Complete mitochondrial genomic sequence of Auricularia delicata (Auriculariaceae), an edible Chinese mushroom.

Auricularia delicata (Mont.) Henn. 1893 is an edible and medicinal jelly mushroom popular in China. Here, we report the assembly and annotation of a complete A. delicata mitochondrial genome based on data sequenced using an Illumina NovaSeq 6000 platform. The length of the complete circular A. delicata mitochondrial genome is 189,696 bp, with a GC content of 34.1%. The A. delicata mitochondrial genome contains 60 genes, including 32 protein-coding genes, 26 tRNA genes, and two rRNA genes. Phylogenetic analysis indicated that A. delicata clustered with the Auricularia group, alongside A. auricula-judae and A. heimuer. Additionally, A. delicata was found to be genetically distant from other species of Polyporales, Russulales, and Agaricales. This genome will provide an invaluable reference for the continued study and utilization of A. delicata and other Auricularia species.

First report of green mold disease caused by Penicillium citrinum on Dictyophora rubrovalvata in China.

Dictyophora rubrovolvata is a saprophytic mushroom widely cultivated in China, including Guizhou Province for its high nutritional, medicinal, and economical values (Chen et al. 2021). In May 2021, green mold disease was observed on the fruiting bodies of D. rubrovolvata, causing its death or preventing it from forming a sporocarp, in an indoor-production facility at Asuo village, Baiyun District Guiyang city, Guizhou Province, China (26°73'51" N, 106°72'88" E). The disease incidence was 60%-70% in the affected 1.33-ha growing area, causing a serious economic loss. To identify the causal agent, a total of 15 samples with symptomatic symptoms were collected. Small pieces (5 mm × 5 mm) were cut from the diseased tissues, surface sterilized in 0.4% NaClO for 5 min, washed three times with sterilized water, placed on potato dextrose agar (PDA) medium, and incubated at 24 °C for 7 days. Twenty-one pure cultures were obtained by single-spore isolation method. The colonies were initially white but after seven days as conidia developed they turned green. Hyphae were hyaline and guttulate. Conidiophores were verrucose stipes, triverticulate, and phialides flask shaped. Conidia were smooth and pale green, with subglobose to globose shape measuring 2.0-2.5 × 1.8-2.5 µm (n=50). Based on these morphological characteristics, the isolates matched the description of the genus Penicillium (Visagie et al. 2014). To confirm the identity, DNA of five representative isolates (QS001, QS005, QS008, QS015, QS017) was extracted according to the manufacturer's instructions (Biomiga Fungal DNA Extraction Kit; CA, USA). Afterwards, PCR was performed to amplify ITS region, calmodulin and ß-tubulin genes using primer pairs ITS1/ITS4 (White et al. 1990), CMD5/CMD6 (Glass et al. 1995), and Bt2a/Bt2b (Hong et al. 2006), respectively. BLASTN analysis of these sequences showed the best matches with Penicillium citrinum CBS 139.45 (ITS region: 98.60% (493/500 bp) identity to accession MH856132.1; CMD: 99.79% (469/470 bp) identity to accession MN969245.1; ß-tubulin:100% (407/407 bp) identity to accession GU944545.1). Representative sequences of the sequenced DNA regions were deposited in GenBank (ITS region: OK446552; CMD: OK492612; ß-tubulin: OK482677). Furthermore, a phylogenetic tree was constructed with MEGA 7 based on the concatenated sequences. Koch's postulates were met to confirm the pathogenicity of the representative isolate (QS001) on D. rubrovolvata. Six discs (5mm×5mm) from actively growing P. citrinum QS001 colonies (5-day-old) were placed on six fruiting bodies of D. rubrovolvata (5-month-old). Mock inoculations were performed using PDA discs only without any fungus. The inoculation sites were wrapped with a sterilized 200-µm nylon mesh. All fruiting bodies were incubated at 23°C ± 2°C under a 0-h/24-h photoperiod and 80% relative humidity (RH) after inoculation. After 14 days, green mold was observed on all P. citrinum QS001 inoculated mushrooms. In contrast, no disease was observed in mock inoculated group. The disease assays were repeated three times. P. citrinum QS001 was isolated from all inoculated D. rubrovolvata and verified via the molecular analysis mentioned above. To the best of our knowledge, this is the first report that P. citrinum causes green mold on D. rubrovalvata in China and further studies should focus on managing this disease to prevent any disease outbreaks.

Wheat embryo globulin nutrients ameliorate d-galactose and aluminum chloride-induced cognitive impairment in rats.

