First report of Sphaerulina azaleae causing leaf spot on Rhododendron pulchrum Sweet in China.

Rhododendron pulchrum Sweet is a popular ornamental evergreen shrub, renowned for its exquisite flowers. In November 2020, leaf spot disease was observed at Qujing Normal University (25.527°N; 103.744°E), Qujing, Yunnan, China. Symptoms were observed on 25-35 % leaves. This led to early leaf drop, significantly reducing its ornamental value. The infected leaves exhibited irregular, dark brown to blackish spots, with random distribution. To investigate the causal agent, ten symptomatic leaves were collected for pathogen isolation. Leaf spots bearing ascomata were picked with a sterile needle, placed on water agar, and then incubated at 26 ℃ for 24 h. Individual germinated spores were transferred onto PDA for further purification and morphological study. Two pure isolates (chl01 and chl02) were obtained. The colonies on PDA were circular, raised, pink-white at the center, dark brown at the margin, and dark brown to black from reverse after four weeks at 26 °C. Moreover, colonies on PDA sporulated following a 5-hour UV light exposure in a laminar flow hood and a subsequent 14 h light: 10 h dark (14:10) cycle for 5 days at 26 °C. The conidiophores are reduced to conidiogenous cells which are 1.5-3 × 1.5-2.5 µm (n = 10), integrated, hyaline, proliferating sympodially near the apex, cylindrical, and widest at the base. Conidia are hyaline, slightly verruculose, guttulate, cylindrical, straight or irregularly curved, subobtuse at the apex, truncate at the base, and 19-28 × 3-4 µm (n= 30), with 1-3 septa. The morphological features were similar to the genus Sphaerulina (Quaedvlieg et al. 2013). To confirm the candidate pathogen, we performed PCR and gene sequencing using specific primers ACT-512F/ACT2Rd, EF1-728F/EF2, ITS4/ITS5, LSU1Fd/LR5 and fRPB2-5F/fRPB2-414R for actin (ACT), the partial translation elongation factor 1-alpha (TEF1), internal transcribed spacer region (ITS), large subunit ribosomal RNA gene (LSU), and RNA polymerase II subunit (RPB2) genes, respectively (Choi et al. 2020). Sequences were deposited in GenBank with accession numbers OR359635 (ACT), OR359637 (TEF1), OR335339 (ITS), OR335299 (LSU) and OR359636 (RPB2). BLAST searches showed 97.34% (ACT), 99.47% (TEF1), 100% (ITS), 100% (LSU) and 100% (RPB2) identity with those of Sphaerulina azaleae CBS 128605 from GenBank, respectively. A multilocus phylogenetic tree was generated using maximum likelihood (ML) based on the concatenated ACT-TEF-ITS-LSU-RPB2 sequence, placing our isolates within the S. azaleae group with high confidence. To fulfill Koch's postulates, three R. pulchrum plants in the landscape (three leaves per plant) were inoculated by pipetting 0.2 ml spore suspension (1 × 106 conidia/ml) onto the surface of each leaf (Khoo et al. 2022). Three additional plants in the landscape were inoculated with sterile water on similar aged leaves. The leaves were enclosed in plastic bags for 72 hours. At 7 days postinoculation, infected leaves exhibited the symptoms as described above, whereas the controls showed no symptoms. This experiment was repeated twice. The same fungus was reisolated from the infected leaves and identified based on morphology and Sanger sequencing. It is worth noting that S. azaleae can cause leaf spot on Rhododendron yedoense f. poukhanense (Choi et al. 2020). To our knowledge, this is the first report of S. azaleae causing leaf spot on R. pulchrum Sweet in China, indicating that appropriate management strategies are needed.

The polymorphisms of ANXA6 influence head and neck cancer susceptibility in the Chinese Han population.

