Average EC50 Value Research Articles

Metabolic activities of five botryticides against Botrytis cinerea examined using the Biolog FF MicroPlate.

Tobacco grey mold caused by Botrytis cinerea is an important fungal disease worldwide. Boscalid, carbendazim, iprodione, pyrimethanil and propiconazole are representative botryticides for grey mold management. This research investigated the sensitivities of B. cinerea from tobacco to these chemicals using the Biolog FF Microplate. All five chemicals showed inhibitory activity, with average EC50 values of 0.94, 0.05, 0.50, 0.61 and 0.31 μg ml−1, respectively. B. cinerea metabolized 96.8% of tested carbon sources, including 29 effectively and 33 moderately, but the metabolic fingerprints differed under pressures imposed by these botryticides. For boscalid, B. cinerea was unable to metabolize many substrates related to tricarboxylic acid cycle. For carbendazim, carbon sources related to glycolysis were not metabolized. For iprodione, use of most carbon substrates was weakly inhibited, and the metabolic profile was similar to that of the control. For propiconazole, no carbon substrates were metabolized and the physiological and biochemical functions of the pathogen were totally inhibited. These findings provide useful information on metabolic activities of these botryticides, and may lead to future applications of the Biolog FF Microplate for examining metabolic effects of other fungicides on other fungi, as well as providing a metabolic fingerprint of B. cinerea that could be useful for identification.

The Fungicidal Activity of Thymol against Fusarium graminearum via Inducing Lipid Peroxidation and Disrupting Ergosterol Biosynthesis.

Thymol is a natural plant-derived compound that has been widely used in pharmaceutical and food preservation applications. However, the antifungal mechanism for thymol against phytopathogens remains unclear. In this study, we identified the antifungal action of thymol against Fusarium graminearum, an economically important phytopathogen showing severe resistance to traditional chemical fungicides. The sensitivity of thymol on different F. graminearum isolates was screened. The hyphal growth, as well as conidial production and germination, were quantified under thymol treatment. Histochemical, microscopic, and biochemical approaches were applied to investigate thymol-induced cell membrane damage. The average EC50 value of thymol for 59 F. graminearum isolates was 26.3 μg·mL−1. Thymol strongly inhibited conidial production and hyphal growth. Thymol-induced cell membrane damage was indicated by propidium iodide (PI) staining, morphological observation, relative conductivity, and glycerol measurement. Thymol induced a significant increase in malondialdehyde (MDA) concentration and a remarkable decrease in ergosterol content. Taken together, thymol showed potential antifungal activity against F. graminearum due to the cell membrane damage originating from lipid peroxidation and the disturbance of ergosterol biosynthesis. These results not only shed new light on the antifungal mechanism of thymol, but also imply a promising alternative for the control of Fusarium head blight (FHB) disease caused by F. graminearum.

PRELIMINARY ECOTOXICOLOGICAL EVALUATION OF ERYTHROSIN B AND ITS PHOTOCATALYTIC DEGRADATION PRODUCTS

The class of xanthene dyes has a complex chemical structure, which showed to be toxic for mixed culture of microorganisms (i.e. anaerobic granular sludge). Because of the unwanted effects of Erythrosin B (Ery B) on environmental components and some food confirmed previously, the dye was chosen in this study to evaluate its ecotoxicity. Also, the Ery B photocatalytic degradation products were assessed in terms of their ecotoxicity. Three-days of seed germination and root growth tests were conducted using a dicotyledonous plant that is the garden cress (Lepidium sativum L.), in the presence of different dye concentration and its photodegradation products. Dye affected mostly the roots of the plant. According to toxic effects on root growth, toxicity of the dye indicated a 72h exposition average Effective Concentration EC50 value corresponding to 25 mgL -1 Ery B. The presence of the Ery B photocatalytic degradation products in the aqueous solution leads to a higher efficiency on Lepidium sativum L. germination, favoring the stem length growth.

