First Report of Botryosphaeria dothidea Causing Stem Blight of Dioscorea oppositifolia in China.

Dioscorea oppositifolia Thunb. (Chinese yam) is one of the traditional foods and medicinal plants in China. It has nutritional and medicinal value and plays an important role in treatment of diabetes and hypertension. In 2018, stem blight was first observed on the stalks of D. oppositifolia in fields of Anguo City (115°27' N; 38°46' E), Hebei Province, China. Over 400 plants were surveyed in four fields, and nearly 30% of the plants were infected. At the initial stage of the disease, there were dark brown spots on the stems and in later stages the leaves and stems withered. To identify the pathogen, 10 symptomatic stalks were collected, and one diseased area was taken out from each sample. Small square stalk pieces (3 to 5 mm) were obtained with sterile scissors from the junction of infected and healthy tissues, sterilized with sodium hypochlorite (10%) for 1 min, followed by washing in sterile water three times, then pieces were transferred to potato dextrose agar (PDA) plates for 7 days at 25°C. The fungal isolates were purified by single-spore isolation. A total of three species of fungi were isolated, and initial pathogenicity tests found that one fungal species could cause the disease symptoms on D. oppositifolia stems. This pathogen was grown on PDA plates in the dark at 25 °C for 10 days. In the beginning, the colonies were white, and as the culture time was extended, the color of the colonies became darker and then became black. Conidia forming on pycnidia were one-celled, hyaline, aseptate, and ovoid, with dimensions of 4.6 to 7.6 × 2.6 to 4.8 µm (n=100). Mycelial DNA was extracted from a 7-day-old culture, and PCR amplifications were performed using primers ITS1/ITS4 and ß-tubF/ß-tubR (Glass and Donaldson 1995; White et al. 1990). BLAST searches at GenBank showed 100.00% nucleotide sequence identity for the ITS sequence with Botryosphaeria dothidea strain sdxf6 (MG282093; 545/545 bp) and for ß-tubulin 99.76% identity with B. dothidea strain SD-B8 (KP183131; 411/412 bp). Sequences from these regions were deposited in GenBank (ITS: OP104323; ß-tubulin: OK669147). Morphological and molecular results confirmed this species as B. dothidea (Angelica et al. 2017; Bernard et al. 2004). To inoculate plants, pathogen was grown on PDA at 25°C in the dark for 15 days, after which a spore suspension (3×105 spores/mL) was prepared by flooding the agar surface with sterilized double-distilled water. Pathogenicity tests were conducted by stem inoculation of 6-month-old healthy D. oppositifolia plants. The stems were wounded by lightly rubbing with a steel sponge, and the wounded stem was wrapped in sterile cotton treated with 1 mL of the spore suspension, then the plants were covered with plastic to maintain a moist environment for 72 h. Control plants were inoculated with sterile water. Inoculated and control plants (ten each) were kept in a moist chamber (25°C, 16-h light and 8-h dark period, 75% relative humidity). After 15 days, all of the inoculated plants showed dark brown spots on the stems, and the symptoms were the same as those in the field, while the controls were healthy. After 30 days, all of the inoculated but none of the control D. oppositifolia plants showed leaf wilting or leaf withering. Isolates from the inoculated and infected leaves were identified as B. dothidea by DNA sequencing with primers ITS1/ITS4 and ß-tubF/ß-tubR, fulfilling Koch's postulates. To our knowledge, this is the first report of B. dothidea causing stem blight on D. oppositifolia. The disease poses a threat to the production of D. oppositifolia, and management strategies need to be developed.

Vacuum-Induced Guest N,N'-Diethylformamide Binding in a Metastable Cd5-Based Metal-Organic Framework.

A three-dimensional (3D) metal-organic framework (MOF) of [Et2NH2]2[Cd5(BTB)4(DEF)2]·4.75DEF (1; H3BTB = benzene-1,3,5-tribenzoic acid and DEF = N,N'-diethylformamide) sustained by symmetrical Z-shaped Cd5 secondary building units (SBUs) with an intrinsically metastable host framework has been prepared and characterized. Upon gentle vacuum (800 Pa) at 50 °C, some encapsulated DEF solvates are released, leading to pore-shape changes and Cd2+ coordination geometry distortion. This is followed by DEF solvate migration to only one end of the SBU with concomitant symmetry breaking. Additional time under vacuum promoted further structural distortion and topology changes as authenticated by single-crystal X-ray diffraction studies. This work was initially inspired by unusual gas adsorption isotherms and points to the potentially complicated, nonspectator role of coordinative solvents such as DEF during MOF activation.