Wheat embryo globulin nutrient (WEGN), with wheat embryo globulin (WEG) as the main functional component, is a nutritional combination that specifically targets memory impairment. In this study, we explored the protective role of WEGN on Alzheimer's disease (AD)-triggered cognitive impairment, neuronal injury, oxidative stress, and acetylcholine system disorder. Specifically, we established an AD model via administration of d-galactose (d-gal) and Aluminum chloride (AlCl3) for 70 days, then on the 36th day, administered animals in the donepezil and WEGN (300, 600, and 900 mg/kg) groups with drugs by gavage for 35 days. Learning and memory ability of the treated rats was tested using the Morris water maze (MWM) and novel object recognition (NOR) test, while pathological changes and neuronal death in their hippocampus CA1 were detected via HE staining and Nissl staining. Moreover, we determined antioxidant enzymes by measuring levels of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) in serum, cortex, and hippocampus, whereas changes in the acetylcholine system were determined by evaluating choline acetyltransferase (ChAT), and acetylcholinesterase (AChE) activities, as well as choline acetylcholine (Ach) content. Results revealed that rats in the WEGN group exhibited significantly lower escape latency, as well as a significantly higher number of targeted crossings and longer residence times in the target quadrant, relative to those in the model group. Notably, rats in the WEGN group spent more time exploring new objects and exhibited lower damage to their hippocampus neuron, had improved learning and memory activity, as well as reversed histological alterations, relative to those in the model group. Meanwhile, biochemical examinations revealed that rats in the WEGN group had significantly lower MDA levels and AChE activities, but significantly higher GSH, SOD, and ChAT activities, as well as Ach content, relative to those in the model group. Overall, these findings indicate that WEGN exerts protective effects on cognitive impairment, neuronal damage, oxidative stress, and choline function in AD rats treated by d-gal/AlCl3.

First Report of Fruit rot Caused by Phytophthora nicotianae on Passion Fruit in Guangxi Province, China.

Passion fruit (Passiflora edulis) is an economically important fruit crop in many tropical and subtropical regions worldwide. In recent years, passion fruit was widely cultivated in Guangxi Province. In 2020, a rot disease occurred on immature fruit of passion fruit in several commercial orchards of Nanning, Guangxi, caused about 50% incidence. The first appeared as small, irregular, water-soaked, brown lesions on immature fruit. As the disease progressed, the lesions rapidly enlarged, causing fruit rot. A layer of sparse white mycelia appeared on the lesions at high humidity. The disease first developed in June, its peak periods from August to September. Five diseased fruits were collected from five different orchards. The edges of symptomatic fleshy mesocarp tissue were cut into pieces (5 mm × 5 mm), surface-sterilized in 75% ethanol solution for 60 s, rinsed three times with sterilized distilled water, and plated on potato dextrose agar (PDA). Plates were incubated at 25°C in the dark. After 5 days, similar white colonies with abundant aerial mycelia developed from all plated tissue samples. Five isolates were obtained, and they were identified as Phytophthora nicotianae based on morphological characteristics and DNA analysis. Spherical hyphal swellings were commonly produced. Numerous sporangia were formed in sterile soil extract. Sporangia were ovoid or obpyriform, papillate, and measured 25 to 58 µm (average 41 µm) × 21 to 45 µm (average 29 µm). Chlamydospores were spherical and 19 to 43 µm in diameter (average 30 µm) (Erwin and Ribeiro 1996). The genomic DNA of a representative isolate Seg2-5 was extracted from mycelia through modified CTAB method (Murray and Thompson 1980). The rDNA internal transcribed spacer (ITS) region, ypt1, and coxII were amplified and sequenced with primers ITS1/ITS4 (White et al., 1990), Yph1F/Yph2R (Schena et al. 2008), and FM75F/FM78R (Villa et al. 2006), respectively. BLAST searches of the ITS, ypt1, and coxII sequences (Accession No. MW470847, MW770870, and MW770871) showed 99 to 100% identity with sequences of P. nicotianae (Accession No. JF792540, MK058408, and MH551183). Based on morphological characteristics and phylogenetic analysis, isolate Seg2-5 was identified as P. nicotianae. To confirm pathogenicity, asymptomatic and immature fruits 'Mantianxing' of passion fruit were previously disinfested in 0.5% sodium hypochlorite. Mycelial plugs of isolate Seg2-5 were placed onto the surface of fruits by nonwounded and pin-prick inoculation. Blank plugs were used as negative controls. Each treatment had five replicates and the test was repeated twice. Fruits were maintained in plastic boxes at 28°C and the initial disease spots appeared at 3 dpi or 5 dpi with wounded or non-wounded inoculation. After 7 to 10 days, all inoculated fruits showed similar symptoms as observed initially in the field, whereas control fruits remained healthy. P. nicotianae was successfully reisolated and identified from the inoculated fruits based on morphological characters and ITS sequence, thus confirming Koch's postulates. P. nicotianae had been previously isolated from passion fruit in South Africa (Van and Huller 1970), Vietnam (Nguyen et al. 2015), and Fujian Province of China (Luo et al. 1993). To our knowledge, this is the first report of P. nicotianae infecting passion fruit in Guangxi Province, China.

First report of Cedecea neteri Causing Yellow rot Disease in Pleurotus pulmonarius in China.