Background: Head and neck cancer (HNC) is the sixth most common malignant tumor worldwide and imposes a serious economic burden on society and individuals. Annexin has been implicated in multiple functions which are essential in HNC development, including cell proliferation, apoptosis, metastasis, and invasion. This study focused on the linkage between ANXA6 variants and HNC susceptibility in Chinese people. Methods: Eight SNPs in ANXA6 from 139 HNC patients and 135 healthy controls were genotyped by the Agena MassARRAY platform. The correlation of SNPs with HNC susceptibility was evaluated using odds ratio and 95% confidence interval calculated by logistic regression using PLINK 1.9. Results: Overall analysis results demonstrated that rs4958897 was correlated with an increased HNC risk (allele: OR = 1.41, p = 0.049; dominant: OR = 1.69, p = 0.039), while rs11960458 was correlated with reduced HNC risk (OR = 0.54, p = 0.030). In age ≤ 53, rs4958897 was related to reduce HNC risk. In males, rs11960458 (OR = 0.50, p = 0.040) and rs13185706 (OR = 0.48, p = 0.043) were protective factors for HNC, but rs4346760 was a risk factor for HNC. Moreover, rs4346760, rs4958897, and rs3762993 were also correlated with increased nasopharyngeal carcinoma risk. Conclusions: Our findings suggest that ANXA6 polymorphisms are linked to the susceptibility to HNC in the Chinese Han population, indicating that ANXA6 may serve as a potential biomarker for HNC prognosis and diagnosis.

Arbuscular mycorrhizal fungi contribute to reactive oxygen species homeostasis of Bombax ceiba L. under drought stress.

Drought stress is one of the major abiotic factors limiting plant growth and causing ecological degradation. The regulation of reactive oxygen species (ROS) generation and ROS scavenging is essential to plant growth under drought stress. To investigate the role of arbuscular mycorrhizal fungi (AMF) on ROS generation and ROS scavenging ability under drought stress in Bombax ceiba, the ROS content, the expression levels of respiratory burst oxidase homologue (Rbohs), and the antioxidant response were evaluated in AMF and NMF (non-inoculated AMF) plants under drought stress. 14 BcRboh genes were identified in the B. ceiba genome and divided into five subgroups based on phylogenetic analysis. The effect of AMF on the expression profiles of BcRbohs were different under our conditions. AMF mainly downregulated the expression of Rbohs (BcRbohA, BcRbohD, BcRbohDX2, BcRbohE, BcRbohFX1, and BcRbohI) in drought-stressed seedlings. For well-water (WW) treatment, AMF slightly upregulated Rbohs in seedlings. AMF inoculation decreased the malondialdehyde (MDA) content by 19.11 and 20.85%, decreased the O2⋅- production rate by 39.69 and 65.20% and decreased H2O2 content by 20.06 and 43.21% compared with non-mycorrhizal (NMF) plants under drought stress in root and shoot, respectively. In addition, AMF inoculation increased the non-enzymatic antioxidants glutathione (GSH) and ascorbic acid (AsA) content in roots by 153.52 and 28.18% under drought stress, respectively. The activities of antioxidant enzymes (SOD, PX, CAT, APX, GPX, GR, MDAR, and DHAR) all increased ranging from 19.47 - 131.54% due to AMF inoculation under drought stress. In conclusion, these results reveal that AMF inoculation can maintain ROS homeostasis by mitigating drought-induced ROS burst, via decreasing ROS generation and enhancing ROS scavenging ability of B. ceiba seedlings.

A chromosome-level genome assembly of Amorphophallus konjac provides insights into konjac glucomannan biosynthesis.

Amorphophallus konjac, a perennial herb in the Araceae family, is a cash crop that can produce a large amount of konjac glucomannan. To explore mechanisms underlying such large genomes in the genus Amorphophallus as well as the gene regulation of glucomannan biosynthesis, we present a chromosome-level genome assembly of A. konjac with a total genome size of 5.60 Gb and a contig N50 of 1.20 Mb. Comparative genomic analysis reveals that A. konjac has undergone two whole-genome duplication (WGD) events in quick succession. Two recent bursts of transposable elements are identified in the A. konjac genome, which contribute greatly to the large genome size. Our transcriptomic analysis of the developmental corms characterizes key genes involved in the biosynthesis of glucomannan and related starches. High expression of cellulose synthase-like A, Cellulose synthase-like D, mannan-synthesis related 1, GDP-mannose pyrophosphorylase and phosphomannomutase fructokinase contributes to glucomannan synthesis during the corm expansion period while high expression of starch synthase, starch branching enzyme and phosphoglucomutase is responsible for starch synthesis in the late corm development stage. In conclusion, we generate a high-quality genome of A. konjac with different sequencing technologies. The expansion of transposable elements has caused the large genome of this species. And the identified key genes in the glucomannan biosynthesis provide valuable candidates for molecular breeding of this crop in the future.

Inoculation With Ectomycorrhizal Fungi and Dark Septate Endophytes Contributes to the Resistance of Pinus spp. to Pine Wilt Disease.