First Report of Rhizoctonia solani AG-4 HG-III Causing Sheath Blight on Foxtail Millet in China.

Foxtail millet (Setaria italica) is an important grain and forage crop. This crop is widely grown in Northern China (Yang et al.2020). In Aug 2021, foxtail millet variety of Jigu42 showing lodging were found in Baoding China with the incidence of 30% and irregular brown lesions were found in sheaths and leaves of infected plants. The center of the lesions was kraurotic and pale, and the edges were gray-brown or dark brown. Twelve samples with typical lesions were collected from the surveyed field to isolate the pathogen. The infected samples were cut into square pieces of about 3 to 5 mm and were immersed into NaOCl (1%) for 1 min followed by washing with sterile water for three times. Then all sterilized tissues were inoculated on potato dextrose agar (PDA) plates and incubated at 25℃. After 3 days, fresh mycelial tips grown from the tissues were transferred to new plates for purification and incubated in the dark at 25°C for 4-5 days until the hyphae covered the whole plates. The colonies of 15 isolates on PDA medium showed similar colonial characteristics, which were fluffy and white initially, gradually turned light brown, and no sclerotia was observed even at 20 days later. Micro-examination revealed that all isolates showed the identical morphological features as Rhizoctonia sp. (Sneh et al. 1991), which contained the septate and right-angled branching hyphae with slight constriction at the base of mycelial branches, and three to seven nuclei per cell (Yang et al. 2013). Total genomic DNA was extracted from 5-day-old cultures, and the internal transcribed spacer (ITS) region of rDNA was amplified with ITS1 and ITS4 as the primers (Garibaldi et al. 2019). The sequencing results showed that the nucleotide sequences of 15 amplicons were identical and shared 100% identity with the corresponding fragments of R. solani AG-4 HG-III from sugar beet (GenBank accession No. MH172666 and MH172663) in Blastn search. The sequencing size of ITS in this study was 3 bp shorter than that of sugar beet, with a length of 722, because the base 'T' in the beginning and 'GA' in the end of the sequences did not detected in our study. Phylogenetic tree of 16 isolates of different AG4 subgroups was created by the software MEGA 7.0 through the NJ method, and the showed that the isolates were clustered to the clade of AG-4 HG-III group. The sequences of three isolates were deposited in GenBank under the accession No. ON810364, ON810365 and ON810366. For pathogenicity test, 5 mm diameters plate of the 5-day-old fungus which cultured on PDA were inoculated to the sheath of 10 foxtail millet plants grown in pots at 5- or 6-leaf stage. Then, the inoculated plants were placed into a growth chamber, and the inoculated sheaths were covered with wet cotton ball for 2 days to keep humidity, while sterile water was inoculated as the control. All plants were cultivated at 26°C with 14 h light and 10 h dark for 14 days. The experiment was repeated for three times. As the result, the same lesions observed in the field appeared on the inoculated plants at 10-14 days post inoculation, whereas the mock was healthy. The pathogen was re-isolated from the infected samples. The morphological characteristics and the nucleotide sequences of ITSs were same as that of the original isolates. All in above, the pathogen cusing sheath blight on foxtail millet was identified as R. solani AG-4 HG-III. To our knowledge, this is the first report of R. solani AG-4 HG-III causing sheath blight on S. italica in China. This finding expands the host range known for R. solani AG-4 HG-III and will be helpful for developing effective control strategies of foxtail millet sheath blight.

First Report of Alternaria tenuissima Causing Brown Spot Disease of Angelica dahurica in China.

