicroRNAs (miRNAs) could regulate the expression of target genes and play important roles in modulation of various metabolic processes. Nevertheless, little is known about the backfat microRNAome (miRNAome) of the Neijiang pig. OBJECTIVES: The primary objective of this study was to analyse miRNAomes of Landrace and Neijiang pig backfat (LPB and NPB resp.). Furthermore, investigating differentially expressed miRNAs participating in lipid metabolism and mining potential biomarker for Neijiang pig breeding. METHODS: Here we used the Landrace pig with different metabolic characteristics as a control to analyse the Neijiang pig-specific backfat miRNAome. A comprehensive analysis of miRNAomes was performed by deep sequencing. RESULTS: Small RNA sequencing identified 326 unique miRNAs, 280 were co-expressed in both libraries. Only 11 and 35 miRNAs were specifically expressed in LPB and NPB respectively. Sixty seven differentially expressed miRNAs were identified by IDEG6. MiR-1-3p were identified that may participate in lipid metabolism. Furthermore, qPCR results revealed that lower expression of miR-1-3p in NPB could increase the expression of LXRα, which is an enzyme important for the synthesis and accumulation of lipid. The double luciferase report experiment suggested that LXRα was the direct target gene of miR-1-3p. In short, miR-1-3p could modulate the synthesis and accumulation of lipid by target LXRα. It may be a potential marker for pig breeding. CONCLUSION: Our investigation has delineated the different miRNAs expression patterns of LPB and NPB, which may help understand the regulatory mechanisms of miRNAs in the lipid metabolism, and provide potential biomarkers for Neijiang pig breeding.

Different fluorescence emitting copper nanoclusters protected by egg white and double-emission fluorescent probe for fast detection of ethanol.

Green emitting copper nanoclusters (G-Cu NCs), yellow emitting Cu NCs (Y-Cu NCs), orange emitting Cu NCs (O-Cu NCs) and red emitting Cu NCs (R-Cu NCs) were prepared using chicken egg white as the stabilizer by changing the reaction conditions. This is a green, facile and cheap method to explore different color emitting CuNCs by the same precursor and stabilizers. The G-Cu NCs were employed for the detection of ethanol due to their aggregation induced emission enhancement (AIEE) effect. The fluorescence emission of Cu NCs at 526 nm under the excitation of 444 nm can be effectively enhanced in the presence of ethanol due to AIEE effect, thus realizing the quantitative determination of ethanol content in the range 5-60%. In addition, a visual dual-emission fluorescence probe with the combination of G-Cu NCs and silicon nanoparticles (Si NPs/G-Cu NCs) was designed to evaluate ethanol content conveniently and rapidly. Desirable linear relationship is observed between ratio of fluorescence intensity (I525/I441) and ethanol content under the excitation of 383 nm. Visible color transformation of this probe is observed in the ethanol content range 2-20%. Moreover, the ethanol sensing platforms were applied to the detection and evaluation of the alcohol content of liquor, and the recoveries in liquor were in the range 99.7% to 113%, broadening the applications of Cu NCs and providing a sensitive detection method for ethanol.

Molecular design of energetic tetrazine-triazole derivatives.

Nitrogen-rich compounds are promising candidates for preparing high energetic density materials (HEDMs) and show the potential in the application of propellants, explosives, and pyrotechnics. Two kinds of typical nitrogen-rich compounds, such as tetrazine and triazole, have attracted the attentions in recent years owing to their high densities, good thermal stabilities, and excellent energetic performances. In this work, four series of innovative energetic compounds based on the conjugates of tetrazine and triazole bearing various substituents (-NH2, -NO2, and -NHNO2) were designed. The optimized structures, crystal densities, heats of formation (HOFs) in gas phase and in condensed phase, detonation properties, bond dissociation energies (BDEs), and impact sensitivity (h50) of these compounds were studied systematically via density functional theory (DFT) method. The detonation velocities of four series of compounds are in the range between 7.03 and 8.59 km s-1 and their detonation pressures are in the range between 20.6 and 33.1 GPa. Results indicated that the linkage of -N=N- bond contributed significantly to HOFs and energy density of the energetic molecules, and 1,2,3-triazole showed better performances than 1,2,4-triazole slightly. As for the same series compounds with different substituents, the compounds with -NHNO2 possessed the highest HOFs (such as A6, B6, C6, D6). In terms of the energetic properties (D and P), four compounds (A7, B7, C7, and D7) exhibited the comparable performance with the widely used hexa-hydro-1,3,5-trinitro-1,3,5-triazine (RDX) and in the meanwhile displayed superior thermal stability and sensitivity to RDX, which indicated their potential application in the insensitive energetic materials.

