Average EC50 Value Research Articles

Baseline sensitivity of Fusarium graminearum from wheat fields in Henan, China, to metconazole and analysis of cross resistance with carbendazim and phenamacril

AbstractThe filamentous plant pathogenic fungus Fusarium graminearum is one of the most important pathogens causing Fusarium head blight (FHB) in wheat in the Henan Province of China. Metconazole is among the demethylation inhibitor (DMI) fungicides with a higher inhibitory activity on the mycelial growth of F. graminearum. In 2016 and 2017, 119 single spore isolates of F. graminearum, prior to being exposed to metconazole, were recovered from 52 wheat fields near 11 cities in Henan Province. The inhibitory activity of metconazole on the mycelia of the Henan F. graminearum population was determined, and EC50 values were calculated. The range of EC50 values of the Henan F. graminearum population to metconazole was 0.0103 to 0.0775 μg/ml with an average EC50 value of 0.0293 ± 0.0114 μg/ml. The sensitivity frequency distribution curve presented a single peak in a narrow range. No cross‐resistance was found between the DMI fungicide metconazole and the benzimidazole fungicide carbendazim or the cyanoacrylate fungicide phenamacril. Therefore, these sensitivity data could be used as the baseline of F. graminearum susceptibility to metconazole in the Henan Province and provide the basis for monitoring metconazole resistance in this area.

Получение стабилизированных наночастиц серебра и изучение их антимикробной и цитотоксической активности в отношении клеток гепатомы человека линии HepG2

Синтезированы наночастицы серебра с использованием для их восстановления и стабилизации арабиногалактана и диоктилсульфосукцината натрия. Средний гидродинамический размер наночастиц, определенный по данным фотонной корреляционной спектроскопии, составлял 30 нм, дзета-потенциал –34.04 ± 1.54 мВ. По данным метода электронной дифракции серебро в образце золя находится в металлической форме. Препарат наночастиц серебра проявлял антибактериальную активность в отношении условно-патогенных грамотрицательных (Escherichia coli) и грамположительных (Bacillus subtilis и B. coagulans) бактерий. Наночастицы серебра также обладали антифунгальной активностью в отношении штаммов фитопатогенных грибов рода Fusarium sporotrichioides и F. solani. Проведено исследование цитотоксической активности наночастиц серебра в отношении клеток гепатомы печени человека линии HepG2. Продемонстрировано ингибирующее действие наночастиц серебра в отношении метаболической активности и жизнеспособности опухолевых клеток. Средние относительные значения EC50 для наночастиц серебра составляли 1.5 ± 0.4 и 41.2 ± 3.9 мкг/мл. Препарат стабилизированных наночастиц серебра может найти применение в медицине в качестве потенциального антимикробного и противоопухолевого средства, а также в сельском хозяйстве в качестве средства подавления роста фитопатогенных грибов.

Baseline Sensitivity and Action Mechanism of Propamidine Against Alternaria brassicicola, the Causal Agent of Dark Leaf Spot on Cabbage.

In the current study, a total of 53 isolates of Alternaria brassicicola collected from Shaanxi Province of China were characterized for their sensitivity to propamidine. The EC50 (50% effective concentration) values for propamidine inhibiting mycelial growth and spore germination ranged from 0.515 to 3.247 µg/ml and 0.393 to 2.982 µg/ml, with average EC50 values of 1.327 ± 0.198 µg/ml and 1.106 ± 0.113 µg/ml, respectively. In greenhouse experiments, propamidine at 100 µg/ml provided >90% efficacy against dark leaf spot on cabbage, which was higher than the efficacy obtained by azoxystrobin at the same concentration. After treatment with propamidine, fungal growth distortions were observed in the form of excess mycelial branching, thickened cell walls, decreased cell membrane permeability, and increased chitin content. Interestingly, colony color faded after treatment with propamidine compared with that of the untreated parental isolates. Importantly, the expressions of melanin biosynthesis-associated genes Amr1, Scd1, Brn1, and Brn2 were downregulated at different levels. The obtained baseline sensitivity and control efficacy data suggested that propamidine inhibited not only growth of A. brassicicola but also melanin biosynthesis, which could reduce the biocompatibility of A. brassicicola in the field. These biological characteristics encourage further investigation of the mechanism of action of propamidine against A. brassicicola.

Effects of SHAM on the Sensitivity of Sclerotinia sclerotiorum and Botrytis cinerea to QoI Fungicides.