Angelica dahurica (Fisch. ex Hoffm.) is an abundantly cultivated Chinese herbal medicine plant in China with about 4000 hectares grown, the annual production is up to 24,000 tons. The medicinal part of A. dahurica is its root, and mainly function for treat cold, headache, toothache, rhinitis, diabetes, etc. Besides, A. dahurica is also used as a spice in Asia. In September 2018, brown spot was observed on the leaves of A. dahurica in fields of Anguo City, Hebei Province, China. In the field investigated, the incidence of brown spot disease reached 15%. The infected leaves showed brown spots surrounded with pale yellow edge, resulting in withered of the whole leaf. It seriously endangers the growth of A. dahurica, reducing the yield and quality of medicinal materials, even leading to the death of plants. We isolated the pathogen from 10 leaves with same lesions, the small square leaf pieces of approximately 3 to 5 mm were obtained with the sterile scissors from the junction of infected and healthy tissues, sterilized with sodium hypochlorite (10%) for 1 min followed by washing in sterile water for 3 times, then incubated on potato dextrose agar (PDA) plates at 25°C for 4 days. The culture was transferred to new PDA plates and was cultivated in dark at 25°C for 10 days. A total of 3 species of fungi were isolated, and only one fungus species has been found to be able to cause the original pathological characteristics of A. dahurica leaves through the back-grafting experiment. The mycelium was black and began to sporulate after 8 days on PDA media by single spore separation. Multiple spores joined together to form spores chain. The spores were spindle-shaped, yellow to yellow brown, and size ranged from 45 to 55 × 15 to 20 µm (n=50), with zero to three longitudinal septa and one to five transverse septa. For pathogenicity tests, the spore suspension (3.5×105 spores/mL) were inoculated to healthy plants grown in experimental field, the test was repeated four times, and 10 leaves were inoculated in each repetition, and the sterile water was inoculated as the blank control. Inoculated leaves were covered with transparent plastic bags for 24 h to keep humidity. Nine days later, it was found that there were lesions on the leaves inoculated with the pathogen, and the traits were the same as those in the field, while the controls are healthy. The fungus was consistently isolated from the inoculated leaves. The similar isolates were re-isolated from the inoculated and infected leaves and identified as Alternaria tenuissima by DNA sequencing, fulfilling Koch's postulates. Fungal genomic DNA was extracted from 7-day-old culture. PCR amplifications were performed using primers ITS1 / ITS4 and TEFF / TEFR respectively (Takahashi et al. 2006, Du 2008). The nucleotide sequence of PCR products, which have been deposited in Genebank under the accession numbers MN153514 and MN735428, showed 99.8%-100% identity with the corresponding sequences of A. tenuissima (MW194297 and MK415954). In order to further identify the pathogen species, we constructed a phylogenetic tree by combining TEF sequence and ITS sequence to distinguish the relationship between the pathogen and other minor species in the genus Alternaria, the isolate was clustered in the Alternaria clade. Therefore, the pathogen was identified as A. tenuissima based on the morphological characteristics and molecular identification. To our knowledge, this is the first report of A. tenuissima causing leaf spot on A. dahurica in China.

The effects of subclinical hypothyroidism on serum lipid level and TLR4 expression of monocyte in peripheral blood of rats.

OBJECTIVE: To observe effect of subclinical hypothyroidism (SCH) on serum lipid level and expression of toll-like receptor 4 (TLR4) in rats' peripheral blood mononuclear cells (PBMC). METHODS: Fifty Wistar female rats were divided into three groups: normal control (NC group; n=10), sham group (n=10), and L-T-4 (L-thyroxine) group (n=30, with thyroidectomy, fed with rich-calcium water after operation. 5 weeks later, abdominal subcutaneous injection of L-T-4: 0.95 µg/100g/d). 8 weeks later, the rats were killed then the peripheral blood was collected to determine the levels of serum thyroid-stimulating hormone (TSH), total thyroid hormone (TT4), total cholesterol (TC) and low density lipoprotein cholesterin (LDL-C). Rats in L-T-4 group were divided into normal lipid (NL) group) and high lipid (HL) group) according to lipid value of NC group. Monocytes were separated from blood to determine TLR4 expression by flow cytometry. RESULTS: In NL and HL groups TSH were higher than in NC and Sham groups (p<0.05). TT4 have no significant differences (p>0.05). TLR4, TLR4 mRNA, NF-κB (p65) were increased (p<0.05). TNF-α, IL-6 and IL-1ß were higher than in NC and sham groups (p<0.01). There were no significant differences of TLR4, TLR4 mRNA, NF-κB (p65), TNF-α, IL-6 and IL-1ß expression between NL and HL groups (p>0.05). CONCLUSION: TLR4, TLR4 mRNA, NF-κB (p65) of PBMC and TNF-α, IL-6, IL-1ß expression in serum were all increased in SCH rats, which was not related to serum dyslipidemia.

