First Report of Agroathelia rolfsii Causing Southern Blight on Lippia (Phyla canescens) in Guangzhou, China.

Lippia (Phyla canescens) is a fast-growing, mat-forming, and prostrate perennial plant well adapted to infertile, high-saline, and drought environments (Leigh, et al. 2004). It arrived in China from Japan as a flowering ground cover in 2001 (Cai, et al. 2004). In June 2022, southern blight appeared in our nursery of the Floriculture Research Institute of Guangdong Academy of Agricultural Sciences. High temperature and damp environment are major factors for this disease. The symptoms of top-layer plants were not easily detected, but they were slightly yellowed. A yellowish-brown water-soak lesion appeared on the stems and lowest leaves exposed to soil. White mycelium appeared in the middle stage. Finally, the surface plants showed water-soak decay, and a mass of beige to black-brown rapeseed-shaped sclerotia appeared on the residue and surrounding soil; these plants died. Sclerotia and mycelia were collected from disease tissue, and after surface sterilization, sclerotia was cultured on potato dextrose agar (PDA) at 28±2°C in an incubator without light. Eight fungal isolates with similar colony morphologies were consistently isolated by purifying from different sampling areas. The isolates exhibited obvious septa and a clamp connection structure within the white mycelium. The average growth rate was 26.86±0.06 mm/day. Numerous white granular sclerotia were produced on the mycelium 6 days later. The sclerotia with a diameter of 1.24±0.07mm (n=189) gradually changed from diage to yellow to brown. A typical strain B1 was selected for further identification, targeting its 18S rRNA and LSU rRNA sequences (Yang, et al. 2011; Xue, et al. 2019). Its 18S rRNA sequence (GenBank Accession No. OR517233, 1626 bp) is 99.63% and 99.57% identical to Athelia rolfsii (AY665774, 1179bp; KC670714, 1775bp; JF819726, 1781bp). Its LSU rRNA sequence (OR539570, 757 bp) is 99.87% identical to Agroathelia rolfsii (OR526537, 904 bp). For Athelia rolfsii, a synonym of Agroathelia rolfsii, by combining the morphological characteristics and molecular identification, the isolate pathogen B1 was confirmed to be Agroathelia rolfsii (the teleomorph of Sclerotium rolfsii). To fullfill Koch's postulates, we inoculated the mycelial plugs to healthy lippia stems and leaves which has grown for one year, with PDA plugs free of mycelium as the control. All the plants were kept in a greenhouse at 28±2°C with a 14-h photoperiod and 80% relative humidity. Each treatment was repeated thrice and vaccinated with 6 points. At 7 d following inoculation, all plants inoculated with B1 showed typical symptoms, but the control group was asymptomatic, and sclerotia appeared 17d after inoculation. Using the same protocol mentioned above, pathogenic fungal was reisolated only from treated groups, but not from the control group. Chose three of the pathogens for 18S rRNA and LSU rRNA sequencing, the results showed 100% identity to B1, the same as its microstructure. There are few reports about the disease on P. canescens. Sosa (2007) investigated the pathogens on P. canescens in Argentina, 16 fungi were found but no A. rolfsii. Sclerotium rolfsii were identified on P. nodiflora or P. lanceolata (Michaux) Greene in America (Farr, et al. 1989). To our knowledge, this is the first report in China. Because this pathogen has wide-ranging hosts and causes serious damage, the results from this study will offer guidance for the prevention and treatment of this disease.

Using rice straw-augmented ecological floating beds to enhance nitrogen removal in carbon-limited wastewater.