It is a common practice to add salicylhydroxamic acid (SHAM) into artificial medium in the in vitro sensitivity assay of fungal phytopathogens to the quinone outside inhibitor (QoI) fungicides. The rationale for adding SHAM is to inhibit fungal alternative oxidase, which is presumed to be inhibited by secondary metabolites of plants. Therefore, the ideal characteristics of SHAM should be almost nontoxic to phytopathogens and have no significant effect on control efficacy of fungicides. However, this study showed that the average effective concentration for 50% inhibition (EC50) of mycelial growth values of SHAM were 97.5 and 401.4 μg/ml for Sclerotinia sclerotiorum and Botrytis cinerea, respectively. EC50 values of the three QoI fungicides azoxystrobin, kresoxim-methyl, and trifloxystrobin in the presence of SHAM at 20 and 80 μg/ml for S. sclerotiorum and B. cinerea, respectively, declined by 52.7 to 78.1% compared with those without SHAM. For the dicarboximide fungicide dimethachlone, the average EC50 values in the presence of SHAM declined by 18.2% (P = 0.008) for S. sclerotiorum and 35.9% (P = 0.012) for B. cinerea. Pot experiments showed that SHAM increased control efficacy of the three QoI fungicides against the two pathogens by 43 to 83%. For dimethachlone, SHAM increased control efficacy by 134% for S. sclerotiorum and 86% for B. cinerea. Biochemical studies showed that SHAM significantly inhibited peroxidase activity (P = 0.024) of B. cinerea and esterase activity (P = 0.015) of S. sclerotiorum. The strong inhibitions of SHAM per se on mycelial growth of B. cinerea and S. sclerotiorum and significant influences on the sensitivity of the two pathogens to both the QoI fungicides and dimethachlone as well as inhibitions on peroxidase and esterase indicate that SHAM should not be added in the in vitro assay of sensitivity to the QoI fungicides.

Preparation of stabilized silver nanoparticles and study of their antimicrobial and cytotoxic activity on the human hepatoma HepG2 cell line

Silver nanoparticles were synthesized using arabinogalactan and sodium dioctyl sulfosuccinate to reduce and stabilize them. The average hydrodynamic size of the nanoparticles, determined by photon correlation spectroscopy, was 30 nm, and the zeta potential was –34.04 ± 1.54 mV. According to the electron diffraction method, silver in the sol sample is in metallic form. The preparation of silver nanoparticles showed antibacterial activity against opportunistic Gram-negative (Escherichia coli) and Gram-positive (Bacillus subtilis and B. coagulans) bacteria. Silver nanoparticles also had antifungal activity against strains of phytopathogenic fungi of the genus Fusarium sporotrichioides and F. solani. A study of the cytotoxic activity of silver nanoparticles was made on human hepatoma cells of the HepG2 line. The inhibitory effect of silver nanoparticles on the metabolic activity and viability of tumor cells has been demonstrated. The average relative EC50 values for silver nanoparticles were 1.5 ± 0.4 μg/ml and 41.2 ± 3.9 μg/mL. The preparation of stabilized silver nanoparticles can find application in medicine, as a potential antimicrobial and antitumor agent, as well as in agriculture as a means of suppressing growth of phytopathogenic fungi.

Antifungal Activity and Action Mechanism of the Natural Product Cinnamic Acid Against Sclerotinia sclerotiorum.

Sclerotinia stem rot caused by Sclerotinia sclerotiorum (Lib.) de Bary spreads worldwide and causes serious economic losses. Considering the development of fungicide resistance and chemical residues, it is urgently necessary to explore alternative fungicides. In this study, the activity of the natural product cinnamic acid was assessed. The EC50 values for cinnamic acid inhibition of mycelial growth of 103 S. sclerotiorum strains ranged from 9.37 to 42.54 µg/ml with an average EC50 value of 18.77 (±3.39) µg/ml. No cross-resistance was detected between cinnamic acid and the commonly used fungicides carbendazim or dimethachlon. After treatment with cinnamic acid, mycelia distorted with more branches, no sclerotia developed, and the oxalic acid content decreased, whereas cell membrane permeability increased significantly. In pot experiments, cinnamic acid at 2,000 µg/ml provided over 95% efficacy against both carbendazim-sensitive and carbendazim-resistant strains of S. sclerotiorum. The expression of the sclerotia development-correlated genes Sac1, Pac1, Smk1, and Pka1 decreased, whereas the Cna1 gene expression increased. Altogether, cinnamic acid shows potential to be a natural alternative to commercial fungicides or a lead compound to develop new fungicides for the control of Sclerotinia stem rot. The biological characteristics contribute to the understanding of the action mechanism of cinnamic acid against S. sclerotiorum.