Pleurotus pulmonarius is a popular edible fungus and widely cultivated in many areas of China. In June 2018, yellow rot (more than 10% incidence) was found on the first crop of P. pulmonarius fruiting bodies in a mushroom factory in Nanning, Guangxi Province, China. At first, yellow water-soaked lesions appeared in the infected fruiting bodies. Lesions then spread and purulent tissues were formed. Severe rot induced production of deformed fruiting bodies and offensive odor. Internal sections of the diseased tissue (approximately 0.5 × 0.5 cm) were sterilized in 75% alcohol for 30 s, rinsed three times with sterilized and deionized water, crushed and suspended in sterilized and deionized water. The suspension was spread on the Luria-Bertani (LB) medium. After incubation at 30°C for 2 days, dominant bacterial colonies were oyster white, smooth, convex, and circular. Individual colonies were transferred two times to LB medium using the conventional streak plate techniques to obtain the pure cultures. The cells were gram-negative, short rods, motile, and no capsules or endospores were observed. Using a BoJian Gram-negative bacteria biochemical analysis kit (5 CARDS, Hopebio, Qingdao, China), data were obtained and analyzed, showing that the isolated strain belongs to the Cedecea genus (positive for ß-galactosidase, citric acid, arginine, sucrose, mannitol, sorbitol, D-glucose, gelatin hydrolysis and VP test but negative for H2S, urease, oxidase, indole, rhamnose, melibiose, amygdalin, lysine, ornithine, lactose, inositol and arabinose). Amplified 16S rDNA gene sequences (1,424 bp, GenBank accession No. MT925570) of the isolate using the universal primers 27f and 1492r (Lane 1991) exhibited 99.86% identity with Cedecea neteri M006 (CP009458.1). Based on its morphological characteristics, 16S rDNA sequences, and biochemical test results, the strain was identified as C. neteri. Pathogenicity tests for this strain were performed with bacterial suspensions (approximately 1 × 108 CFU/ml) after growing for 24 h in LB medium at 30°C. Mycelia of P. pulmonarius were cultivated for 60 days in plastic bags. Then young fruiting bodies were formed after induced with low temperature stimulation to serve as a host source. The prepared bacterial suspensions were directly sprayed onto the surface of three bags of fruiting bodies; another three bags were sprayed with sterilized and deionized water as negative control. All inoculated fruiting bodies were then incubated at 20°C with 90 to 95% relative humidity. All experiments were repeated three times. After 2 days, all the fruiting bodies inoculated with the bacterial suspensions showed yellow water-soaked lesions, and the normal growth of the fruiting bodies was inhibited. An offensive odor then developed along with a severe soft rot that was similar to the disease symptoms observed under natural conditions. The fruiting bodies of negative control were growing healthily with no symptoms. Koch's postulates were fulfilled by isolating bacteria from lesions on artificially inoculated fruiting bodies that were identical to the original isolates based on morphological characteristics, 16S rDNA sequences and biochemical test results. C. neteri was formally reported as a pathogen to humans that could cause bacteremia (Farmer et al. 1982). Recently, it has also been reported causing soft rot disease on mushrooms of Pholiota nameko (Yan et al. 2018) and yellow sticky disease on mushrooms of Flammulina velutipes (Yan et al. 2019). However, to the best of our knowledge, this is the first report of C. neteri-induced yellow rot disease of P. pulmonarius in China.

Long-term outcome of microvascular decompression for hemifacial spasm.

AIM: To investigate the long term outcomes of microvascular decompression (MVD) for hemifacial spasm (HFS) and to identify any prognostic factors. METHODS: A retrospective analysis of 189 consecutive patients with typical HFS who underwent MVD. Multiple logistic regression analysis of variables at various time points including at least immediate time point and one at no less than six years was performed. RESULTS: Short-term follow-up showed a cure rate of 91%, including 51 cases of delayed resolution (27%). At two years or more information was available in 148 (out of 189) cases of patients. 101 cases (68% - of 148 cases) had complete recovery, 28 cases (19%) achieved a partial though worthwhile recovery, so that the effective rate of symptoms relief at six years was 87%. Complications were found (66/189, 34.92%) and cured within the follow-up period (cure rate of 100%). In both the univariate and multivariate analyses, the postoperative findings of clinical outcomes showed that preoperative illness duration, compressive pattern, the intraoperative indentation of the root exit zone (REZ) of the facial nerve and intraoperative AMR disappearance were negative predictors and age considered to be positive, which significantly predicted the clinical outcome of patients following MVD. CONCLUSIONS: MVD may be a safe and effective strategy for HFS patients in view of relatively higher cure rates and lower complication risks within follow-up. Besides, patients' age, duration of disease, intraoperative indentation of the REZ of the facial nerve, and disappearance of AMR were the major influential variables may be useful for the prediction of prognosis in the patients underwent MVD.