Pine wilt disease (PWD) is a deadly disease to pines (Pinus spp.) worldwide. The occurrence of PWD can reduce the relative abundance of root ectomycorrhizal fungi (ECMF) and dark septate endophytes (DSE). However, the effects of exogenous ECMF/DSE inoculation on the rhizosphere microbial community structure of Pinus tabulaeformis infected by pine wood nematode (PWN) is little known. Here, we tested how ECMF/DSE may improve resistance to PWD by quantifying microbial carbon biomass and soil enzymatic activity among different treatments at 6 and 9 months after PWN infection. Denaturing gradient gel electrophoresis (DGGE) was used to study the microbial community structure at 3, 6, and 9 months after PWN infection in the rhizosphere of P. tabulaeformis seedlings inoculated with ECMF/DSE. The results showed that exogenous ECMF/DSE inoculation reduced the disease severity caused by PWN infection. After PWN infection, the rhizosphere microbial carbon of seedlings inoculated with Amanita vaginata, Suillus bovinus, Gaeumannomyces cylindrosporus, and Paraphoma chrysanthemicola was 38.16, 49.67, 42.11, and 96.05% higher than that of the control group, respectively. Inoculation of ECMF/DSE inhibited the decrease of rhizosphere microbial biomass caused by PWN infection. The richness and diversity of P. tabulaeformis rhizosphere fungi at 9 months were reduced by PWN infection but partially recovered by the exogenous fungi (ECMF/DSE) inoculation except for P. chrysanthemicola, which indicates a role of ECMF/DSE in maintaining stability of the microbial community. Inoculation with ECMF/DSE increased the beneficial bacterial (Thauera sp., Mesorhizobium sp., etc.) and fungal groups (Tomentella ellisii, Wilcoxina mikolae, etc.) of in the rhizosphere. In summary, exogenous ECMF/DSE inoculation could increase P. tabulaeformis resistance to PWD probably by improving the rhizosphere microenvironment.

[Advances in studies on genetics of syndromes combining sensorineural hearing loss with scoliosis].

Sensorineural hearing loss and scoliosis are common in several disease groups, such as hereditary connective tissue syndrome, hereditary motor and sensory neuropathy, lysosomal storage syndrome and endocrine disorders. These diseases have significant phenotypic diversity and genetic heterogeneity, different subtypes show inconsistent characteristics of deafness. Moreover, subtypes with similar clinical manifestations have different genetic mechanisms. Using new generation sequencing technology, considerable progress has been achieved in these diseases. This paper reviews clinical manifestations and genetic mechanism of syndromes combining sensorineural hearing loss and scoliosis.

Discovery of sensorineural hearing loss and ossicle deformity in a Chinese Li nationality family with spondyloepiphyseal dysplasia congenita caused by p.G504S mutation of COL2A1.

BACKGROUND: Spondyloepiphyseal dysplasia congenita (SEDC) is an autosomal dominant chondrodysplasia characterized by disproportionate short stature, abnormal epiphyses, and flattened vertebral bodies. COL2A1 has been confirmed as the pathogenic gene. Hearing loss represents an infrequent manifestation for 25-30% of patients with SEDC. The characteristics of the hearing impairment were rarely documented. METHODS: Audiological, ophthalmic, imaging examinations were conducted on the family members. The whole exome sequencing (WES) was performed to detect the candidate gene, and the Sanger sequencing was used to confirm the causative variation. RESULTS: COL2A1 c.1510G>A (p.G504S), a hot spot variation, was identified as the disease-causing mutation of the Chinese Li nationality family with SEDC. This variation was co-segregated with the SEDC phenotype in the family and was absent in the 1000 Genomes Project, ESP and ExAC. Clinically, several manifestations were first demonstrated in SEDC patients caused by p.G504S, including sensorineural hearing loss, auditory ossicles deformity, retinal detachment, sacrum cracked and elbow and wrist joints deformity. Other classical SEDC manifestations such as bones and joints pain, midfacial dysplasia, disproportionate short stature, spinal deformity, thoracocyllosis, coxa arthropathy, myopia and waddling gait were also showed in the family patients. CONCLUSION: We first identified the mutation p.G504S in COL2A1 gene as the pathogenesis in a Chinese Li nationality family and reported the correlation between p.G504S and atypical clinical phenotypes including sensorineural hearing loss, auditory ossicles deformity, retinal detachment, sacrum cracked and elbow and wrist joints deformity. Our findings would extend the phenotypic spectrum of SEDC and deepen clinicians' understanding of genotype-phenotype correlation of the disease.

