First Report of Monosporascus cannonballus causing Root Rot and Vine Decline in Watermelon (Citrullus lanatus) in China.

In June 2023, a sudden outbreak root rot and vine decline symptoms was observed during a watermelon (Citrullus lanatus T.) variety demonstration trial located in Taizhou City, Zhejiang Province, China, with an incidence rate ranging from 75% to 100% and an affected area of nearly 2,000 square meters. The disease initially appeared with a rapid and alarming invasion of root rot and vine decline symptoms within watermelon plants. Affected plants exhibited rapid deterioration, showing symptoms of wilting, yellowing and eventual demise, predominantly during the pre-harvest stage. Notably, numerous black, spherical, erumpent perithecia were clearly visible on the watermelon's root epidermis, a characteristic trait of the disease. Symptomatic plant samples were rigorously disinfected with 75% ethanol, and plated on potato dextrose agar medium for incubation at 25°C, successfully isolate two potential strains. These isolates were inoculated in oatmeal agar and incubated in a 25℃ light incubator. After 30 days, mature perithecia, the same as those found on the watermelon's root epidermis, reached a diameter of 500 µm. Each perithecium contained several pear-shaped asci, 56 to 108.5 µm in length and 30.5 to 46.4 µm in width, typically holding 1, rarely 2 ascospores. These characteristics align precisely with the typical strains of Monosporascus cannonballus Pollack and Uecker (1974). Additionally, sequencing the internal transcribed spacer region of ribosomal DNA (ITS) gene (White et al., 1990), large subunit ribosomal RNA (LSU) gene (Rehner and Samuels 1995), and beta-tubulin (TUB) gene (Glass and Donaldson, 1995) were performed. BLAST analysis indicated the highest nucleotide sequence identity with M. cannonballus CBS 586.93 reference sequence (ITS: 100%, JQ771930; TUB: 98.99%, JQ907292). Representative sequences of isolate ZJUP0990-2 from these regions were deposited in GenBank (Accession No.: OR357656 for ITS; OR474500 for LSU; OR365762 for TUB). A multigene phylogenomic analysis (ITS-LSU-TUB) was undertaken to ascertain the exact phylogenetic position of M. cannonballus within the genus Monosporascus. The amalgamation of both morphological and molecular insights consistently reaffirmed the accurate classification of the causative agent as M. cannonballus. To validate the pathogenicity of M. cannonballus, a controlled greenhouse experiment was conducted using watermelon (cv. Nabite) as the subject. Mycelium fragments, harvested from the edge of the colony ZJUP0990-2, were inoculated into oat liquid medium and cultivated under dark conditions at a consistent temperature of 30°C for 7 days. After 20 days, the inoculated plants exhibited root rot and wilting, mirroring the symptoms observed during the field outbreak. In contrast, the control plants did not exhibit any signs of disease. M. cannonballus was successfully re-isolated from the symptomatic roots of the inoculated plants, satisfying Koch's postulates. This experiment was repeated three times. This pathogenic fungus has previously been documented as a menace to melons in various regions including Mexico (Chew-Madinaveitia et al., 2012) and Brazil (Sales et al., 2004), as well as watermelons in Brazil (Sales et al., 2010), northern Mexico (Gaytan-Mascorro et al., 2012), and Saudi Arabia (Karlatti et al., 1997). To our knowledge, this is the first reported presence of M. cannonballus on watermelons in China. This new disease poses a serious threat to watermelon production, potentially leading to severe economic losses and impacting food security.

First report of Trunk Canker Caused by Botryosphaeria dothidea on Cinnamomum camphora in China.