Activity of a novel bactericide, zinc thiazole against Xanthomonas oryzae pv. oryzae in Anhui Province of China

AbstractBacterial leaf blight of rice (BLB), caused by Xanthomonas oryzae pv oryzae, is one of the most serious bacterial diseases in China. Presently, bismerthiazol has been the major bactericide for the control of BLB, however, bismerthiazol‐resistant strains of X. oryzae pv. oryzae have appeared in the field in China. Zinc thiazole is a novel bactericide with strong antibacterial activity against Xanthomonas spp. In this study, sensitivity of 109 X. oryzae pv. oryzae strains to zinc thiazole was determined. The EC50 values for zinc thiazole in inhibiting bacterial growth of the 109 X. oryzae pv. oryzae strains were 0.53–9.62 µg mL−1 with the average EC50 value of 4.82 ± 1.86 µg/ml. The minimum inhibitory concentration (MIC) values of zinc thiazole against the 109 X. oryzae pv. oryzae strains were assessed and the results showed that the MIC values of zinc thiazole for completely inhibiting the growth of these 109 strains ranged from 5.0 to 40.0 µg mL−1. In the evaluation of protective and curative activity test, zinc thiazole exhibited great activity against BLB and provided over 88% control efficacy (at 300 µg mL−1) 1 and 3 days before or after inoculations, which was also higher that that of bismerthiazol in the corresponding treatments. Our field trials showed that zinc thiazole at 375 g.a.i ha−1 provided over 70% control efficacy in 2012 and over 80% control efficacy in 2013 at both sites. Moreover, in all the four field trials, zinc thiazole at 250 g.a.i ha−1 provided higher control efficacy than that of bismerthiazol at 250 g.a.i ha−1. Taken together, zinc thiazole is therefore an alternative tool for the management of BLB.

Activity of a novel strobilurin fungicide benzothiostrobin against Sclerotinia sclerotiorum

Benzothiostrobin is a novel strobilurin fungicide. In this study, baseline sensitivity of Sclerotinia sclerotiorum (Lib.) de Bary to benzothiostrobin was determined using 100 strains collected during 2012 and 2013 from different geographical regions in Jiangsu Province of China, and the average EC50 value was 0.0218 (±0.0111)μg/mL for mycelial growth. After benzothiostrobin treatment, hyphae were contorted with offshoot of top increasing and cell membrane permeability increased markedly, while sclerotial production and oxalic acid content significantly decreased. Benzothiostrobin strongly inhibited mycelial respiration within 12h and the oxygen consumption of the mycelia could not be inhibited after 24h. On detached rapeseed leaves, the protective and curative activity test of benzothiostrobin suggested that benzothiostrobin had good control efficiency against S. sclerotiorum, and protective activity was better than curative activity. These results will contribute to us evaluating the potential of the new strobilurin fungicide benzothiostrobin for management of diseases caused by S. sclerotiorum and understanding the mode of action of benzothiostrobin against S. sclerotiorum.

Sensitivity of Rhizoctonia solani causing rice sheath blight to fluxapyroxad in China

Fluxapyroxad,3-(difluoromethyl)-1-methyl-N-(3’,4’,5’-trifluorobiphenyl-2-yl) pyrazole-4-carboxamide (C18H12F5N3O, Fig. 1), is a SDHI fungicide, which is a new active ingredient that interferes with succinate ubiquinone reductase in the electron transport chain of fungi. Between 2008 and 2010, a total of 128 isolates of Rhizoctonia solani from Anhui Province of China were characterized for the baseline sensitivity to fluxapyroxad. The isolates collected between 2008 and 2010 all showed similar sensitivity to fluxapyroxad. Baseline sensitivity was distributed as unimodal curves with an average EC50 value of 0.054 ± 0.014 μg ml−1. However, EC50 values of boscalid for inhibition of mycelial growth of R. solani isolates ranged from 1.89 to 2.68 μg/ml and the average (±SE) EC50 value was 2.212 ± 0.228 μg/ml, indicating that the R. solani isolates were less sensitive to boscalid than that of fluxapyroxad. Fluxapyroxad exhibited excellent protective and curative activity against rice sheath blight and provided 82.6–94.2 % protective or curative control efficacy. In field trials, control efficacy of fluxapyroxad at 100 g a.i/ha 15 days and 30 day after second application was 83.4–88.0 %, suggesting excellent activity against sheath blight. Control efficacy of boscalid at a dosage of 600 g a.i/ha 15 days and 30 day after second application was about 51.7–57.0 %. There was a significant difference in the efficacy between fluxapyroxad and boscalid or jinggangmycin. These results suggested that fluxapyroxad is a good alternative fungicide to jinggangmycin for the control of rice sheath blight.

First Report of Dimethachlon Resistance in Field Isolates of Sclerotinia sclerotiorum on Oilseed Rape in Shaanxi Province of Northwestern China.