Dermal bioaccessibility of plasticizers in indoor dust and clothing.

Several studies indicate that human exposure to plasticizers via dermal pathway is not negligible, but the dermal bioaccessibility of phthalates and alternative plasticizers from the important environmental matrix including indoor dust and clothing and the importance weight of dermal exposure to those pollutants have been poorly studied. An in vitro physiologically based extraction test was employed to investigate the dermal bioaccessibility of target phthalates and alternative plasticizers from indoor dust and clothing. Temperature, incubation time, sweat/sebum ratio and solid/liquid ratio were selected to study their effects on the bioaccessibility. The bioaccessibility of Diethyl phthalates (DEP), dibutyl phthalate (DBP), bis-2-ethylhexyl phthalate (DEHP), Acetyl tributyl citrate (ATBC), bis-2-ethylhexyladipate (DEHA) and bis-2-ethylhexyl terephthalate (DEHT) in indoor dust were 66.20 ±â€¯1.93%, 94.27 ±â€¯1.31%, 80.37 ±â€¯8.09%, 75.02 ±â€¯2.12%, 94.50 ±â€¯3.42% and 74.09 ±â€¯3.79%, respectively, under the condition of 1:1 sweat/sebum ratio, 1/100 solid/liquid ratio (indoor dust), 1:1 area/area ratio (1:1, clothing) and 90 min incubation time at 36.3 °C which are chosen based on the experimental results and human physical conditions. DBP showed the highest bioaccessibility in all samples. The time course of the plasticizer release was fitted to a first-order one-compartment model. DBP showed the highest release rate (k1) calculated from the model, which was consistent with the bioaccessibility result. Risk assessment indicated that dermal exposure of DBP was an important exposure route, accounting for about 21.58% of total intake, and indoor dust was an important exposure media when considering the dermal bioaccessibility.

Ultrastructure of antennal sensilla of three fruit borers (Lepidoptera: Crambidae or Tortricidae).

Three fruit borers Conogethes punctiferalis (Guenée) (Crambidae), Grapholita molesta Busck (Tortricidae), and Spilonota albicana Motschulsky (Tortricidae) are serious pests of fruit trees. In this study, their antennal morphology, types of sensilla, and distributions were observed by using SEM (Scanning Electron Microscope). Nine types of sensilla were found on the antennae of C. punctiferalis, while eight types of sensilla were presented on each of G. molesta and S. albicana. The sensilla trichodea with two subtypes were the most abundant sensilla among three fruit borers. Two subtypes of sensillum coeloconica (type I with spines and type II without spines) were observed on the antennae of C. punctiferalis and G. molesta. However, sensilla coeloconica (type I) were only found in S. albicana. Although the sensilla campaniformia were only found on the antennae of C. punctiferalis, our observations confirm sensilla campaniformia presence in the moths. In addition, the functions of these sensilla were discussed based on previously reported lepidopteran insects. As a result, our study may provide useful information for advanced electrophysiological and behavioral experiments to better understand the mechanisms related to pests control.

Identification, Expression Patterns, and Functional Characterization of Chemosensory Proteins in Dendroctonus armandi (Coleoptera: Curculionidae: Scolytinae).

The Chinese white pine beetle, Dendroctonus armandi Tsai and Li (Coleoptera: Curculionidae: Scolytinae), is a serious pest of coniferous forests in China. Thus, there is considerable interest in developing eco-friendly pest-control methods, with the use of semiochemicals as a distinct possibility. Olfaction is extremely important for fitness of D. armandi because it is the primary mechanism through which the insect locates hosts and mates. Thus, here we characterized nine full-length genes encoding chemosensory proteins (CSPs) from D. armandi. The genes were ubiquitously and multiply expressed across different developmental stages and adult tissues, indicating various roles in developmental metamorphosis, olfaction, and gustation. Ligand-binding assays implied that DarmCSP2 may be the carrier of D. armandi pheromones and various plant host volatiles. These volatiles were identified through RNA interference of DarmCSP2 as: (+)-α-pinene, (+)-ß-pinene, (-)-ß-pinene, (+)-camphene, (+)-3-carene, and myrcene. The systematic chemosensory functional analysis of DarmCSP2 in this study clarified the molecular mechanisms underlying D. armandi olfaction and provided a theoretical foundation for eco-friendly pest control.

