First report of Neopestalotiopsis clavispora causing leaf spot disease on banana (Musa acuminate L.) in China.

Banana (Musa acuminate L.) is an important tropical fruit in China. In October 2020, a new leaf spot disease was observed on banana plants at an orchard of Zhenkang county (23°45'23.46″ N, 98°48'46.52″ E), Lincang city, Yunnan province, China. The disease incidence was about 1%. The leaf spots occurred sporadically and the percentage of the leaf area covered by lesions was less than 5%. Symptoms on the leaves were initially small, irregular, reddish-brown spots that gradually expanded to fusiform-shaped lesions with a yellow halo and eventually become necrotic, dry, and cracked. To isolate the pathogen, thirty symptomatic leaves (15 mm2) from five plants were surface disinfected in 70% ethanol (10 s) and 0.8% NaClO (2 min), rinsed in sterile water three times, and transferred to potato dextrose agar (PDA) at 28°C for 5 days. Twenty-five colonies formed on the PDA plates were white with cottony aerial mycelium, round with a light orange underside. Abundant black globular acervuli semi-immersed on PDA were observed after a week. Conidia were straight or slightly curved, clavate to spindle, five cells, four septa with dimensions of 17.49 to 34.51 µm × 4.24 to 7.28 µm (avg. 23.83 × 5.62 µm; n=50). The apical and basal cells were hyaline, whereas the three median cells were dark brown. Conidia had a single basal appendage with lengths of 2.95 to 17.7 µm (avg. 7.18 µm; n=50) and two to three apical appendages with lengths of 10.7 to 53.84 µm (avg. 17.36 µm; n=50). These morphological characteristics are consistent with those of Neopestalotiopsis spp. (Maharachchikumbura et al. 2014). To confirm species, single-spore cultures of two representative isolates CATAS-102001 and CATAS-102002 were selected for further identification. The internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF1-α) and ß-tubulin (TUB2) genes of the two isolates were amplified with primers ITS1/ITS4 (White et al. 1990), EF1-728/EF2 (O'Donnell et al. 1998; Carbone and Kohn, 1999) and T1/Bt2b (Glass and Donaldson, 1995; O'Donnell and Cigelnik, 1997), respectively, and sequenced. The sequences were deposited in GenBank (ITS: OM281005 and OM281006; TEF1-α: OM328820 and OM328821; TUB2: OM328818 and OM328819). A maximum likelihood phylogenetic tree was constructed using the MEGA 7.0 (Kumar et al. 2016) based on the concatenated sequences ITS region, EF1-α and TUB2 gene, and the cluster analysis placed the representative isolates CATAS-102001 and CATAS-102002 within a clade comprising Neopestalotiopsis clavispora. The pathogenicity of two isolates was conducted on six 7-leaf-old banana seedlings. Two leaves from each potted plants were stab inoculated by puncturing into 1-mm using a sterilized needle, and stabbing three points at both sides of leaf midrib, and then placing 10 µl conidial suspension (1×106 conidia/ml) on one side of wounded points and the other side of wounded points were inoculated with sterile water as control. Inoculated plants were kept inside a plastic bag for 72 h and maintained in the greenhouse (12 h/12 h light/dark, 28°C, 90% relative humidity). The experiments were repeated twice. Irregular necrotic lesions on inoculated leaves appeared 7 days after inoculation, whereas controls were asymptomatic. The fungus was recovered from inoculated leaves, and its taxonomy was confirmed morphologically and molecularly, fulfilling Koch's postulates. Neopestalotiopsis clavispora has been reported to cause leaf spot on Mangifera indica (Shu et al. 2020), Macadamia integrifolia (Santos et al. 2019) and Ligustrum lucidum (Chen et al. 2020). To our knowledge, this is the first report of N. clavispora on banana in China. The identification of N. clavispora as the causal agent of the observed leaf spot disease on banana is critical to the prevention and control of this disease in the future.

Rheinheimera lutimaris sp. nov., a marine bacterium isolated from coastal sediment.

A Gram-stain-negative, aerobic, rod-shaped bacterium, designated strain YQF-2T, was isolated from coastal sediment sampled in Jiangsu Province and characterized phylogenetically and phenotypically. Optimal bacterial growth occurred at 28 °C (range 4-38 °C) and pH 7 (pH 6-10). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YQF-2T was related to members of the genus Rheinheimera and shared the highest sequence identities with Rheinheimera pacifica KMM 1406T (98.6%), followed by Rheinheimera aestuarii H29T (98.4%), Rheinheimera japonica KMM 9513T (98.3%), Rheinheimera aquimaris SW-353T (98.3%), Rheinheimera hassiensis E48T (97.8%) and Rheinheimera muenzenbergensis E49T (97.7%). The 16S rRNA gene sequence identities between strain YQF-2T and other members of the genus Rheinheimera were below 97.2%. The digital DNA-DNA hybridization value between strain YQF-2T and R. pacifica KMM 1406T was 23.3±2.3%. The average nucleotide identity value between strain YQF-2T and R. pacifica KMM 1406T was 79.7%. The unique respiratory quinone was ubiquinone-8. Phosphatidylethanolamine and phosphatidylglycerol were identified as the major polar lipids. The strain had summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), summed feature 8 (C18:1 ω7c and/or C18:1 ω6c), C16:0, C12:0 3-OH and iso-C17:0 3-OH as major fatty acids. The G+C content of the genomic DNA was 50.0 mol%. On the basis of phenotypic, genotypic and phylogenetic evidence, strain YQF-2T represents a novel species of the genus Rheinheimera, for which the name Rheinheimera lutimaris sp. nov. is proposed, with the type strain YQF-2T (=KCTC 72184T=MCCC 1K03663T).