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic fungal pathogen causing diseases in a wide range of plants, including oilseed rape (3). Substantial economic losses caused by S. sclerotiorum have been reported in the United States, Canada, Brazil, South Africa, Hungary, India, Nepal, and Japan (1). Application of fungicides is the principal tool for controlling S. sclerotiorum because of lack of high level of host resistance. Dicarboximide fungicides such as dimethachlon have been widely used to control S. sclerotiorum in recent years in China and field isolates with reduced sensitivity to dimethachlon have been reported in Jiangsu Province of eastern China (2). In order to understand the current status of dimethachlon resistance in S. sclerotiorum isolates of northwestern China, 196 and 344 isolates of S. sclerotiorum collected from oilseed rape fields in 10 counties throughout Shaanxi Province in 2011 and 2012, respectively, were assayed for sensitivity to dimethachlon using 5 μg ml-1 dimethachlon as a discriminatory dose. Mycelial plugs (6 mm in diameter) cut from the margin of a 48-h-old colony were placed in the center of petri dishes containing potato dextrose agar (PDA) amended with 5 μg ml-1 dimethachlon; PDA without fungicide served as the control. Cultures were incubated at 26°C and colony growth was measured after 72 h of incubation. Isolates that showed growth on PDA amended with fungicide were tentatively considered resistant to dimethachlon, whereas the completely inhibited isolates were considered sensitive. Results showed that 1.02% or 2 isolates of the 196 isolates collected in 2011 and 3.78% or 13 isolates of the 344 isolates collected in 2012 were resistant to dimethachlon. For all the isolates considered resistant and 42 randomly selected sensitive isolates, 50% effective concentrations (EC50) were determined on PDA amended with a series of dimethachlon concentrations. The average EC50 value of dimethachlon for sensitive isolates was 0.29 ± 0.02 μg ml-1 Resistance ratios (EC50 of resistant isolate / average EC50 of sensitive isolates) for the two resistant isolates detected in 2011 were 10.28 and 23.83, respectively, whereas resistance ratios for the 13 resistant isolates detected in 2012 ranged from 24.90 to 101.97. The average EC50 value of dimethachlon for the 13 resistant isolates detected in 2012 was 19.05 μg ml-1, and EC50 values for the two resistant isolates detected in 2011 were 2.98 and 6.91 μg ml-1, respectively. These results indicated that both resistance frequency and resistance level increased from 2011 to 2012. Bioassay results of three resistant isolates indicated that there was positive cross-resistance between dimethachlon and other dicarboximide fungicides such as iprodione and procymidone. To our knowledge, this is the first report of dimethachlon resistance in S. sclerotiorum in Shaanxi Province of northwestern China. The molecular mechanism of dimethachlon resistance in field isolates of S. sclerotiorum remains to be studied. Although resistance frequency is low at present, dimethachlon resistance should be kept in mind and fungicide resistance management tactics such as use of biological control agents, fungicide tank-mixing, or alternating dimethachlon with other fungicides having different modes of action is recommended in controlling S. sclerotiorum.

Polarity of extracts and fractions of four Combretum (Combretaceae) species used to treat infections and gastrointestinal disorders in southern African traditional medicine has a major effect on different relevant in vitro activities