Cinnamomum camphora, an essential woody plant in China, experienced a severe outbreak of trunk canker affecting an area of 540,000 square meters in the Xiaoshan District of Zhejiang province during the early summer of 2022. The observed symptoms included stem canker, dieback, twig blight, and extensive vascular discoloration, with an incidence rate ranging from 45% to 70%. To investigate the etiology of the disease, symptomatic plant samples were subjected to rigorous surface sterilization involving washing with running tap water, followed by surface sterilization using 75% ethanol. The samples were then rinsed twice with sterile distilled water, plated on potato dextrose agar (PDA) medium, and incubated at a temperature of 28°C. The isolated fungi exhibited characteristics consistent with the species Botryosphaeria dothidea. Fungal isolates displayed abundant white aerial mycelium, which darkened to grey after five to seven days, eventually giving rise to black pycnidia. Single hyphal tip cultures of putative two isolates were stored at the Agricultural Experiment Station of Zhejiang University. Conidia formed on pycnidia were one-celled, hyaline, aseptate, and fusiform, with dimensions of 18.34-27.12 µm x 3.77-6.84 µm (average 22.90 µm x 5.20 µm) (one hundred conidia were measured). To determine the fungal species, genomic DNA was extracted from individual isolates ZJUP0868, and subjected to DNA sequence analysis of four gene regions: the internal transcribed spacer (ITS) using the primer-pair ITS4 and ITS1 (White et al., 1990), the large subunit (LSU) gene with LR0R and LR5 (Rehner and Samuels, 1995), the small subunit (SSU) gene with SR1R and SR7 (Zoller et al., 1999), and the translation elongation factor 1-alpha (tef1) with EF1-983F and EF1-2218R (Rehner and Buckley, 2005). Furthermore, DNA sequence analysis of above four genes was performed. BLAST analysis indicated the highest nucleotide sequence identity with Botryosphaeria dothidea CBS 115476 reference sequence (ITS: 99.59%, KF766151; LSU: 99.88%, DQ377852; SSU: 100%, NG_062738; tef1: 98.93%, AY236898). Representative sequences of isolate ZJUP0868 from these regions were deposited in GenBank (ITS: Accession No. OR192838; LSU: Accession No:OR248147; SSU: Accession No:OR248174; tef1: Accession No. OR262053). A phylogenomic analysis was conducted to determine the phylogenetic position of Botryosphaeria dothidea in the family Botryosphaeriaceae. The combined morphological and molecular findings confirmed the identification of the pathogen as Botryosphaeria dothidea. Pathogenicity tests were conducted by stem inoculation of two-year-old Cinnamomum camphora baranches. Mycelial plugs (2-3 mm in diameter) from actively growing colonies of B. dothidea (PDA) were applied to bark of similar size on the middle point of the stems. Inoculated barks were wrapped with Parafilm, while control branches received sterile PDA plugs. Inoculated and control branches (7 each) were maintained in a greenhouse at 28°C. After two weeks, all inoculated plants exhibited dark vascular stem tissue, whereas the control plants remained healthy. B. dothidea was re-isolated from symptomatic tissues, thus fulfilling Koch's postulates. No symptoms were observed in the control branched, and B. dothidea was not re-isolated from their tissues. B. dothidea , the type species of Botryosphaeria (Botryosphaeriaceae, Botryosphaeriales), is commonly associated with cankers and dieback in woody plants. Previous reports have identified Botryosphaeria dothidea as a pathogen causing stem dieback and trunk canker on Glycine max (Chen et al., 2020), Camellia oleifera (Hao et al., 2022), and Gleditsia sinensis (Huang et al., 2020). Additionally, B. dothidea has been reported to cause leave wilt on various plant species in China, including Daimyo oak (Liu et al., 2023) and Cornus officinalis (Zhang et al., 2022). To the best of our knowledge, this is the first report of B. dothidea on C. camphora in China. This findings contribute to a better understanding of the pathogens affecting Cinnamomum camphora in the China.

Effects of different technical substitutions on reducing replant disease of Radix pseudostella-riae and the underlying mechanism.