Isolation and expression of HMG-CoA synthase and HMG-CoA reductase genes in different development stages, tissues and treatments of the Chinese white pine beetle, Dendroctonus armandi (Curculionidae: Scolytinae).

We isolated two full-length cDNAs encoding 3-hydroxy-3-methyl-glutaryl coenzyme A synthase (HMG-S) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-R) from the Chinese white pine beetle (Dendroctonus armandi), and carried out some bioinformatic analysis on the full-length nucleic acid sequences and deduced amino acid sequences. Differential expression of the DaHMG-S and DaHMG-R genes was observed between sexes (emerged adults), and within these significant differences among development stage, tissue distribution, fed on phloem of Pinus armandi and topically applied juvenile hormone (JH) III. Increase of DaHMG-S and DaHMG-R mRNA levels in males suggested that they may play a role in mevalonate pathway. Information from the present study might contribute to understanding the relationship between D. armandi and its semiochemical production.

The CYP51F1 Gene of Leptographium qinlingensis: Sequence Characteristic, Phylogeny and Transcript Levels.

Leptographium qinlingensis is a fungal associate of the Chinese white pine beetle (Dendroctonus armandi) and a pathogen of the Chinese white pine (Pinus armandi) that must overcome the terpenoid oleoresin defenses of host trees. L. qinlingensis responds to monoterpene flow with abundant mechanisms that include export and the use of these compounds as a carbon source. As one of the fungal cytochrome P450 proteins (CYPs), which play important roles in general metabolism, CYP51 (lanosterol 14-α demethylase) can catalyze the biosynthesis of ergosterol and is a target for antifungal drug. We have identified an L. qinlingensis CYP51F1 gene, and the phylogenetic analysis shows the highest homology with the 14-α-demethylase sequence from Grosmannia clavigera (a fungal associate of Dendroctonus ponderosae). The transcription level of CYP51F1 following treatment with terpenes and pine phloem extracts was upregulated, while using monoterpenes as the only carbon source led to the downregulation of CYP5F1 expression. The homology modeling structure of CYP51F1 is similar to the structure of the lanosterol 14-α demethylase protein of Saccharomyces cerevisiae YJM789, which has an N-terminal membrane helix 1 (MH1) and transmembrane helix 1 (TMH1). The minimal inhibitory concentrations (MIC) of terpenoid and azole fungicides (itraconazole (ITC)) and the docking of terpenoid molecules, lanosterol and ITC in the protein structure suggested that CYP51F1 may be inhibited by terpenoid molecules by competitive binding with azole fungicides.

Corrective surgery for deformity of the upper cervical spine due to ankylosing spondylitis.

Rotational and flexion deformity of C1-C2 due to ankylosing spondylitis is rare. We did surgical correction in one such case by lateral release, resection of the posterior arch of C1 and mobilization of the vertebral arteries, wedge osteotomy of the lateral masses of C1 and internal fixation under general anesthesia. There were no vascular and neurological complications during the surgery. After operation the atlantoaxial rotational deformity was corrected and the normal cervical lordosis was restored. At 1 year followup his visual field and feeding became normal and internal fixation was stable.

Investigation of competitive binding of ibuprofen and salicylic acid with serum albumin by affinity capillary electrophoresis.

Ibuprofen and salicylic acid, two typical non-steroidal anti-inflammatory drugs, are used commonly as analgesic drug in clinical medicine and sometimes are co-administered. When the drugs are co-administered, the drug-drug interactions may occur, and can lead to alter the safety and efficacy of drugs, resulting in variations in drug response of the co-administered drugs. Affinity capillary electrophoresis (ACE) was employed to investigate the competitive binding of ibuprofen and salicylic acid on serum albumin. Mobility ratio, derivatives from mobility shift method, was used to deduce the binding constant (K(b)). The binding constants of ibuprofen with HSA are 2.97×106 M⁻¹ and 7.07×104 M⁻¹, respectively; while for salicylic acid, the binding constant is 5.99×104 M⁻¹. The competitive binding of the two drugs was investigated by addition of excessive ibuprofen into the solutions containing constant concentrations of salicylic acid and serum albumin. The results confirmed that ibuprofen and salicylic acid have different high-affinity binding site, but share some low-affinity binding sites on the serum albumin; and ibuprofen is able to partially replace salicylic acid from the preformed binary complexes of serum albumin and salicylic acid.