Ethnopharmacological importanceGastrointestinal disorders and infections are the major pathoaetiologies of diarrhoea causing many problems in human health and animal production. Many Combretum species are used in traditional medicine to treat infectious diseases including diarrhoea and many other ailments by rural people in Africa and Asia. Much of the work done to date on this genus was on the non-polar or intermediate polarity components. Some parameters that may cause diarrhoea and the evaluation of more polar extracts have apparently not been investigated. AimsThe polar components were extracted and fractionated by solvent–solvent fractionation to yield fractions with different polarities. The activity of these fractions on different parameters that could be involved in factors associated with diarrhoea was investigated. The cytotoxic activities of the extracts were also determined to evaluate the potential of these extracts to combat diarrhoea in production animals. Materials and methodsPhenolic-enriched leaf extracts of Combretum bracteosum (COB), Combretum padoides (COP), Combretum vendae (COV) and Combretum woodii (COW) were obtained by extracting with a mixture of 70% acetone acidified with 1% HCl and n-hexane. Acetone was removed from a portion of the 70% acetone extract and it was sequentially treated by solvent–solvent fractionation with dichloromethane, ethyl acetate, and butanol to yield fractions with a large variation in polarity. The phenolic constituents of the extracts and fractions were determined using standard procedures The antioxidant activities were determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH); 2,2′-azino-bis (3-ethylbenzothiazoline)-6-sulphonic acid (ABTS+) radical scavenging, ferric reducing antioxidant power (FRAP) methods and lipid peroxidation inhibitory capacity standard methods. The ferric reducing antioxidant activities of the fractions were also determined. The minimum inhibitory concentrations (MICs) of the crude extracts and fractions against four bacterial and three fungal strains were assessed with a microplate serial dilution method. Cyclooxygenase (COX) and lipoxygenase (LOX) enzyme inhibitory assays and cytotoxicity studies against Vero cells were also carried out. ResultSome of the fractions had much higher antioxidant activity than the positive controls. The average EC50 values of the extracts for the DPPH and ABTS antioxidant assays were 0.21–12µg/ml (COP), 0.25–16µg/ml (COV), 0.33–9.41µg/ml (COW) and 4.97–85µg/ml (COB) respectively while the mean EC50 values for the positive controls ascorbic acid and trolox were 1.28–1.51 and 1.02–1.19µg/ml respectively. All the crude extracts inhibited lipid peroxidation of linoleic acid by more than 80% at a concentration of 64 µg/ml. COP had the highest antibacterial activity with MICs ranging between 19–2500µg/ml, followed by COV with MICs ranging between 39–625µg/ml; COW and COB had similar MICs ranging between 39–2500µg/ml. COP also had the highest antifungal activity with MICs between 19–625µg/ml. The MIC for COW and COV ranged from 19 to 1250 µg/ml. COB had the lowest antifungal activity (MIC values were between 39 and 625 µg/ml). In general non-polar fractions had a high antimicrobial activity and polar fractions had a high antioxidant activity. The extracts had no activity against COX 1 and 2 enzymes in the anti-inflammatory assay but had good lipoxygenase inhibition. The crude extracts had high concentration of hydrolysable tannin (gallotannin). A good correlation (R2= 0.99) was found between the antioxidant activity and total tannin content indicating that, gallotannins may be responsible for the antioxidant activity. ConclusionThe results obtained in this study with more polar extracts indicate that the use of extracts of these plant species as antidiarrhoeal agents may have a scientific basis. The extractant used here extracted a much higher percentage of the phytochemicals than acetone. It was better for isolating antioxidant compounds (polar) but not good for isolating antimicrobial compounds (non-polar) from the same species compared to acetone, ethyl acetate, dichloromethane, and hexane.

Baseline sensitivity and control efficacy of flutolanil in Rhizoctonia solani

Rice sheath blight is one of the most important rice diseases in the world and the succinate dehydrogenase inhibitor fungicide flutolanil has potential for its control. In this study, a total of 112 Rhizoctonia solani isolates from Jiangsu province in China were characterized for their baseline sensitivity to flutolanil. The baseline sensitivity curve was unimodal with an average EC50 value of 0.0736 ± 0.0331 μg mL−1 for inhibition of mycelial growth. Flutolanil exhibited excellent acropetal movement in rice, and better protective and curative activity than validamycin against rice sheath blight. In field trials in 2011 and 2012, flutolanil used at a concentration of 150 g a.i. ha−1 provided control efficacy ranging from 63.7 to 88.9 %, and its control efficacy could reach 83.9 % ~ 96.5 % at a concentration of 300 g a.i. ha−1. These results suggested that flutolanil will be a good alternative fungicide to validamycin for control of rice sheath blight.

Abstract C262: Malignant mesothelioma lacking merlin shows enhanced sensitivity to the FAK inhibitor defactinib (VS-6063): Elucidation of the merlin-FAK relationship.