Agricultural biomass used as solid carbon substrates in ecological floating beds (EFBs) has been proven to be applicable in nitrogen removal for carbon-limited wastewater treatment. However, the subtle interactions among plants, rhizosphere microorganisms, and supplementary carbon sources have not been thoroughly studied. This study combined rice straw mats with different aquatic macrophytes in EFBs to investigate denitrification efficiency in carbon-limited eutrophic waters. Results showed that rice straw significantly enhanced the nitrogen removal efficiency of EFBs, while enriching nitrogen-fixing and denitrifying bacteria (such as Rhizobium, Rubrivivax, and Rhodobacter, etc.). Additionally, during the denitrification process in EFBs, rice straw can release humic acid-like fraction as electron donors to support the metabolic activities of microorganisms, while aquatic macrophytes provide a more diverse range of dissolved organic matters, facilitating a sustainable denitrification process. These findings help to understand the synergistic effect of denitrification processes within wetland ecosystems using agricultural biomass.

Interspecific differences in growth response and tolerance to the antibiotic sulfadiazine in ten clonal wetland plants in South China.

Pollution caused by residual antibiotics is a worldwide environmental issue. Antibiotic residues often occur in aquatic ecosystems, posing threats to the health of aquatic organisms. The effects of antibiotic residues on the growth of crop plants and on human health are reasonably well known. However, less is known about antibiotic effects on wetland plants. Therefore, we studied the response and tolerance of ten clonal wetland plants grown in soil spiked with sulfadiazine at 10 mg kg(-1) (an environmentally relevant concentration) and 100 mg kg(-1). At 10 mg kg(-1), ramet number was the least affected trait, while root number was the most affected among plant species. Plant shoot and total biomass were reduced in all species except in Cyperus malaccensis var. brevifolius and Panicum repens. Chlorophyll content was reduced in Alocasia macrorrhiza, Saururus chinensis, and Commelina diffusa. In general, Panicum paludosum and C. malaccensis var. brevifolius showed the least reduction of growth parameters, whereas growth of both A. macrorrhiza and S. chinensis was severely reduced. At 100 mg kg(-1), negative responses occurred in all species. Comprehensive tolerance analysis revealed that P. paludosum and C. malaccensis var. brevifolius were the species most resistant to sulfadiazine. These species are potential candidates for sulfadiazine polluted wetland restoration. A. macrorrhiza and S. chinensis were the most susceptible species and they should be protected from sulfadiazine pollution. Relative plant shoot biomass and height were the most useful indicators for evaluating plant tolerance to sulfadiazine. Plant tolerance to sulfadiazine was associated with the differences of plants in height and shoot biomass.

Proteasome inhibitor MG132 enhances TRAIL-induced apoptosis and inhibits invasion of human osteosarcoma OS732 cells.

MG132 as a proteasome inhibitor could induce apoptosis in various cancer cells. This study aimed to discuss the effect of proteasome inhibitor MG132 on the TRAIL-induced apoptosis of human osteosarcoma OS732 cells. MG132 and TRAIL were applied on OS732 cells respectively or jointly. Cell survival rates, changes of cellular shape, cell apoptosis and cell invasion were analyzed, respectively, by 3-(4,5)-dimethylthiahiazo(-z-y1)-2,5-di-phenytetrazoliumromide (MTT) assay, inverted phase contrast microscope, flow cytometry, and transwell invasion chamber methods. The protein levels of DR5, caspase-3, caspase-8, p27(kip1) and MMP-9 were measured by Western blot analysis. The results indicated that combination of MG132 and TRAIL had the effect of up-regulating expression of DR5, caspase-3, caspase-8 and p27(kip1), down-regulating expression of MMP-9 and inducing apoptosis as well as suppressing the ability of invasion of OS732 cells. The survival rate of combined application of 10 µM MG132 and 100 ng/ml TRAIL on OS732 cells was significantly lower than that of the individual application (p<0.01). Changes of cellular shape and apoptotic rates also indicated the apoptosis-inducing effect of combined application was much stronger than that of individual application. Cell cycle analysis showed combination of MG132 and TRAIL mostly caused OS732 cells arrested at G2-M-phase. The invasion ability of OS732 cells was restrained significantly in the combined group compared with the individual group and control group.