Stem Rot Disease of Juglans sigillata Dode Caused by Fusarium fujikuroi in China.

Juglans sigillata Dode is an endemic species in the southwest China, and is an important nut and woody oil tree. The shell of its fruit is hard and can be used to make various crafts. From 216 to 2019, typical stem rot symptoms of 8-year-old J. sigillata were observed in cultivated fields in a 600-ha orchard in Zigong, Sichuan province, China. At this orchard, approximately 35% of the trees have been seriously damaged over the past few years. The typical symptoms were water-soaking on the stem, rotting, wilting, and blighting, eventually leading to the death of the plant. In June, ten diseased tissues were collected and surface-sterilized by 3% NaClO and 75% alcohol. Morphological observations were made from the isolates grown on Potato dextrose agar (PDA) and incubated at 25°C for 3 to 9 days. Morphological characteristics were made on pure cultures grown on Synthetic low nutrient agar (SNA). Five isolates with similar morphology were isolated from single spores. Colonies on PDA reached 8.3 cm in diameter after 6 days at 25 °C, aerial mycelia were white to cream and wol-like, later turning violet and dark purple with age. The hyphae of the strain were colorless and septate. There were two types of conidia on SNA, microconidia and macroconidia. Microconidia (n = 50) were oval, elliptic or clavate, no septate, 2.2 to 3.8 × 7.6 to11.7 µm. Conidiophores were branched or unbranched, solitary or in groups, phialides cylindrical to flask-shaped, monophialidic and polyphialidic. Macroconidia (n = 50) were long slender with a curved apical cell and foot-like basal cell, 3 to 4 septate and 2.1 to 3.9 × 26.2 to 53.4 µm. For molecular identification, the internal transcribed spacer (ITS), ß-tubulin (TUB2), translation elongation factor (TEF1) and large subunit (LSU) were amplified with the corresponding primer pairs ITS1/ITS4 (White et al. 1990), BT2A/BT2B, EF1/EF2 (O'Donnell et al. 1997), and LROR/LR5 (Rehner and Samuels 1994), respectively. BLAST search results indicated that the ITS, TUB2, TEF1, LSU sequences (GenBank acc. nos. MT791384, MT786729, MN853324, and MT705246) showed 99 to100% identity with Fusarium fujikuroi sequences at NCBI (GenBank acc. nos. MG798789, MH398245, MK604519 and KJ954504). The results were confirmed by multilocus phylogenetic analysis. Based on the morphological characteristics and molecular analysis of the isolates, the fungus was identified as F. fujikuroi (Leslie and Summerell 2006). Koch's postulates were checked under controlled conditions. Fifteen 2-year-old healthy potted J. sigillata were inoculated by pricking the epidermis of stem with a needle and applying 150 µl of a microconidial suspension (1 × 106 spores/ml) to the wounded surface with a brush. Sterilizd distilled water was used as the control. The experiment was repeated three times. All the plants were incubated at 25 ± 2°C after inoculation for daily observation of disease development. After 12 days, the inoculated plants showed the same symptoms as observed in the original diseased plants, while the control plants were asymptomatic. The fungus was re-isolated from the symptomatic stems and was completely identical to the isolates used to inoculate the plants. Thus, we confirmed that F. fujikuroi caused the stem rot of J. sigillata. To our knowledge, this is the first report of this fungus causing stem rot in J. sigillata in China. Our results can help identify stem rot disease of J. sigillata and develop control measures for the disease.

Brown leaf spot of Cycas debaoensis Caused by Colletotrichum siamense in Sichuan, China.