Radix pseudostellariae is a traditional Chinese medicinical herb, with tuberous roots being used as a medicine. Serious continuous monoculture problems were suffered from process of artificial and intensive cultivation. To explore the effective technical methods to overcome the monoculture problems, the effects of different technical substitution patterns on soil environment remediation, photosynthetic physiology and yield performance of R. pseudostellariae were assessed under continuous cropping system with four technical substitution treatments in the phase between two crops after the newly harvested R. pseudostellariae (first crop): fallow (RP-F-RP), fallow treated with microbial fertilizer (RP-F-BF), water flooding (RP-WF), and water flooding treated with specific microbial fertilizer (RP-WF-BF). Results showed that RP-WF-BF pattern was the single one that could effectively restore R. pseudostellariae yield under two-year monoculture and three-year monoculture to more than 90% and 70% of the newly planted respectively. All the other patterns did not significantly improve R. pseudostellariae yield under two-year monoculture. The contents of polysaccharide and total saponin in R. pseudostellariae under RP-WF-BF treatment were significantly increased by 15.3% and 16.5% compared with those of the newly planted, respectively. The abundance of beneficial microorganisms in the rhizosphere soil of R. pseudostellariae significantly increased. A reverse pattern occurred for pathogens under RP-WF-BF pattern. Moreover, soil nitrogen cycling was improved. The expression of AOB, nosZ and nirK was increased by 931%, 124% and 100% compared with those in the RP-F-RP pattern, respectively. Soil acidification under RP-WF-BF pattern was alleviated. The alleviation of soil biological and abiotic stress enhanced the stability of the antioxidant enzyme system, thereby improving the growth and development of R. pseudostellariae at the seedling and the early expand stages. The chlorophyll content, leaf area index and photosynthesis rate of leaves were increased, with the dry matter translocation improved and accumulation of underground dry matter accelerated, which ultimately increased yield and quality under RP-WF-BF pattern. In this study, the separate water flooding treatment (RP-WF) and microbial fertilizer treatment (RP-F-BF) failed to significantly reduce the continuous cropping obstacles of R. pseudostellariae, while the combination of them could produce a multiplication effect of sustainable strengthening on rhizosphere environment. The findings suggested that effective technical substitution could reduce replant disease of R. pseudostellariae.

[Back arm flexion and extension combined with manipulation for the treatment of patient with adhesive scapulohumeral periarthritis].

OBJECTIVE: To observe clinical efficacy of back arm flexion and extension combined with manipulation in treating adhesive scapulohumeral periarthritis. METHODS: From March 2016 to April 2018, 53 patients with adhesive scapulohumeral periarthritis were treated with back arm flexion and extension combined with manipulation, the main symptoms were shoulder pain and limited activity. There were 22 males and 31 females aged from 45 to 71 years old with an average of (51.63±5.79) years;the courses of disease ranged from 3 to 24 months with an average of (8.62±3.73) months. Manipulation treatment was carried out once a week, and back arm flexion and extension were performed for 3 times a day with15 movements each time, totally 4 weeks for a single course. Visual analogue scale (VAS) and Constant-Murley shoulder function score were observed and compared before treatment and one week, one and three months after treatment. RESULTS: All patients were followed up from 3 to 12 months, with an average of (5.91±3.55) months. VAS score before treatment was 4.02±1.46, and was improved to 3.15±1.54, 1.98±1.37, 1.12±0.86 respectively at one week, one and three months after treatment. Constant-Murley score before treatment was 42.70 ±5.72, and improved to 54.25 ±6.34, 67.45 ±6.84, 82.40 ±6.63 at one week, one and three months after treatment respectively;19 patients got excellent result, 22 good, 9 fair and 3 poor. CONCLUSION: Back arm flexion combined with manipulation for the treatment of adhesive scapulohumeral periarthritis could effectively relieve pain, improve shoulder function, which is a simple effective treatment strategy.

The therapeutic value of the SphK1-targeting microRNA-3677 in human osteosarcoma cells.