Pseudoperonospora cubensis in China: Its sensitivity to and control by oxathiapiprolin

The novel fungicide oxathiapiprolin has potential for the control of downy mildew of cucumber, which is caused by Pseudoperonospora cubensis. In this study, an in vitro bioassay with detached leaves was used to determine the baseline sensitivity to oxathiapiprolin for 77 Ps. cubensis isolates from 11 provinces in China. The baseline sensitivity curve was continuous, and the average EC50 value was 2.23 × 10−4 μg ml−1. In field trials, the control of downy mildew of cucumber was greater with oxathiapiprolin at 20 or 30 g a.i. ha−1 than with dimethomorph at 262.5 g a.i. ha−1. Oxathiapiprolin was taken up by cucumber roots and transported upwards to stems and leaves. The full length of PscORP1, the gene that encodes the target protein of oxathiapiprolin in Ps. cubensis, was sequenced for the first time. Our results suggested that oxathiapiprolin will be an excellent alternative fungicide for control of cucumber downy mildew. However, as Ps. cubensis is a high-risk pathogen, resistance development to oxathiapiprolin should be monitored and managed.

Activity of a novel succinate dehydrogenase inhibitor fungicide pyraziflumid against Sclerotinia sclerotiorum

Pyraziflumid is a novel member of succinate dehydrogenase inhibitor fungicides (SDHI). In this study, baseline sensitivity of Sclerotinia sclerotiorum (Lib.) de Bary to pyraziflumid was determined using 105 strains collected during 2015 and 2017 from different geographical regions in Jiangsu Province of China, and the average EC50 value was 0.0561 (±0.0263)μg/ml for mycelial growth. There was no cross-resistance between pyraziflumid and the widely used fungicides carbendazim, dimethachlon and the phenylpyrrole fungicide fludioxonil. After pyraziflumid treated, hyphae were contorted with offshoot of top increasing, cell membrane permeability increased markedly, oxalic acid content significantly decreased and mycelial respiration was strongly inhibited. But the number and dry weight of sclerotia did not change significantly. The protective and curative activity test of pyraziflumid suggested that pyraziflumid had great control efficiency against S. sclerotiorum on detached rapeseed leaves, and protective activity was better than curative activity. These results will contribute to us on evaluating the potential of the new SDHI fungicide pyraziflumid for management of diseases caused by S. sclerotiorum and understanding the mode of action of pyraziflumid against S. sclerotiorum.

Evaluation of Phenamacril and Ipconazole for Control of Rice Bakanae Disease Caused by Fusarium fujikuroi.

This study evaluated the use of phenamacril and ipconazole, alone and in mixtures, for the control of rice bakanae disease caused by Fusarium fujikuroi. Mixtures were studied with the goal of reducing the selection of fungicide-resistant field isolates of the fungus. When tested alone, both phenamacril and ipconazole exhibited high antifungal activity against F. fujikuroi mycelial growth; the average EC50 value for 19 field isolates was 0.1544 μg/ml for phenamacril and 0.0472 μg/ml for ipconazole. A 2:1 mixture of phenamacril and ipconazole caused a slightly synergistic (greater than additive) inhibition of mycelial growth. Inhibition of F. fujikuroi sporulation was highest for ipconazole alone, intermediate with the 2:1 mixture, and lowest for phenamacril alone. Inhibition by phenamacril and ipconazole alone or by the 2:1 mixture was substantially lower for spore germination than for mycelial growth or sporulation. When the total fungicide concentration was <24 g of a.i./100 kg of treated rice seeds, the fungicides, whether alone or in the 2:1 mixture, were not phytotoxic to seeds or seedlings of two rice cultivars. In a greenhouse experiment, the 2:1 mixture of phenamacril and ipconazole at 6 g of a.i./100 kg of treated seeds provided 100% control of rice bakanae disease on two cultivars. Overall, the results indicate that the use of a 2:1 mixture of phenamacril and ipconazole should control rice bakanae disease while reducing the occurrence of fungicide resistance in F. fujikuroi.