Research progress and perspective in metabolism and metabolomics of psoriasis.

ABSTRACT: Psoriasis is considered a systemic disease associated with metabolic abnormalities, and it is important to understand the mechanisms by which metabolism affects pathophysiological processes both holistically and systematically. Metabolites are closely related to disease phenotypes, especially in systemic diseases under multifactorial modulation. The emergence of metabolomics has provided information regarding metabolite changes in lesions and circulation and deepened our understanding of the association between metabolic reprogramming and psoriasis. Metabolomics has great potential for the development of effective biomarkers for clinical diagnosis, therapeutic monitoring, prediction of the efficacy of psoriasis management, and further discovery of new metabolism-based therapeutic targets.

[Molecular cloning and characterization of calcineurin A in Setosphaeria turcica].

A Setosphaeria turcica gene encoding the catalytic subunit of calcineurin was cloned using degenerated primers corresponding to conserved domains of Ser/Thr protein phosphatases and its complete cDNA (GenBank accession No. EF 407562) was obtained with RACE method. It's validated single copied model by southern hybridization. Furthermore, the CNA inhibitor Cyclosporin A (CsA) exhibited potent antifungal activity against conidial germination and appressorium formation of S. turcica. The inhibition ratio was positively correlated to CsA concentration. However, appressorium formation was more sensitive than conidium germination to the inhibitor at the same concentration. It was suggested that CNA might play an important role in the pathogenicity of S. turcica.

Evaluating the component contribution to nonlinear optical performances using stable [Ni4O4] cuboidal clusters as models.

Five stable clusters sharing the cuboidal [Ni4O4] skeleton are subjected to third-order nonlinear optical (NLO) property measurements. Preliminary results suggest that the NLO property is largely defined by the cluster core skeleton and the directly coordinated atoms, with limited contribution from the heavy atoms peripherally attached to the aromatic ligands.

[Association of QT dispersion, ST/heart rate slope and ST-segment depression in exercise test to evaluate restenosis after percutaneous coronary intervention].

OBJECTIVE: To evaluate the sensitivity and specificity of QT dispersion (QTd) and ST/heart rate slope (ST/HRs) at the end of ECG exercise test plus ST-segment depression on diagnosing restenosis after percutaneous coronary intervention (PCI). METHODS: Between November 2001 and December 2003, 129 patients underwent PCI successfully, and they were examined 3-6 months later. At the end of treadmill exercise, QTd and ST/HRs were measured. All patients also accepted coronary angiography to ascertain whether he/she had restenosis. The results of QTd and ST/HRs plus ST-segment depression were then evaluated. RESULTS: The sensitivity and specificity of QTd and ST/HRs plus ST-segment depression were 84.6% and 80.4% respectively. Both of them were significantly higher than conventional ST-segment depression standard (sensitivity was 53.3% and specificity was 66.7%, P<0.05). CONCLUSION: Measuring QTd and ST/HRs at the end of ECG treadmill exercise test plus ST-segment depression can be used for the diagnosis of restenosis after PCI.

A cuboidal [Ni4O4] cluster as a precursor for recyclable, carbon-supported nickel nanoparticle reduction catalysts.

Cuboidal [Ni4O4] clusters supported by a pyridine alkoxide ligand have been developed. One of these clusters was selected as a precursor for carbon-hosted Ni nanoparticles (NiNPs/C) which were efficient catalysts for the conversion of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP) at room temperature.

StPBS2, a MAPK kinase gene, is involved in determining hyphal morphology, cell wall development, hypertonic stress reaction as well as the production of secondary metabolites in Northern Corn Leaf Blight pathogen Setosphaeria turcica.