Sphingosine kinase 1 (SphK1) is a potential therapeutic target for human osteosarcoma (OS). SphK1-targeting microRNAs (miRNAs) could have important therapeutic value for OS. We discovered that micorRNA-3677 (miR-3677) is a SphK1-targeting miRNA, inhibiting OS cell progression. The results of RNA-Pull down assay confirmed direct binding between biotinylated-miR-3677 and SphK1 mRNA in primary human OS cells. In established and primary human OS cells forced overexpression of miR-3677, by a lentiviral construct, decreased SphK1 3'-UTR (untranslated region) activity and downregulated SphK1 expression. Both were however enhanced with miR-3677 inhibition in OS cells. Function studies demonstrated that OS cell growth, proliferation and migration were inhibited with miR-3677 overexpression, but augmented with miR-3677 inhibition. MiR-3677 overexpression-induced anti-OS cell activity was reversed with re-expression of the 3'-UTR-depleted SphK1. Additionally, in SphK1 knockout OS cells (by CRISPR/Cas9 strategy), altering miR-3677 expression failed to further alter cell functions. Finally, we show that miR-3677 expression was significantly downregulated in primary human OS tissues, correlating with SphK1 mRNA upregulation. We conclude that targeting SphK1 by miR-3677 inhibits human OS cell progression.

A study on the epidemiological characteristics and infectious forecast model of malaria at Guangzhou Airport among Chinese returnees from Africa.

BACKGROUND: Cross-border malaria transmission in China is a major component of Chinese imported malaria cases. Such cases mostly are travellers returning from malaria endemic countries in Africa. By investigating malaria infectious status among Chinese worker in Africa, this study analysed the malaria risk factors, in order to establish infectious forecast model. METHODS: Chinese returnees data from Africa were collected at Guangzhou Baiyun International Airport, Guangzhou, China between August 2015 and March 2016 and were included in the cross-sectional and retrospective survey. RESULTS: A total of 1492 respondents were included in the study with the majority consisting of junior middle school educated male. Most of them are manual and technical workers hired by companies, with average of 37.04 years of age. Overall malaria incidence rate of the population was 8.98% (134/1492), and there were no significant differences regarding age, gender, occupation, or team. Forecast model was developed on the basis of malaria risk factors including working country, local ecological environment type, work duration and intensity of mosquito bite prevention. CONCLUSIONS: The survey suggested that malaria incidence was high among Chinese travellers who had worked in Africa countries of heavy malaria burden. Further research on the frequency and severity of clinical episodes among Chinese travellers having worked in Africa is needed.

Antioxidant and anticancer aporphine alkaloids from the leaves of Nelumbo nucifera Gaertn. cv. Rosa-plena.

Fifteen compounds were extracted and purified from the leaves of Nelumbo nucifera Gaertn. cv. Rosa-plena. These compounds include liriodenine (1), lysicamine (2), (-)-anonaine (3), (-)-asimilobine (4), (-)-caaverine (5), (-)-N-methylasimilobine (6), (-)-nuciferine (7), (-)-nornuciferine (8), (-)-roemerine (9), 7-hydroxydehydronuciferine (10) cepharadione B (11), ß-sitostenone (12), stigmasta-4,22-dien-3-one (13) and two chlorophylls: pheophytin-a (14) and aristophyll-C (15). The anti-oxidation activity of the compounds was examined by antiradical scavenging, metal chelating and ferric reducing power assays. The results have shown that these compounds have antioxidative activity. The study has also examined the antiproliferation activity of the isolated compounds against human melanoma, prostate and gastric cancer cells. The results shown that 7-hydroxydehydronuciferine (10) significantly inhibited the proliferation of melanoma, prostate and gastric cancer cells. Together, these findings suggest that leaves of Nelumbo nucifera Gaertn. cv. Rosa-plena are a good resource for obtaining the biologically active substances with antioxidant properties.

The effect of butanolides from Cinnamomum tenuifolium on platelet aggregation.

This study investigated the effects of isotenuifolide and tenuifolide B from the stems of Cinnamomum tenuifolium on adenosine diphosphate (ADP)-induced human platelet aggregation. Treatment of human platelet-rich plasma with isotenuifolide (1 and 2 µg/µL) and tenuifolide B (1, 2 and 4 µg/µL) did not have any significant effect on human platelet aggregation in vitro, however, treatment of human platelet-rich plasma with isotenuifolide (4 µg/µL) resulted in an inhibitory effect on platelet aggregation, suggesting the potential of this compound as an anti-atherosclerogenic agent in humans. Isotenuifolide is a new butanolide compound, whose structure was characterized by spectral analyses.

The pharmacological activities of (-)-anonaine.