Abstract Malignant pleural mesothelioma (MPM) is an aggressive tumor in the lining of the lung often resulting from prior exposure to asbestos. Median overall survival with the standard of care chemotherapy is only 12 months from diagnosis. Thus, new therapeutic modalities are urgently needed to improve the prognosis of MPM patients. ∼50% of MPM patients exhibit biallelic inactivation of the NF2 tumor suppressor gene by mutation and/or deletion resulting in lack of expression of functional merlin protein. Merlin inactivation is a critical step in MPM tumorigenesis and is related, at least in part, to up-regulation of focal adhesion kinase (FAK) activity. FAK has also been demonstrated to play a role in maintenance of cancer stem cells (CSCs), a subpopulation of tumor cells associated with drug resistance and tumor recurrence. Therefore, therapeutic targeting of FAK using defactinib (VS-6063), a potent, orally active FAK kinase inhibitor, may benefit cancer patients through targeting of both CSCs and bulk tumor cells. Among a panel of MPM cell lines in 3-dimensional Matrigel culture, MPM lines lacking expression of merlin were found to be especially sensitive to defactinib. In contrast, MPM cell lines with wild-type merlin expression were less sensitive with average EC50 values of 5.1 μM. Oral dosing of defactinib induced significant tumor growth inhibition in a merlin-negative MPM model pre-implanted in the lungs of mice. Using Aldefluor activity as a marker for CSCs in MPM, we find that chemotherapeutic agents enriched for Aldefluor-positive CSCs, while the FAK inhibitor defactinib reduced the proportion of CSCs. When used in combination with pemetrexed or cisplatin, defactinib blocked the enrichment of CSCs cells by these chemotherapeutic agents. Disruption of cell-cell or cell-extracellular matrix (ECM) contacts with blocking antibodies allowed us to propose a molecular model to account for the observed merlin-FAK inhibitor synthetic lethal relationship. We find that weak cell-cell adhesions in merlin-negative MPM cells lead to a greater dependence of tumor cell survival and proliferation on cell-ECM-induced FAK signaling rendering merlin-negative cells especially vulnerable to FAK inhibitor treatment. Taken together, these data support the clinical development of defactinib in an ongoing Phase 2 registration-directed clinical trial evaluating the activity of defactinib in MPM patients (stratified by merlin status) with a confirmed response (PR or SD) following first line platinum/pemetrexed therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C262. Citation Format: Irina M. Shapiro, Vihren N. Kolev, Yuwaraj Kadariya, Christian M. Vidal, Quentin G. Wright, Jennifer E. Ring, Craig Menges, Joseph R. Testa, Qunli Xu, Jonathan A. Pachter. Malignant mesothelioma lacking merlin shows enhanced sensitivity to the FAK inhibitor defactinib (VS-6063): Elucidation of the merlin-FAK relationship. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C262.

The Antioxidant Property of Chitosan Green Tea Polyphenols Complex Induces Transglutaminase Activation in Wound Healing

The present study examined, for the first time, the in vitro wound healing potential of chitosan green tea polyphenols (CGP) complex based on the activation of transglutaminase (TGM) genes in epidermal morphogenesis. Response surface methodology was applied to determine the optimal processing condition that gave maximum extraction of green tea polyphenols. The antioxidant activity, scavenging ability, and chelating ability were studied and expressed as average EC50 values of CGP and other treatments. In silico analysis and gene coexpression network was subjected to the TGM sequences analysis. The temporal expressions of TGMs were profiled by semi-quantitative reverse transcription (RT)-PCR technology within 10 days after wounding and 2 days postwounding. CGP showed the effectiveness of antioxidant properties, and the observations of histopathological photography showed advanced tissue granulation and epithelialization formation by CGP treatment. In silico and coexpression analysis confirmed the regulation via TGM gene family in dermatological tissues. RT-PCR demonstrated increased levels of TGM1-3 expression induced by CGP treatment. The efficacy of CGP in wound healing based on these results may be ascribed to its antioxidant properties and activation of the expression of TGMs, and is, thus, essential for the facilitated repair of skin injury.

Frequency distribution of sensitivity of Ustilaginoidea virens to four EBI fungicides, prochloraz, difenoconazole, propiconazole and tebuconazole, and their efficacy in controlling rice false smut in Anhui Province of China

False smut, caused by Ustilaginoidea virens, is an important emerging disease of rice (Oryza sativa L.) in China. Up to now, as most varieties with high yielding and good quality are susceptible or even highly susceptible to false smut in most rice-growing ecological regions, especially in Anhui Province, chemical control with fungicides would be an important measure for the control of this disease. The ergosterol biosynthesis inhibitor (EBI) fungicides, such as prochloraz, difenoconazole, propiconazole and tebuconazole, are extensively used in China for the control of rice diseases, such as rice sheath blight and rice blast. In this study, a total of 102 U. virens isolates (from Anhui Province of China) were tested for their sensitivity to these four EBI fungicides during the stage of mycelial growth. The EC50 ranges of values for prochloraz, difenoconazole, propiconazole and tebuconazole inhibiting mycelial growth of the 102 U. virens isolates were 0.04–0.75, 0.04–1.08, 0.04–0.38 and 0.03–0.57 μg ml−1, with the average EC50 values of 0.32 ± 0.08, 0.45 ± 0.08, 0.19 ± 0.03 and 0.21 ± 0.06 μg ml−1, respectively. These values suggested that the tested U. virens isolates were very sensitive to these four EBI fungicides. Results of field trials showed that two sprays of three of the fungicides exhibited greater control efficacy than a single spray for the control of rice false smut. Two sprays of each was better than a single spray for the control of rice sheath blight. Two sprays of 50% propiconazole EC at 300 g a.i. ha−1 gave the best control of rice false smut at both two sites during the two consecutive years, 2010 and 2011, with the control efficacy ranging from 71.5 to 74.3%. Sensitivity of the field U. virens isolates to EBI fungicides should be monitored. Mixtures, as well as alternation with other fungicides with different modes of action, should be tested.