Transcriptome Analysis Reveals the Mechanism of Fungicidal of Thymol Against Fusarium oxysporum f. sp. niveum.

Fusarium oxysporum: f. sp. niveum (Fusarium oxysporum) causes watermelon wilt, drastically reducing its yield. Thymol (5-methyl-2-(1-methylethyl) phenol, PubChem CID: 6989) has been extensively reported to have antimicrobial activity against pathogenic microorganisms. In this study, the growth of F. oxysporum was significantly inhibited by thymol in vitro. The dry weight of F. oxysporum was decreased significantly. Thymol-induced cell membrane damage was observed at 24 hpi (hours post-incubation). Therefore, the changes in the gene expression level of F. oxysporum after treatment with 80μg/mL (average EC50 value) of thymol were analyzed using RNA-Seq to reveal the underlying fungicidal mechanisms of thymol. Among the 5057 differentially expressed genes (DEGs), 2440 and 2617 were up-regulated and down-regulated, respectively. Bioinformatics analysis demonstrated that the expression levels of most genes, including glycosphingolipid biosynthesis and sphingolipid metabolism, were down-regulated. However, the genes involved in antioxidant activity, chitin biosynthesis, and cell wall modification were up-regulated. These results were verified by quantitative reverse transcription PCR (qRT-PCR). According to these results, thymol produces reactive oxygen species (ROS) accumulation and destroys the integrity of the cell wall and cell membrane by inhibiting the genes involved in cell wall and cell membrane synthesis.

Four azoles’ profile in the control of Septoria, yellow rust and brown rust in wheat across Europe

Leaf diseases cause major yield losses in winter wheat every year across Europe. Septoria leaf blotch – STB (Zymoseptoria tritici) is the most serious leaf disease in Northern Europe, but also yellow rust (Puccinia striiformis) and brown rust (Puccinia triticina) are known to cause major problems in some regions and seasons. Problems with fungicide resistance in the populations of Z. tritici have caused concerns for future control options. With the aim of investigating the differences in azole performances against STB, yellow rust and brown rust, 40 field trials were carried out during two seasons (2015 and 2016) in 10 different countries across Europe covering a diversity of climatic zones and agricultural practices. Four single triazoles (epoxiconazole, prothioconazole, tebuconazole and metconazole) and two mixtures of azoles (epoxiconazole + metconazole; prothioconazole + tebuconazole) were tested at full and half rates. Regarding control of yellow rust and brown rust similar control patterns were seen across Europe and treatments with epoxiconazole and tebuconazole provided between 80 and 100% control. In contrast lower levels of control and major variations in azole performances against STB were seen across Europe, with ranking of the azoles tested varying significantly across the continent. Similarly, the CYP51 mutation frequencies varied greatly across Europe with a clear pattern of decreasing frequencies from west to east of all investigated mutations except I381V and A379G. Azoles were most effective against STB when used as mixtures, either as epoxiconazole + metconazole or prothioconazole + tebuconazole. This was especially clear in the western parts of Europe with high frequencies of CYP51 mutations D134G, V136C and S524T in local Z. tritici populations. Effectiveness of all single azoles decreased from 2015 to 2016 except for tebuconazole and azole mixtures, the mixtures providing more robust control across all sites and sensitivities. The average EC50 values for Z. tritici from the trial sites measured for the four azoles showed different levels of cross-resistance and similarly did the efficacy ranking from the azoles. Across all trials full rates of azole mixtures were best at increasing yields, by up to 20%. Single azoles increased yields between 14 and 18%. The greatest yield responses were measured at the sites where yellow rust was the primary disease controlled.