Rheinheimera sediminis sp. nov., a marine bacterium isolated from coastal sediment.

A Gram-stain-negative, aerobic, rod-shaped bacterium, designated strain YQF-1T, was isolated from coastal sediment in Jiangsu Province (PR China) and characterized phylogenetically and phenotypically. Bacterial optimal growth occurred at 28 °C (range 4-40 °C) and pH 7 (range pH 6-11). Phylogenetic analysis based on 16S rRNA gene sequence indicated that YQF-1T was related to members of the genus Rheinheimera and shared the highest sequence identities with Rheinheimera mesophila DSM 29723T (98.5 %), followed by Rheinheimera tangshanensis DSM 19460T (98.4 %), Rheinheimera tilapiae Ruye-90T (97.9 %), Rheinheimera soli BD-d46T (97.9 %), Rheinheimera aquatica GR5T (97.4 %), Rheinheimera coerulea TAPG2T (97.3 %) and Rheinheimera texasensis A62-14BT (97.1 %). The 16S rRNA gene sequence identities between YQF-1T and other members of the genus Rheinheimera were below 97.0 %. The digital DNA-DNA hybridization value between YQF-1T and Rheinheimera mesophila DSM 29723T was 25.1±2.3 %. The average nucleotide identity (ANI) value between YQF-1T and Rheinheimera mesophila DSM 29723T was 81.4 %. The major respiratory quinone was Q-8. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phospholipid, two unidentified aminolipids and three unidentified lipids. The strain had summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), C16 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), iso-C15 : 0, and anteiso-C17 : 1 ω9c as the major fatty acids. The G+C content of the genomic DNA was 46.2 mol%. On the basis of phenotypic, genotypic and phylogenetic evidence, strain YQF-1T represents a novel species of the genus Rheinheimera, for which the name Rheinheimera sediminis sp. nov. is proposed, with the type strain YQF-1T (=KCTC 72183T=MCCC 1K03646T).

[Genetic diversity analysis of Fusarium oxysporum f. sp. cubense populations from China using Inter-Simple Sequence Repeats-PCR (ISSR-PCR) technique].

OBJECTIVE: We used Inter-Simple Sequence Repeats (ISSR) markers to reveal the genetic diversity of 95 Fusarium oxysporum f. sp. cubense ( FOC ) isolates from banana in China, for the rational control of the disease. METHODS: Eight primers were chosen for analyzing FOC isolates to study their genetic diversity by ISSR-PCR. All isolates were clustered using Unweighted Pair-Group Method with Arithmetic means (UPGMA) analysis by NTSYSpc v2.10e software. RESULTS: A total of 52 sites were generated, among them 92.3% were polymorphic. Genetic distance was 0.57 to 1.00 based on the Nei's standard. Isolates were grouped into six distinct clusters (A, B, C, D, E and F) based on ISSR analysis using a genetic distance threshold of 0.68, the proportion of 51.06%, 39.58%, 5.20%, 2.08%, 1.04%, and 1.04%, respectively. CONCLUSION: There were high levels of genetic variation among the FOC isolates, and the ISSR clustering groups had obvious correlation with hosts and races of the pathogen.

Fibrinogen improves liver function via promoting cell aggregation and fibronectin assembly in hepatic spheroids.

Many key functions performed by the liver depend on the interaction between parenchymal cells and the microenvironment comprised of neighboring cells and extracellular matrix. The biological macromolecules in the matrix, which are dynamically changing, participate in various physiological processes through interactions with cell surface receptors, antigens, and ion channels. We found the rat liver biomatrix scaffold (LBS) prepared from adult rats is more effective in enhancing the function of hepatic spheroids than those derived from newborn or senile rats. Combined with matrisome and bioinformatics analyses, we further found that the glycoproteins, fibronectin and fibrinogen may have special potential for improving hepatocyte function. Human primary hepatocyte organoids and HepaRG spheroids showed more mature hepatocyte phenotype after adding fibronectin and fibrinogen to the culture system. During the cultivation of hepatic spheroids, fibrinogen resulted in an increase in cell-cell junction by promoting cell aggregation and helping fibronectin to assemble on cell surface, which resulted in activation of Wnt/ß-catenin pathway. Fibronectin-integrin αVß1-Wnt/ß-catenin may be the axis of signal transduction in parenchymal cell microenvironment. Importantly, fibrinogen enhances the signal transduction. These results suggest that the addition of fibronectin and fibrinogen to the 3D culture system is a new strategy for inducing parenchymal cell functional maturation.