Cycas debaoensis Y. C. Zhong et C. J. Chen is an endemic species in China that is listed among China's national key preserved wild plants (Class I) (Xie et al. 2005). It is mainly distributed in south China (Guangxi, Guizhou, and other regions). In April 2017, a new leaf disease of C. debaoensis was found in Chengdu (30°35'32″ N; 104°05'11″E) in China with an incidence over 40%. Symptoms on C. debaoensis initially appeared as brown necrotic lesions on the margin or in the center of leaves. The lesions then enlarged gradually and developed into brown spots, necrotic lesions with dark brown margins. Many small and black dots were observed on necrotic lesions. Eventually, the diseased leaves withered and died. Ten samples were collected and surface-sterilized by 3% NaClO and 75% ehanol respectively for 60s and 90s, rinsed with autoclaved distilled water and then blot-dried with autoclaved paper towels. Five isolates from diseased leaves with similar morphology were isolated from single spores. Morphological characteristics were recorded from pure cultures grown on potato dextrose agar (PDA) incubated at 25°C for 3-9 days. Initially, the colonies grown on PDA were white, then, became pale gray with concentric zones and greenish black beneath. Conidia were single-celled, smooth-walled, straight, colorless, cylindrical with both ends bluntly rounded,13.0-16.5 × 4.7-5.8 µm in size (n = 100 spores). For molecular identification, the genomic DNA of the isolates was extracted using a DNeasyTM Plant Mini Kit (Qiagen). The internal transcribed spacer (ITS) (ITS1/ITS4 White et al., 1990), ß-tubulin (TUB2) (BT2A/BT2B (O'Donnell et al., 1997)), actin (ACT) (ACT512F/ACT (Carbone & Kohn, 1999)), calmodulin (CAL) (CL1C/CL2C (Weir et al., 2012)), mating type protein and chitin synthase (CHS-1) (CHS-1) (CHS-9 79F/CHS-345R (Carbone & Kohn, 1999)) were amplified. BLAST results indicated that the ITS, TUB2, ACT, CAL, CHS-1 sequences (GenBank MN305712, MN605072, MT478663, MT465591 and MT478664) showed 99-100% identity with C. siamense sequences at NCBI (GenBank JF710564, MK341542, MK855094, MH351155 and MK471373). The Phylogenetic tree inferred from the combined dataesets (TEF, TUB and ACT) show that the isolate belongs to C. siamense clade with a credibility value of 99%. Two-year-old potted plants of C. debaoensis (10 plants) were used for pathogenicity test. On each plant, 5 leaves were sprayed with a conidial suspension (1 × 106 conidia/ml) on both sides of the leaves. Autoclaved distilled water was used as negative control (10 plants). Plants were kept in the greenhouse at 25 °C under 16h/8h photoperiod and 70-75% relative humidity (RH). The symptoms observed on the inoculated plants were similar to those observed in the field, while the controls remained asymptomatic. C. siamense was re-isolated from all diseased inoculated plants, and the culture and fungus characteristics were the same as the original isolate. The morphological characteristics and molecular analyses of the isolate matched the description of C. siamense (Prihastuti et al., 2009). C. siamense was previously reported infecting Citrus reticulata (Cheng et al. 2013), but this is the first report of brown leaf spot on C. debaoensis caused by C. siamense in China. This finding provides important basis for further research on the control of the disease.

Comparison of stomach microRNA transcriptomes of Tibetan and Yorkshire pigs by deep sequencing.

MiRNAs regulate the expression of target genes in diverse cellular processes and hence play important roles in different physiological processes, yet little is known about the stomach microRNAome (miRNAome) of the Tibetan pig. The objective of this experiment was to investigate differentially expressed stomach miRNAs participating in digestion. Firstly, we isolated total RNA by Trizol reagent from three Tibetan and three Yorkshire purebred pigs stomach samples at 90-day-old. Secondly, a comprehensive analysis of Tibetan and Yorkshire pig stomach miRNAomes was performed by small RNA sequencing in the Illumina HiSeq 2000 system. Finally, SYBR Green Real-time RT-PCR was performed to validate the differentially expressed miRNAs. We identified 318 unique miRNAs, 260 were co-expressed in both libraries, 17 and 31 miRNAs were specifically expressed in Tibetan and Yorkshire pigs respectively. Fifty six differentially expressed miRNAs were identified by the identifying differentially expressed genes 6 (IDEG6). Kyoto encyclopedia of genes and genomes analysis revealed that some of the differentially expressed miRNAs were associated with protein and fat digestion. Two differentially expressed miRNAs (miR-214-3p and ssc-un39) participating in the digestion of lipid were identified. Additionally, qRT-PCR results suggested that a higher expression of miR-214-3p in the Tibetan pig stomach could lead to relatively lower expression of calcium-dependent phospholipase A2, which is an enzyme important for the digestion of glycerol phospholipid. This study has delineated the different stomach miRNAs expression patterns of Tibetan and Yorkshire pigs, which would help explain the regulatory mechanisms of miRNAs in digestion of Tibetan pigs, and contribute to utilize a the unique digestion merits of Tibetan pig in future porcine hybridization breeding.