Effect of epoxiconazole on rice blast and rice grain yield in China

Sensitivity to epoxiconazole of 90 single- spore isolates of Magnaporthe oryzae was determined. The EC50 values for epoxiconazole in inhibiting mycelial growth of the 90 M. oryzae isolates were 0.11- 0.86 μg/ml, with an average EC50 value of 0.260± 0.082 μg/ml. There was no correlation between sensitiv- ity to epoxiconazole and sensitivity to carbendazim or iprobenfos. In protective and curative tests, epoxiconazole applied at 200 μg/ml provided over 70 % control efficacy, while tricyclazole exhibited better protective than curative activity. The results of four field trials performed in 2010 and 2011 at two sites showed that epoxiconazole at 112.5 g a.i/ha provided over 75 % control efficacy, which was similar to tricyclazole with 300 g a.i./ha and better than carbendazim with 562.5 ga .i./ha.

Baseline sensitivity and efficacy of thifluzamide in Rhizoctonia solani

Thifluzamide is a SDHI (succinate dehydrogenase inhibitor) fungicide, which interferes with succinate ubiquinone reductase in the mitochondrial electron transport chain of fungi. Presently, jinggangmycin is the major fungicide extensively used for the control of rice sheath blight caused by Rhizoctonia solani and resistance to jinggangmycin was first reported to occur in China. A total of 128 isolates of R. solani from Anhui Province of China were characterised for the baseline sensitivity to thifluzamide. The isolates were very sensitive to thifluzamide and the baseline sensitivity curve was unimodal with an average EC50 value of 0.058 ± 0.012 µg mL−1. However, EC50 values of boscalid (another SDHI fungicide) for inhibition of mycelial growth of 22 arbitrarily selected R. solani isolates ranged from 1.89 to 2.68 µg mL−1. Thifluzamide applied at 110 µg mL−1 exhibited excellent protective and curative activity against rice sheath blight and provided 81.1–91.0% protective or curative control efficacy. In field trials in 2010 and 2011, control efficacies of thifluzamide at 82 g.a.i ha−1 15 and 30 days after second application were 84.2% and 86.7%, respectively, suggesting excellent activity against sheath blight. There was a statistically significant difference in the efficacy between thifluzamide and boscalid or jinggangmycin. These results suggested that thifluzamide should be a good alternative fungicide to jinggangmycin for the control of rice sheath blight.

Sensitivity of Fusarium verticillioides isolates from rice to a novel cyanoacrylate fungicide

The baseline sensitivity of Fusarium verticillioides to the novel cyanoacrylate fungicide 2-cyano-3-amino-3-phenylancryic acetate (experimental code JS399-19) was established, and its impact on the germination of rice seeds and efficacy in controlling rice bakanae disease by seed treatment were evaluated. The EC50 values for JS399-19 inhibiting mycelial growth of wild-type F. verticillioides isolates ranged from 0.162 to 0.559 μg/ml (a 3.5-fold difference between the lowest and highest value), and the average (±SE) EC50 value was 0.316 ± 0.072 μg/ml. The sensitivity curve was unimodal over the sensitive range and the sensitivity data can be used as a baseline for monitoring any future changes in sensitivity to JS399-19 by F. verticillioides populations. Germination of healthy seeds was greater than 77% for all the selected cultivars, and was unaffected by treatment with JS399-19. Treatment of infected seeds (harvested from diseased plants) with all concentrations of JS399-19 in this study significantly decreased the abnormal elongation and damping-off of seedlings in plastic bottles in the greenhouse, indicating that JS399-19 has excellent efficacy in controlling rice bakanae disease, and was safe to rice seeds. It could be used as a disinfectant of rice seeds infected by F. verticillioides. The results also suggested that the F. verticillioides isolates exhibited little sensitivity shift to this fungicide after a single application. If properly applied, JS399-19 might be used as a major fungicide or a good companion fungicide for the control of rice bakanae disease in China.