Resistance risk assessment for fluazinam in Sclerotinia sclerotiorum

In the current study, sensitivity distribution of Sclerotinia sclerotiorum populations to fluazinam was determined using 103 strains collected from the fields of Jiangsu Province of China in 2016–2017 and the resistance risk of fluazinam was assessed. The average EC50 (50% effective concentration) values and MIC (minimum inhibitory concentration) values of 103 S. sclerotiorum strains against fluazinam were 0.0073±0.0045μg/ml and <0.3μg/ml for mycelial growth, respectively. Nine mutants with low resistance level were obtained from wild type sensitive strains exposed on PDA medium amended with fluazinam and the resistance was stable after their ten transfers on PDA without the fungicide. Compared with the parental strains, the nine fluazinam-resistant mutants decreased in mycelial growth, sclerotial production, pathogenicity and were more sensitive to 0.7M NaCl. In addition, cell membrane permeability of resistant mutants was higher than that of their parental strains. Cross resistance assay showed that there was no cross-resistance between fluazinam and fludioxonil, dimetachlone, prochloraz, tebuconazole, azoxystrobin, or procymidone in S. sclerotiorum. The above results indicated that there was a low resistance risk for fluazinam in S. sclerotiorum. However, the sensitivity of all fluazinam-resistant mutants to fludioxonil decreased. Sequencing alignment results showed that there were no mutations in the two-component histidine kinase gene (Shk1) of the resistant mutants and the expression levels of Shk1 of three resistant mutants were significantly up-regulated while others were almost the same as their parental strains. These results will contribute to evaluating the resistance risk of fluazinam for management of diseases caused by S. sclerotiorum and further increase our understanding about the mode of action of fluazinam.

New 2-aryl-7,8-dimethoxy-3,4-dihydroisoquinolin-2-ium salts as potential antifungal agents: synthesis, bioactivity and structure-activity relationships

The title compounds can be considered as simple analogues of quaternary benzo[c]phenanthridine alkaloids (QBAs). In order to develop potent QBA-like antifungal agents, as our continuing study, a series of new title compounds were synthesized and evaluated for bioactivity against five plant pathogenic fungi by the mycelium growth rate method in this study. The SAR were also derived. The majority of the compounds showed good to excellent inhibition activity with average EC50 values of 7.87–20.0 μM for the fungi, superior to sanguinarine and cherythrine (two QBAs) and the commercial fungicide azoxystrobin. Part of the compounds were more active than commercial fungicides thiabendazole or carbendazim against F. solani, F. graminearum and C. gloeosporioides. Six compounds with average EC50 of 3.5–5.1 μg/mL possessed very great potential for development of new antifungal agents. SAR found that substitution patterns of the two aryl-rings significantly affect the activity. There exists a complex interaction effect between substituents of the two aryl-rings on the activity. Generally, the presence of electron-withdrawing groups on the C-ring can significantly increase the activity. These findings will be of great importance for the design of more potent antifungal isoquinoline agents.

Activity and biochemical characteristics of plant extract cuminic acid against Sclerotinia sclerotiorum

Sclerotinia stem rot caused by Sclerotinia sclerotiorum (Lib.) de Bary is present worldwide and causes large economic losses. As more and more serious resistance of S. sclerotiorum against commonly used fungicides, development of novel biorational fungicides to control this fungal has become more and more urgent. In this study, the activity of cuminic acid isolated from the seed of Cuminum cyminum L, was assessed. The median effective concentration (EC50) values for inhibition of mycelial growth of 78 S. sclerotiorum strains ranged from 5.46 to 29.34 μg/ml, with an average EC50 value of 17.36 ± 2.28 μg/ml. Meanwhile, both in pot experiments and on detached leaves, cuminic acid exhibited significant control effects against S. sclerotiorum. After treated with cuminic acid, mycelia were clustered seriously and the surface were collapsed and shriveled. In S. sclerotiorum, cell membrane permeability would be increased while oxalic acid content would be decreased and glycerol content would be increased significantly treated with cuminic acid. Above all, cuminic acid showed the potential as a natural alternative to commercial fungicides or a lead compound to develop new fungicides for the control of Sclerotinia stem rot.

Bioactivity and structure-activity relationship of cinnamic acid esters and their derivatives as potential antifungal agents for plant protection.

A series of cinnamic acid esters and their derivatives were synthesized and evaluated for antifungal activities in vitro against four plant pathogenic fungi by using the mycelium growth rate method. Structure−activity relationship was derived also. Almost all of the compounds showed some inhibition activity on each of the fungi at 0.5 mM. Eight compounds showed the higher average activity with average EC50 values of 17.4–28.6 μg/mL for the fungi than kresoxim-methyl, a commercial fungicide standard, and ten compounds were much more active than commercial fungicide standards carbendazim against P. grisea or kresoxim-methyl against both P. grisea and Valsa mali. Compounds C1 and C2 showed the higher activity with average EC50 values of 17.4 and 18.5 μg/mL and great potential for development of new plant antifungal agents. The structure−activity relationship analysis showed that both the substitution pattern of the phenyl ring and the alkyl group in the alcohol moiety significantly influences the activity. There exists complexly comprehensive effect between the substituents on the phenyl ring and the alkyl group in the alcohol moiety on the activity. Thus, cinnamic acid esters showed great potential the development of new antifungal agents for plant protection due to high activity, natural compounds or natural compound framework, simple structure, easy preparation, low-cost and environmentally friendly.