Sweat gland organoids contribute to cutaneous wound healing and sweat gland regeneration.

Sweat glands perform a vital thermoregulatory function in mammals. Like other skin components, they originate from epidermal progenitors. However, they have low regenerative potential in response to injury. We have established a sweat gland culture and expansion method using 3D organoids cultures. The epithelial cells derived from sweat glands in dermis of adult mouse paw pads were embedded into Matrigel and formed sweat gland organoids (SGOs). These organoids maintained remarkable stem cell features and demonstrated differentiation capacity to give rise to either sweat gland cells (SGCs) or epidermal cells. Moreover, the bipotent SGO-derived cells could be induced into stratified epidermis structures at the air-liquid interface culture in a medium tailored for skin epidermal cells in vitro. The SGCs embedded in Matrigel tailored for sweat glands formed epithelial organoids, which expressed sweat-gland-specific markers, such as cytokeratin (CK) 18 and CK19, aquaporin (AQP) 5 and αATP. More importantly, they had potential of regeneration of epidermis and sweat gland when they were transplanted into the mouse back wound and claw pad with sweat gland injury, respectively. In summary, we established and optimized culture conditions for effective generation of mouse SGOs. These cells are candidates to restore impaired sweat gland tissue as well as to improve cutaneous skin regeneration.

Analysis of the 16S-23S rRNA gene internal transcribed spacer region in Klebsiella species.

The 16S-23S rRNA gene internal transcribed spacer (ITS) regions of Klebsiella spp., including Klebsiella pneumoniae subsp. pneumoniae, Klebsiella pneumoniae subsp. ozaenae, Klebsiella pneumoniae subsp. rhinoscleromatis, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrigena, and Klebsiella ornithinolytica, were characterized, and the feasibility of using ITS sequences to discriminate Klebsiella species and subspecies was explored. A total of 336 ITS sequences from 21 representative strains and 11 clinical isolates of Klebsiella were sequenced and analyzed. Three distinct ITS types-ITS(none) (without tRNA genes), ITS(glu) [with a tRNA(Glu (UUC)) gene], and ITS(ile+ala) [with tRNA(Ile (GAU)) and tRNA(Ala (UGC)) genes]-were detected in all species except for K. pneumoniae subsp. rhinoscleromatis, which has only ITS(glu) and ITS(ile+ala). The presence of ITS(none) in Enterobacteriaceae had never been reported before. Both the length and the sequence of each ITS type are highly conserved within the species, with identity levels from 0.961 to 1.000 for ITS(none), from 0.967 to 1.000 for ITS(glu), and from 0.968 to 1.000 for ITS(ile+ala). Interspecies sequence identities range from 0.775 to 0.989 for ITS(none), from 0.798 to 0.997 for ITS(glu), and from 0.712 to 0.985 for ITS(ile+ala). Regions with significant interspecies variations but low intraspecies polymorphisms were identified; these may be targeted in the design of probes for the identification of Klebsiella to the species level. Phylogenetic analysis based on ITS regions reveals the relationships among Klebsiella species similarly to that based on 16S rRNA genes.

Establishment of an ex Vivo Model of Nonalcoholic Fatty Liver Disease Using a Tissue-Engineered Liver.

The prevalence of nonalcoholic fatty liver disease (NAFLD), a common cause of chronic liver disease, continues to increase in parallel with that of obesity. Currently, there are no preclinical models to study its complex pathogenesis nor to assess candidate therapies. We have established a tissue-engineered (TE) liver by seeding cells into liver-derived matrix scaffolds and then perfusing the scaffolds with a medium that dynamically provides requisite nutrients, vitamins, minerals, and hormones. Liver-specific biomatrix scaffolds, comprised of almost all of the liver's known extracellular matrix (ECM) components and matrix-bound soluble signals (e.g., growth factors/cytokines), were recellularized with human hepatic cell line HepG2 and perfused with a complete medium enabling the cells to form functioning liver tissue. By perfusing the system with medium with a high fat content, the cells established a TE fatty (TEF) liver model paralleling that of livers in NAFLD patients. The high fat medium containing 500 µM of free fatty acids (FFAs) (oleic acid:palmitic acid = 2:1) caused the TEF livers to accumulate 2-times more fat than those in the control medium over an 8 day culture period and significantly influenced the capacity of fatty acid synthesis and metabolism. PDK4, CYP2E1, and CYP7A1 genes associated with NAFLD and other liver diseases were all up-regulated, and the metabolic activity of CYP3A4 was significantly impaired. Excess FFAs also induced alterations in transporters and key enzymes in the lipid biosynthesis pathway. The TEF liver was used to test if an antisteatotic drug, Metformin, used in patients with NAFLD, would be able to provide effects paralleling those observed in some patients. Metformin treatment of the TEF liver model caused reduced cellular triglycerides, activated AMPK molecule, inhibited mTORC1 signaling pathway, which thus affected the synthesis and metabolism of FFAs. Overall, the TEF liver offers a stable and reproducible model to study the NAFLD development process and antisteatotic drug effects.

Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3) spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.