Mitogen activated protein kinase kinase (MAPKK) is a crucial component in the MAPK signaling pathway. However, the functions of MAPKKs in foliar pathogens remain poorly understood. In the current study, a MAPKK gene designated as StPBS2 was cloned from Setosphaeria turcica and the functions of this gene were investigated by RNAi technology. Four independent StPBS2 gene silence transformants with different efficiencies were confirmed by real time PCR. Compared to the wild type strain (WT), these transformants showed decreased colony growth, shortened hyphae cell length, broadened cell width and an obvious reduction in conidium yield. Moreover, the cell wall of the transformants was thicker and they were also more sensitive to substances that interfere with cell wall biosynthesis than WT. Additionally, the transformants displayed higher sensitivity to hypertonic stress than WT and the sensitivity was associated with the level of silencing of StPBS2. They were also resistant to the fungicides iprodione, procymidone and fludioxonil, to which WT almost completely sensitive. The transformants produced more red secondary metabolites than WT and the production was enhanced with increasing silencing level and increased glucose content in PDA medium. Our results suggest that StPBS2 is involved in morphogenesis, condiogenesis, cell wall development, hypertonic stress reaction and resistance to fungicides, as well as in the biosynthesis of secondary metabolites in S. turcica.

StSTE12 is required for the pathogenicity of Setosphaeria turcica by regulating appressorium development and penetration.

In filamentous fungi, the pathogenic mitogen-activated protein kinase (PMK) pathway performs an important function in plant infection. STE12-like genes found in higher eukaryotes encode transcription factors and are regulated by the PMK pathway. However, the functions of STE12-like genes in foliar pathogens remain poorly understood. In this study, we cloned StSTE12 from Setosphaeria turcica and investigated its functions by RNA interference. Transformants ste12-3, ste12-2 and, ste12-1, in which the StSTE12 silencing efficiency increased in order, were confirmed by real time PCR. Compared with the wild-type (WT) strain, the transformants showed reduced growth rate, lighter colony color, and obviously decreased conidium production. More importantly, different to WT strain and ste12-3 with lower StSTE12silencing efficiency, ste12-1 and ste12-2 with higher StSTE12 silencing efficiency were nonpathogenic on intact leaves, but pathogenic on wounded leaves. However, the biological activity of HT-toxin from all transformants showed no difference on corn leaves. Furthermore, ste12-1 and ste12-2 did not penetrate artificial cellophane membrane and showed abnormal and delayed development appressoria. Although it could penetrate the cellophane membranes, ste12-3 formed appressoria after 48 h of inoculation more than WT. Therefore, StSTE12 was involved in vegetative growth, conidiation, appressorial development, penetration as well as the pathogenicity, but it was not related to HT-toxin biosynthesis. More interestingly, all the results suggested that StSTE12 was crucial for pathogenicity due to involvement in regulating appressoria development and penetration.

StRas2 regulates morphogenesis, conidiation and appressorium development in Setosphaeria turcica.

The proteins of Ras family are a large group of monomeric GTPases and act as molecular switches transducing extracellular signals into the cell in higher eukaryotes. However, little is known about roles of Ras family in the foliar pathogens. In this research, we cloned the gene named StRas2 encoding Ras in Setosphaeria turcica and investigated its function by RNA interference technology. We found that the growth rate of RNAi transformants named as R1, R2, R3, R4, R5 and R6, in which the StRas2 silencing efficiency fell in turn. With the highest silencing efficiency, the transformant R1 showed anomalistic hyphae morphology, indicating its growth was significantly affected. The transformants with a middle-silencing efficiency, such as R3, R4, displayed a delay when forming appressoria and invasive hyphae. R1 could not form conidia and appressoria. However, the conidial formation in R5 and R6 was significantly reduced, and these two transformants could form appressoria and penetrate the artificial cellophane, only that its invasive hyphae were fascicular and rarely branched. The HT-toxin biological activity of all transformants showed no difference. All results suggested that StRas2 is involved in the morphogenesis, conidiation, and appressorium development and is not related to the biosynthesis of HT-toxin.