Synergistic apoptotic effect of crocin and cisplatin on osteosarcoma cells via caspase induced apoptosis.

Crocin is well-known traditional Chinese medicine which is extracted from saffron. However, its role in osteosarcoma has not been well understood. Therefore, we used crocin and cisplatin individually or jointly on MG63 and OS732 cells so as to explore whether crocin could induce cellular apoptosis and suppress the ability of invasion of osteosarcoma cells. Cell survival rates, changes of cellular shape, cell apoptosis and cell invasion were analyzed, respectively, by 3-(4,5)-dimethylthiahiazo (-z-y1)-2,5-di- phenytetrazoliumromide (MTT) assay, inverted phase contrast microscope and fluorescence microscope, flow cytometry, and Transwell invasion chamber methods. The expressions of caspase-3 and caspase-8 were detected by Western blot. The survival rate of combined application was significantly lower than that of the individual application. Apoptosis-inducing effect of combined application was much stronger than that of individual application. The invasion ability of MG63 and OS732 cells was restrained significantly in the combined group compared with the individual group and control group. Combined group has the effect of up-regulating the expressions of cleaved-caspase-3 and caspase-8. The results suggested that combination of crocin and cisplatin has a strong killing effect on osteosarcoma cells and suppresses the ability of invasion of MG63 and OS732 cells which might be related to up-regulate the expression of caspase-3 and caspase-8.

Surgical treatment of an osteopetrotic patient with postoperative fractures: lessons from siblings with osteopetrosis.

Osteopetrosis is a rare, inherited disease characterized by dense and brittle bones, and it is associated with an increased risk of femoral fractures. However, the surgical treatment of the fracture patients with osteopetrosis is a subject of controversy, because it is difficult to compare the treatment effects between surgical treatment and conservative treatment in part due to the rarity of siblings with osteopetrosis. In fact, the genetic background and the environmental factors, such as living conditions and daily work, may influence the prognosis of the fracture patients with osteopetrosis. Here we describe siblings with osteopetrosis, a 23-year-old female patient and her older brother, both of whom had suffered from bilateral femoral fractures at their childhood. They do not have other brothers or sisters, and their parents are healthy. The younger sister had undergone surgical treatment for femoral fractures at the age of 8 years and recovered very well. In contrast, her brother had received the conservative treatment for femoral fractures at the age of 6 years, and thereafter he is badly disabled due to the malunion of the fractured femurs. Apparently, the surgical treatment is superior to the conservative treatment for the fracture patient with osteopetrosis. Unfortunately, fifteen years later, the sister fractured again at both femurs and underwent another operation. She recovered quickly from the second operation, indicating that second operation should be considered in osteopetrosis patients with postoperative fractures. The present report provides valuable information for the choice of treatment of femoral fractures associated with osteopetrosis.

Enhancement of osteosarcoma cell sensitivity to cisplatin using paclitaxel in the presence of hyperthermia.

PURPOSE: This paper aimed to evaluate the effects of a combination of paclitaxel and cisplatin on osteosarcoma (OS) cell lines in the presence of hyperthermia and to investigate the related mechanism. MATERIALS AND METHODS: Two types of OS cell lines (OS732 and MG63) were treated with paclitaxel and cisplatin in the presence of hyperthermia. The survival rate was measured by MTT assay, and the clonogenic rate was measured by a clonogenic assay. The cellular changes were observed with an inverted phase contrast microscope and a fluorescence microscope. The apoptotic effect was analysed with flow cytometry (FCM). Fas expression by the OS cell lines was measured by western blot. Fas expression in OS tissue was measured by immunohistochemistry. RESULTS: Our study indicated that 1 h after the application of a combination of 10 µg/mL paclitaxel and 5 µg/mL cisplatin to OS cells at 43 °C, the survival rate of the OS cells was 11.96%, which was significantly lower than when either 10 µg/mL paclitaxel (45.02%) or 5 µg/mL cisplatin (48.69%) was applied alone (p < 0.01). Additionally, the clonogenic assay demonstrated that the clonogenic survival rate in the OS cells of the combination group was lower than that in the individual groups. Moreover, the cellular changes and apoptosis rates indicated that apoptosis in the combined application group was much greater than when either drug was applied individually. Fas expression by OS cell lines was increased by the combination of paclitaxel and cisplatin under hyperthermic conditions. More importantly, our study revealed low Fas expression in OS, which better explained the up-regulation of Fas achieved by the combination of paclitaxel and cisplatin in the presence of hyperthermia. CONCLUSIONS: The combination of paclitaxel and cisplatin increases the effects of thermochemotherapy on OS cell lines, primarily through the induction of apoptosis by the up-regulation of Fas expression.