In vitro inhibition of Sclerotinia sclerotiorum by mixtures of azoxystrobin, SHAM, and thiram

The necrotrophic fungal phytopathogen Sclerotinia sclerotiorum (Lib.) de Bary has a broad host range and frequently causes destructive diseases. The extensive use of common fungicides to control these diseases has selected for resistance in populations of S. sclerotiorum. In this study, 105 isolates of S. sclerotiorum from different geographical regions in Jiangsu Province of China were characterized for baseline sensitivity to azoxystrobin, and the average EC50 value was 0.2932μg/mL for mycelial growth. Of the mixtures of the fungicides thiram and azoxystrobin that were tested using an in vitro mycelial growth assay, the 1:4 ratio provided the greatest inhibition of S. sclerotiorum. When tested against nine isolates, the 1:4 mixture resulted in a mean synergy ratio of 2.31, indicating synergistic inhibition. Mycelial respiration was inhibited for about 2h by azoxystrobin alone but for 48h by the mixture of thiram and azoxystrobin. Salicylhydroxamic acid (SHAM, a known inhibitor of alternative respiration) also increased the inhibition of mycelial growth and respiration caused by azoxystrobin. These results suggest the need for further study of effects of combinations of azoxystrobin with thiram or SHAM in planta to evaluate their potential for management of diseases caused by S. sclerotiorum.

Abstract 4727: Antitumor activity of the MDM2 antagonist, RG7112

Abstract The p53 tumor suppressor is a transcription factor which plays a central role in prevention of tumor development by inducing cell cycle arrest or apoptosis in response to diverse stresses. In unstressed cells, p53 level is tightly controlled by MDM2 which binds p53 and negatively regulates its activity and stability. MDM2 is overproduced in many human cancers as a mechanism to impair p53 function. Antagonists of p53-MDM2 interaction could reactivate p53 and may offer a novel approach to cancer therapy. The first potent and selective small-molecule inhibitors of p53-MDM2 binding, the Nutlins, provided preclinical proof-of-concept for MDM2 antagonists as a new therapeutic option for patients with tumors expressing wild-type p53. RG7112 is a member of the Nutlin family of MDM2 antagonists with improved potency, selectivity and pharmacological properties which is currently in Ph1 clinical evaluation. Here, we describe its biological activity in models of human cancer in vitro and in vivo. RG7112 binds to MDM2 with high affinity in vitro (KD ∼11 nM) and effectively blocks its interaction with p53. Crystal structures of RG7112-MDM2 complexes revealed that this imidazoline molecule interact with the p53-binding pocket of MDM2 by projecting functional groups that mimic critical amino acid residues from the p53 molecule. Treatment of cancer cells expressing wild-type p53 with RG7112 stabilized p53 and activated the p53 pathway as revealed by the induction of multiple p53-regulated genes. This led to cell cycle arrest in G1 and G2 phases followed by apoptosis. It showed potent antitumor activity in vitro against a panel of solid tumor and leukemia cells expressing wild-type p53 with EC50 in the 0.2 - 2.2 µM range. The overall selectivity between the panels of seven mutant and fifteen wild-type p53 lines, expressed as fold difference in the average EC50 values was 14-fold. As previously shown with nutlin-3, RG7112 demonstrated variable apoptotic activity in a panel of solid tumor cells with the best apoptotic response in cells with mdm2 gene amplification. Interestingly, inhibition of caspase activity did not change the kinetics of p53-induced cell death. Oral administration of RG7112 to nude mice bearing established human tumor xenografts caused dose-dependent tumor inhibition and tumor regression at non-toxic concentration. The in vivo mode of action was confirmed by pharmacodynamic markers, indicating p53 pathway activation and induction of growth arrest and apoptosis in tumor tissues. Our results showed that RG7112 is a potent p53 activator and antitumor agent in vitro and in vivo that may have therapeutic utility in the treatment of solid tumors and leukemia with wild-type p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4727. doi:1538-7445.AM2012-4727

The Chimeric Approach Reveals That Differences in the TRPV1 Pore Domain Determine Species-specific Sensitivity to Block of Heat Activation