Several species of Magnoliaceae and Annonaceae are used in Traditional Chinese Medicine. (-)-Anonaine, isolated from several species of Magnoliaceae and Annonaceae, presents antiplasmodial, antibacterial, antifungal, antioxidation, anticancer, antidepression, and vasorelaxant activity. This article provides an overview of the pharmacological functions of (-)-anonaine.

Alkaloids from Cinnamomum philippinense.

A new pyridine, 2-(4'-hydroxypyridin-3'-yl)-acetic acid (1), along with five known alkaloids, cinnaretamine (2), crykonisine (3), corydaldine (4), glaziovine (5) and zenkerine (6), were isolated from the roots of Cinnamomum philippinense (Lauraceae). Their structures were characterized and identified by spectral analysis.

Inhibitory action of methadone and its metabolites on erg-mediated K+ current in GH3 pituitary tumor cells.

Methadone (Mtd) is a widely used opioid drug associated with the side effect of hyperprolactinemia. The mechanism of how Mtd induces prolactin secretion remains unclear. The effects of Mtd and its two main metabolites (EDDP: (±)-2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium percholarate and EMDP: 2-ethyl-5-methyl-3,3-dipnehyl-1-pyrroline) on ion currents were investigated in GH3 pituitary tumor cells. Hyperpolarization-elicited K+ currents in GH3 cells bathed in a high-K(+), Ca(2+)-free solution were studied to evaluate the effects of Mtd and other related compounds on the ether-à-go-go-related-gene (erg) K(+) current (I(K(erg))). Mtd suppressed the amplitude of I(K(erg)) in a concentration-dependent manner with an IC(50) value of 10.4 µM. With the aid of a minimal binding scheme, the inhibitory action of Mtd on I(K(erg)) was estimated with a dissociation constant of 8.2 µM. Mtd tended to increase the rate of I(K(erg)) deactivation in a voltage-dependent fashion. EDDP (10 µM) had no effect on I(K(erg)), while EMDP (10µM) slightly suppressed it. In GH3 cells incubated with naloxone (30 µM), the Mtd-induced inhibition of I(K(erg)) remained unaltered. Under cell-attached voltage-clamp recordings, Mtd increased the frequency of spontaneous action currents with no change in current amplitude. Similarly, Mtd can suppress I(K(erg)) in differentiated NG108-15 cells; dynorphin A(1-13) did not reverse Mtd-induced inhibition of I(K(erg)). This study shows that Mtd has a depressant effect on I(K(erg)), and suggests its ability to affect membrane excitability and prolactin secretion. The cyclization of Mtd, in which EDDP and EMDP are formed, tends to be critical in removal of the Mtd binding to erg K+ channel.

Thymol-evoked Ca+ mobilization and ion currents in pituitary GH3 cells.

In this study, an attempt was made to elucidate the effects of thymol, a monocyclic phenolic compound, on Ca2+ mobilization and ion currents in pituitary GH3 cells with the aid of fura-2 fluorimetry and the whole-cell voltage-clamp technique. Thymol increased intracellular Ca2+ concentrations ([Ca2+]i) in GH3 cells loaded with Ca2+-sensitive dye fura-2. Removing extracellular Ca2+ reduced the thymol-induced [Ca2+]i rise. In Ca2+ -free solution, thymol-evoked [Ca2+]i rise was unchanged by depleting the Ca2+ store with thapsigargin (1 microM), while the thapsigargin-induced [Ca2+]i rise was reduced by pretreatment with thymol. These results imply that the Ca2+ stores depleted by thymol comprise thapsigargin-sensitive and thapsigargin-insensitive pools. In addition, after depletion of the internal Ca2+ store with 100 microM thymol in Ca2+ -free solution, a subsequent application of Ca2+ greatly induced a [Ca2+]i increase. The results indicate that, similar to thapsigargin, 100 microM thymol may activate the capacitative calcium entry (CCE) channel. However, thymol (100 microM) had a slight depressant action in L-type calcium current (I(CaL)). The stimulatory actions of thymol on Ca2+ signaling may partly be responsible for the underlying cellular mechanisms through which it affects neuroendocrine functions.