Efficient killing effect of osteosarcoma cells by cinobufacini and cisplatin in combination.

PURPOSE: To study the killing effects on osteosarcoma cells of cinobufacini and cisplatin in combination and the related mechanisms so as to explore the chemotherapeutic method with integrated traditional Chinese and Western medicines. METHODS: Cinobufacini and cisplatin were applied to OS732 cells singly or jointly and survival rates were measured by MTT assay. Changes in cellular shape were observed with inverted phase contrast and fluorescence microscopy and apoptosis rates were analyzed with flow cytometry (FCM). Immunocytochemistry were used to examine the Fas expression of OS732 cells. RESULTS: The combination of cinobufacini and cisplatin had the effect of up-regulating Fas expression and inducing apoptosis. The survival rate of combined application of 100 µg/ml cinobufacini and 1 µg/ml cisplatin on OS-732 cells was significantly lower than with either of the agents alone (p<0.01). Changes in cellular shape and apoptotic rates also indicated the apoptosis-inducing effects of combined application were much enhanced. CONCLUSION: The combination of cinobufacini and cisplatin demonstrated strong killing effects on OS-732 cells which might be related to up-regulation of Fas expression.

Oxaliplatin sensitizes OS cells to TRAIL-induced apoptosis via down-regulation of Mcl1.

PURPOSE: To investigate the killing effect on OS cells of a combination of oxaliplatin and TRAIL and related molecular mechanisms. METHODS: TRAIL and oxaliplatin were applied to OS732 cells singly or jointly and survival inhibition rates were measured by MTT assay, changes of cellular shape being assessed with inverted phase contrast and fluorescence microscopy. Apoptotic rates were analyzed by flow cytometry (FCM) and immunocytochemistry was used to examine Mcl1 expression of OS732 cells. RESULTS: The survival inhibition rate of combined application of 100 µg/ml TRAIL and 1 µg/ml oxaliplatin on OS-732 cells was significantly higher than that of either agent singly (p<0.01). Changes of cellular shape and apoptotic rates also indicated apoptosis-inducing effects of combined application to be much stronger than those of individual application. Oxaliplatin had the effect of down-regulating Mcl1 expression and sensitizing OS cells to TRAIL-induced apoptosis. CONCLUSION: A combination of TRAIL and oxaliplatin exerts strong killing effects on OS-732 cells which might be related to down-regulation of Mcl1 expression.

Induction of apoptosis by a combination of paclitaxel and carboplatin in the presence of hyperthermia.

PURPOSE: To study enhancing effects of paclitaxel in the thermochemotherapy of osteosarcoma cell lines and related mechanisms. MATERIALS AND METHODS: Paclitaxel and carboplatin were used alone or jointly on OS732 cell lines in the presence of hyperthermia. Inhibition of proliferation was measured by MTT assay and cellular changes were assessed with inverted phase contrast and fluorescence microscopy. Apoptosis was analyzed with flow cytometry (FCM) and Fas expression by immunocytochemistry. RESULTS: At 43 degrees C, one hour after the application of 10 µg/ml paclitaxel and 5 µg/ml carboplatin on OS732 cells jointly, the survival rate was 15.8% which was significantly lower than with 10 µg/ml paclitaxel (45.8%) and 5 µg/ml carboplatin (47.7%) respectively (P<0.01). Moreover, changes of morphology and apoptotic rates indicated that the apoptosis-inducing effect of combined application was also much enhanced, as evident also regarding Fas expression. CONCLUSION: Paclitaxel is conducive to thermochemotherapy of osteosarcoma cell lines, possibly accomplished by up-regulation of Fas expression with induction of apoptosis.