Dissemination of macrolides, fusidic acid and mupirocin resistance among Staphylococcus aureus clinical isolates.

As an increasingly common cause of skin infections worldwide, the prevalence of antibiotic-resistant Staphylococcus aureus (S. aureus) across China has not been well documented. This literature aims to study the resistance profile to commonly used antibiotics, including macrolides, fusidic acid (FA) and mupirocin, and its relationship to the genetic typing in 34 S. aureus strains, including 6 methicillin-resistant S. aureus (MRSA), isolated from a Chinese hospital. The MIC results showed 27 (79.4%), 1 (2.9%) and 6 (17.6%) isolates were resistant to macrolides, FA and mupirocin, respectively. Among 27 macrolide-resistant S. aureus isolates, 5 (18.5%) were also resistant to mupirocin and 1 (3.7%) to FA. A total of 13 available resistant genes were analyzed in 28 antibiotic-resistant strains using polymerase chain reaction (PCR). The positive rates of macrolide-resistant ermA, ermB, ermC, erm33 and low level mupirocin-resistant ileS mutations were 11.1%, 25.9%, 51.9%, 7.4% and 100%, respectively. Other determinants for FA- and high level mupirocin-resistance were not found. The results of multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE) revealed 13 sequence types (STs) and 18 clusters in 23 resistant gene positive S. aureus isolates. Among these STs, ST5 was most prevalent, accounting for 18.2%. Notably, various clusters were found with similar resistance phenotype and genotype, exhibiting a weak genetic relatedness and high genetic heterogeneities. In conclusion, macrolides, especially erythromycin, are not appropriate to treat skin infections caused by S. aureus, and more effective measures are required to reduce the dissemination of macrolides, FA and mupirocin resistance of the pathogen.

Comparison of liver microRNA transcriptomes of Tibetan and Yorkshire pigs by deep sequencing.

MicroRNAs (miRNAs) play an important role in the modulation of various metabolic processes in the liver, yet little is known about the liver microRNAome (miRNAome) of the Tibetan pig. Here we used the Yorkshire pig as a control to analyze the Tibetan pig-specific liver miRNAome, and for preliminary investigation of differentially expressed miRNAs participating in metabolism. A comprehensive analysis of Tibetan and Yorkshire pig liver miRNAomes by small RNA sequencing identified 362 unique miRNAs. Among these, 304 were co-expressed in both libraries, and 10 and 48 miRNAs were specifically expressed. Differential expression analysis of miRNAs, miRNA target prediction and KEGG analysis revealed that differentially expressed miRNAs were associated mainly with the metabolism of glucose, lipid and protein. Six differentially expressed miRNAs (miR-34a, miR-326, miR-1, miR-335, miR-185 and miR-378) participating in the metabolism of glucose and lipid were identified. Additionally, qPCR results revealed that a lower expression of miR-34a in Tibetan pig liver may promote gluconeogenesis by increasing the expression of Sirtuin type 1 (Sirt1); a lower expression of miR-1 in Tibetan pig liver may promote the synthesis and accumulation of lipid by increasing the expression of Liver X receptor α (LXRα); and a lower expression of miR-185 in Tibetan pig liver may promote the uptake of cholesterol from blood and secretion of bile by increasing the expression of the scavenger receptor class B type I (SR-BI). Our results provide new information and understanding of porcine miRNA profiles, which may help explain the regulatory mechanisms of miRNAs in the metabolic functions of Tibetan pig liver, and provide new biomarkers to assist in the development of Tibetan pig breeding characteristics.