In vivo electroporation in DNA-VLP prime-boost preferentially enhances HIV-1 envelope-specific IgG2a, neutralizing antibody and CD8 T cell responses

Although in vivo electroporation (EP) has been utilized to improve immunogenicity in DNA vaccines alone or in prime-boost regimens with both proteins and viral-vectors, no studies on in vivo EP in DNA-VLP prime-boost regimens against HIV-1 have been reported. Previously we developed stably transfected Drosophila S2 clones to produce HIV-1 virus-like particles (VLP) and demonstrated that priming mice twice with DNA plasmids encoding HIV-1 gp120 and gag and boosting twice with HIV-1 VLP (i.e. DDVV immunization) elicited both envelope-specific antibody and envelope and gag-specific CD8 T cell responses. However, the potency and the breadth of immunogenicity still need to be improved. In this study we tested the effect of in vivo EP during DNA priming on immunogenicity of this DNA-VLP prime-boost regimen. Here we report that although both DDVV and DDVV+EP elicited gp120-specific ELISA-binding antibody responses, average EC50 values of gp120-specific ELISA-binding total IgG, IgG2a, but not IgG1, antibody responses were significantly higher in DDVV+EP than in DDVV. Moreover, while DDVV elicited neutralizing antibody responses against autologous, but not other five, strains tested, DDVV+EP not only elicited significantly higher anti-autologous neutralizing antibody responses, but also cross-neutralizes four of five other HIV-1 strains tested, including two tier 2 strains. Finally, although CD4 and CD8 T cells from both DDVV and DDVV+EP immunizations secreted IFN-γ, IL-2, TNF-α upon HIV-1 envelope peptide stimulation, average HIV-1 envelope-specific CD8 T cells that secreted IFN-γ, IL-2 and/or TNF-α were significantly higher in DDVV+EP than in DDVV. Thus we conclude that DDVV+EP immunization preferentially increases HIV-1 envelope-specific TH1 cytokine-mediated IgG2a responses and significantly enhances the potency and the breadth of neutralizing antibody responses including tier 2 viruses. Further study on this heterologous regimen in large animals is warranted.

Potential use of cuminic acid as a botanical fungicide against Valsa mali.

Valsa canker caused by Valsa mali is commonly present in eastern Asia and cause large economic losses. Because of limited agricultural measures and chemical residues of commonly used fungicides there is an urgent need of alternative plant protecting agents. On this background the activity of cuminic acid, a plant extract from the seed of Cuminum cyminum L, was assessed. The median effective concentration (EC50) values for inhibition of mycelial growth of seven V.mali strains ranged from 3.046 to 8.342μg/mL, with an average EC50 value of 4.956±0.281μg/mL. The antifungal activity was the direct activity of cuminic acid instead of the influence on the pH of media by cuminic acid. After treated with cuminic acid, mycelia dissolved with decreased branches and swelling; cell membrane permeability increased while pectinases activity decreased significantly. Moreover, peroxidase (POD) activity of the apple leaves increased after treated with cuminic acid. Importantly, on detached branches of apple tree, cuminic acid exhibited both protective and curative activity. These results indicated that cuminic acid not only showed the antifungal activity, but also could improve the defense capacity of the plants. Taken together, cuminic acid showed the potential as a natural alternative to commercial fungicides or a lead compound to develop new fungicides for the control of Valsa canker.

Sodium chloride as a reference substance for the three growth endpoints used in the Lemna minor L. (1753) test

Lemna sp. growth inhibition test standardized protocols suggest the use of compounds such as 3,5-dichlorophenol as reference substances for checking the test organism’s sensitivity routinely. However, this and other recommended chemicals present risks to human health and to the environment. Sodium chloride (NaCl) appears as a less toxic alternative reference substance which has been successfully used in routine ecotoxicological tests. However, the evaluation of this compound in multiple growth endpoints used in the L. minor test, which is required for recommending it as a reference substance for this test organism, has not yet been reported. In the present study, NaCl was tested with L. minor for the growth endpoints frond number, total frond area and fresh weight. Results showed acceptable sensitivity and reproducibility (coefficient of variance &lt; 15.0%) for all three of the measured endpoints. Statistically significant differences were observed between the EC50 values calculated based on the three endpoints (p &lt; 0.05). Total frond area was the most sensitive one, with average EC50 value of 2742.80 ± 245.7 mg L-1. It was anticipated that NaCl can be a suitable alternative reference substance and that total frond area should be the endpoint of choice for sensitivity toxicity tests using NaCl.