The capsaicin-, heat-, and proton-activated ion channel TRPV1, a member of the transient receptor potential cation channel family is a polymodal nociceptor. For almost a decade, TRPV1 has been explored by the pharmaceutical industry as a potential target for example for pain conditions. Antagonists which block TRPV1 activation by capsaicin, heat, and protons were developed by a number of pharmaceutical companies. The unexpected finding of hyperthermia as an on-target side effect in clinical studies using polymodal TRPV1 antagonists has prompted companies to search for ways to circumvent hyperthermia, for example by the development of modality-selective antagonists. The significant lack of consistency of the pharmacology of many TRPV1 antagonists across different species has been a further obstacle. JYL-1421 for example was shown to block capsaicin and heat responses in human and monkey TRPV1 while it was largely ineffective in blocking heat responses in rat TRPV1. These findings suggested structural dissimilarities between different TRPV1 species relevant for small compound antagonism for example of heat activation. Using a chimeric approach (human and rat TRPV1) in combination with a novel FLIPR-based heat activation assay and patch-clamp electrophysiology we have identified the pore region as being strongly linked to the observed species differences. We demonstrate that by exchanging the pore domains JYL-1421, which is modality-selective in rat can be made modality-selective in human TRPV1 and vice-versa.

Fungicide Sensitivity of Colletotrichum cereale Isolated from Turfgrasses in the Northeastern United States

Twenty-seven isolates of Colletotrichum cereale collected from the northeastern United States between 1993 and 1995 and twenty-three isolates collected in 2007 were screened for resistance to fosetyl-Al, chlorothalonil, polyoxin-D, iprodione, fludioxonil, triadimefon, azoxystrobin, and thiophanate-methyl. The comparison between isolates collected from 1993 to 1995 to isolates from 2007 demonstrated significant increases in EC50 values for azoxystrobin, polyoxin-D, thiophanate-methyl, and triadimefon. Significant increases in EC90 were observed for all fungicides except chlorothalonil and fosetyl-Al. Both EC50 and EC90 values for chlorothalonil decreased significantly between the two sampling periods. Most importantly, our results indicate that the occurrence of C. cereale isolates resistant to azoxystrobin and thiophanate-methyl has increased in the Northeast over the past 12 to 14 years based on the isolates tested and that overall resistance to some commonly used turf fungicides has increased as reflected in significantly higher average EC50 and EC90 values.

Occurrence and Characterization of Dimethachlon Insensitivity in Sclerotinia sclerotiorum in Jiangsu Province of China

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is the main fungal disease of oilseed rape (Brassica napus) in China. Numerous fungicide applications are required for control. Dimethachlon, one of the dicarboximide fungicides, has been the major fungicide for disease control after benzimidazole resistance became widespread. Fungal populations were collected throughout Jiangsu Province between 2006 and 2007 in order to determine their sensitivity to dimethachlon. A total of 1,066 single-sclerotium isolates of S. sclerotiorum were collected, and most of the isolates were considered sensitive to dimethachlon. Five isolates collected in Yancheng and Changzhou showed normal growth at 5 μg/ml dimethachlon with the resistance factor ≈10 (resistance factor was estimated as ratios between the EC50 values of resistant isolates and the average EC50 values of sensitive ones) compared to the sensitive isolates (EC50 is the concentration of fungicide causing 50% reduction in growth). Through in vitro selection for resistance to the fungicide, 25 dimethachlon-resistant mutants were derived from 10 wild-type isolates of S. sclerotiorum. The resistance factors for the isolates ranged from 198 to 484, and the isolates were considered highly resistant to dimethachlon. Therefore, at least two different mechanisms of resistance seem to be involved: one that may provide a moderate resistance (insensitivity) and a second that may give a high resistance level under laboratory conditions. There was positive cross-resistance between dimethachlon and other dicarboximide fungicides, such as iprodione and procymidone, in these S. sclerotiorum isolates. The field dimethachlon-insensitive and the laboratory-induced dimethachlon-resistant isolates appeared to have mycelial growth, sclerotial production, and pathogenicity comparable to their wild-type parental isolates. Also, results of osmotic tests showed that there were no significant difference in mycelial radial growth between the field dimethachlon-sensitive and field dimethachlon-insensitive isolates on potato dextrose agar plates amended with 2, 4, 6, or 8% (wt/vol) NaCl, but the laboratory-induced dimethachlon-resistant isolates grew significantly more slowly than their wild-type sensitive parents under all concentrations of NaCl. Because these studies yielded a high frequency of laboratory resistance in S. sclerotiorum, together with the occurrence of field insensitivity, appropriate precautions against resistance development in natural populations should be taken.