[Simple osteotomy for correct hallux valgus with increased I, II intermetatarsal angle].

OBJECTIVE: To investigate efficacy of simple osteotomy for correct hallux valgus,and explore its scope and condition. METHODS: From December 2009 to April 2011, 20 patients (32 feet) with hallux valgus were treated by simple osteotomy. There were 1 male (1 foot), 19 females (31 feet) with an average age of 40 years (ranged, 22 to 64 years). The course of disease ranged from 2 to 31 years(mean 12 years). Among them, 6 feet were mild, 20 feet were moderate, 6 feet were serious. Patients had symptoms of metatarsophalangeal joint pain, but tensity of lateral soft tissue were normal. Hallux Valgus Angle (HVA) and Intermetatarsal Angle (IMA) were examined before and after treatment. The criteria of hallux valgus was used to evaluate the effects from valgus deformity, activity of metatarsophalangeal joint, satisfaction of patients. RESULTS: Twenty patients were followed up from 6 to 18 months with an average of 8.5 months. The wounds were healed well, no infection and metatarsal head necrosis occurred, 95.1% of patients were satisfied with the efficacy. Average AOFAS score increased from preoperative (53.1 +/- 7.5) points to the final follow-up (93.1 +/- l.9) points (P<0.05), the average correct HVA increased from preoperative (33.4 +/- 7.8) degrees to postoperative (11.9 +/- 3.6) degrees (P<0.05), the average IMA were decreased from preoperative (12.3 +/- 3.0) degrees to postoperative (6.3 +/- 1.9) degrees (P<0.05), tibial sesamoid position improved from 1.9 to 0.9 (P<0.05). CONCLUSION: the main pathological changes of hallux valgus patients with normal tension of lateral soft tissue is metatarsal adduction, simple osteotomy can get satisfactory results, no need to cut adductor muscle.

[Observation on therapeutic effect of muscular needling combined with scarring moxibustion on active stage of rheumatoid arthritis].

OBJECTIVE: To observe the therapeutic effect of muscular needling combined with scarring moxibustion on active stage of rheumatoid arthritis (RA). METHODS: Sixty cases of RA were randomly divided into a muscular needling group and a medication group, 30 cases in each group. The muscular needling group was treated by muscular needling on Quchi (LI 11), Sanyinjiao (SP 6), etc. combined with scarring moxibustion on Dazhui (GV 14), Zusanli (ST 36) etc., while the medication group was treated by oral administration of Diclofenac sodium and intramuscular injection of Methotrexate. The therapeutic effects, main symptoms and signs, erythrocyte sedimentation rate (ESR) and rheumatoid factor were observed in two groups before and after treatment. RESULTS: The total effective rate of muscular needling group was 76.7%, and that of medication group was 73.3%, there was no significant difference between two groups (P > 0.05). The clinical symptoms, signs, and E8R of two groups were improved obviously compared with those before treatment (P < 0.01, P < 0.05), however there were no significant differences between the two groups after treatment (all P > 0.05). The adverse reactions of medication group were more eminent compared to the muscular needling group. CONCLUSION: Muscular needling can obviously relieve the symptoms and signs of active stage rheumatoid arthritis and the effect is equivalent to oral administration of western medicine, the incidence of adverse reactions in the muscular needling group is obviously lower than that of western medication. Muscular needling is a safe and effective method for treatment of RA.