Sensitivity to Fludioxonil of Botrytis cinerea Isolates from Tomato in Henan Province of China and Characterizations of Fludioxonil‐resistant Mutants

AbstractGrey mould, caused by Botrytis cinerea Pers ex Fr., is one of the most common diseases of tomato worldwide. Fludioxonil belongs to the phenylpyrrole fungicides, which have high activity against B. cinerea. The sensitivity of fludioxonil was evaluated on the basis of the level of inhibition of mycelium growth in 274 B. cinerea isolates collected from different locations (untreated with this fungicide) in Henan Province, China. The EC50 values for fludioxonil ranged from 0.0033 to 0.0415 mg/l, and the average EC50 values were 0.0156 ± 0.0078 mg/l. Three fludioxonil‐resistant mutants were obtained by subculturing fludioxonil‐sensitive wild‐type isolates on continuously increasing fludioxonil concentrations. For the cross‐resistance assay, fludioxonil revealed positive cross‐resistance with procymidone but did not reveal cross‐resistance with pyrimethanil, boscalid and trifloxystrobin. Mycelial growth, conidial production, hyphal dry weight and pathogenicity were diminished significantly between the fludioxonil‐resistant mutants and their sensitive wild‐type parental isolates. This study shows for the first time that fludioxonil‐resistant isolates of B. cinerea are still not present in Henan Province because this fungicide is an attractive and effective fungicide for chemical control. Recommendations can be made to growers to use fludioxonil to control grey mould and to consider the potential moderate resistance risk of using this fungicide.

Development of a New BCMAxCD3 Duobody® Antibody for Multiple Myeloma

B-cell maturation antigen (BCMA) is a tumor necrosis factor (TNF) family surface protein predominantly expressed on terminally differentiated B-cells. BCMA signals through P38/NF-κB pathway upon binding to its ligands; a proliferation inducing ligand (APRIL) and B-cell activator of the TNF family (BAFF) and promote anti-apoptotic gene expression. BCMA expression is elevated in plasma blasts, plasma cells from spleen and bone marrow and correlates with disease progression in multiple myeloma (MM). BCMA expression in premalignant MM settings such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) also gives an opportunity for early cancer interception. To target cancer cells expressing BCMA, we developed a BCMAxCD3 bispecific antibody using the Genmab DuoBody® technology (Ab-957) to recruit T cells to BCMA-expressing MM cells so that T cells could be activated and induced to kill BCMA+ cancer cells. This antibody can induce cytotoxicity of BCMA+ MM cell lines in vitro (H929 cells: EC50=0.15nM, MM1.R cells: EC50=0.06nM, RPMI8226 cells: EC50=0.45nM) with a concomitant T cell activation (H929 cells: EC50=0.21nM, MM1.R cells: EC50=0.1nM, RPMI8226 cells: EC50=0.28nM). In contrast, this antibody was unable to kill BCMA- cancer cell line (MV4-11), demonstrating the specificity of the cytotoxicity. Ab-957 also inhibited tumor development or growth in two BCMA+ MM murine xenograft models inoculated with human T cells. Furthermore, this antibody could deplete BCMA+ cells in bone marrow samples from MM patient’s in an ex-vivo assay with an average EC50 value of 2.5 nM. Lastly, Ab-957 is well-tolerated in cynomolgus monkey and is being developed for Phase I clinical trial in patients with multiple myeloma. DisclosuresPillarisetti:Janssen: Employment. Baldwin:Janssen: Employment. Babich:Janssen: Employment. Majewski:Janssen: Employment. Barone:Janssen: Employment. Li:Janssen: Employment. Zhang:Janssen: Employment. Chin:Janssen: Employment. Luistro:Janssen: Employment. Mendonça:Janssen: Employment. Nanjunda:Janssen: Employment. Rudnick:Janssen Pharmaceuticals R&D: Employment. Bellew:Janssen: Employment. Elsayed:Janssen: Employment, Other: stock options. Attar:Janssen: Employment. Gaudet:Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued.