In vitro cytotoxic potential of Solanum nigrum against human cancer cell lines

Plants have natural products which use to possess antiproliferative potential against many cancers. In the present study, six isolated fractions (ethyl acetate, petroleum ether, chloroform, n-butanol, ethanol and aqueous) from Solanum nigrum were evaluated for their cytotoxic effect on different cell lines. Hepatic carcinoma cell line (HepG2), cervical cancer cell line (HeLa) and baby hamster kidney (BHK) used as normal non-cancerous cells were evaluated for cytotoxicity against isolated fractions. Cell viability assay was performed to evaluate the cytotoxicity of all fractions on different cell lines followed by the lactate dehydrogenase and vascular endothelial growth factor assays of most active fraction among all screened for cytotoxic analysis. HPLC analysis of most active fractions against cytotoxicity was performed to check the biological activity of compounds. Results displayed the potent cytotoxic activity of ethyl acetate fraction of S. nigrum against HepG2 cells with IC₅₀ value of 7.89 μg/ml. Other fractions exhibited potent anticancer activity against HepG2 cells followed by HeLa cells. Fractions in our study showed no cytotoxicity in BHK cells. Cytotoxic activity observed in our current study exposed high antiproliferative potential and activity of ethyl acetate fraction against HepG2 cells. The results demonstrated that S. nigrum fractions exhibited anticancer activity against hepatic and cervical cancer cell lines with non-toxic effect in normal cells. These results reveal significant potential of S. nigrum for the therapeutic of cancers across the globe in future.     

In vitro antimicrobial activity of propolis samples from different geographical origins against certain oral pathogens

Propolis is an agent having antimicrobial properties, however, its composition can vary depending on the area where it is collected. In the present study, the antimicrobial activity of five propolis samples, collected from four different regions in Turkey and from Brazil, against nine anaerobic strains was evaluated. Ethanol extracts of propolis (EEP) were prepared from propolis samples and we determined minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of EEP on the growth of test microorganisms by using agar dilution method. All strains were susceptible and MIC values ranged from 4 to 512 microg/ml for propolis activity. Propolis from Kazan-Ankara showed most effective MIC values to the studied microorganisms. MBC values of Kazan-Ankara EEP samples were ranged from 8 to 512 microg/ml. Death was observed within 4 h of incubation for Peptostreptococcus anaerobius and micros and Lactobacillus acidophilus and Actinomyces naeslundii, while 8 h for Prevotella oralis and Prevotella melaninogenica and Porphyromonas gingivalis, 12 h for Fusobacterium nucleatum, 16 h for Veillonella parvula. It was shown that propolis samples were more effective against Gram positive anaerobic bacteria than Gram negative ones. The organic chemical compositions of EEPs were determined by high-resolution gas chromatography coupled to mass spectrometry (GC-MS). The main compounds of EEPs were flavonoids such as pinobanksin, quercetin, naringenin, galangine, chrysin and aromatic acids such as cafeic acid. Because of increased antimicrobial resistance, propolis may be kept in mind in the treatment of oral cavity diseases.     

Synergistic anticancer action of quercetin and curcumin against triple-negative breast cancer cell lines

Women with the breast cancer type 1 susceptibility protein (BRCA1) mutation and loss of BRCA1 expression are reported to have an increased risk of triple-negative breast cancer (TNBC). Targeting BRCA1 modulation might offer a therapeutic option to treat TNBC patients. Our studies detected that BRCA1 is poorly expressed in TNBC cell lines and highly expressed in ER⁺ breast cancer cell lines. To modulate BRCA1 expression, we tested two different dietary components to find out if any would induce tumor suppressor genes. We detected that quercetin and curcumin dose-dependently enhanced the BRCA1 expression. Further, a synergistic action of quercetin and curcumin was observed in modulating the BRCA1 level and in inhibiting the cell survival and migration of TNBC cell lines. Quercetin and curcumin appeared to induce BRCA1 promoter histone acetylation. Furthermore, BRCA1 knockdown induced cell survival and cell migration in ER ⁺ cells were significantly decreased by the combined treatment of quercetin and curcumin. Our present study concluded that the combination treatment of quercetin and curcumin acts synergistically to induce anticancer activity against TNBC cells by modulating tumor suppressor genes.     

Potent anticancer activities of novel aminophenol analogues against various cancer cell lines

Novel aminophenol analogues were synthesized based on the structure of fenretinide (N-(4-hydroxyphenyl)retinamide, 5), which is a potent anticancer agent. Our findings showed that the anticancer activities of 5 were due to the side chain attached to the aminophenol moiety. A p-octylaminophenol (p-OAP) provided the most potent anticancer activity among p-alkylaminophenols examined. In this study, we investigated anticancer activities against various cancer cell lines by the new aminophenols, p-dodecylaminophenol (1), p-decylaminophenol (2), N-(4-hydroxyphenyl)dodecananamide (3), and N-(4-hydroxyphenyl)decananamide (4), which exhibits a side chain as long as 5. Cell growth of breast cancer (MCF-7, MCF-7/Adr(R)), prostate cancer (DU-145), and leukemia (HL60) cells was suppressed by 1 and 2 in a fashion dependent on the length of the alkyl chain attached to the aminophenol. In contrast, 3 and 4 were extremely weak. Compound 5 was less potent than 1. Cell growth of liver cancer (HepG2) was not markedly affected by these compounds. In addition, apoptosis of HL60 cells was induced by 1 and 2 in a chain length-dependent manner, but not by 3 and 4. Incorporation of compounds into HL60 cells was in the order 1>2=3>4. These results indicated that anticancer activities for 1 and 2 are correlated with their incorporation into cancer cells and their capability to induce apoptosis, but not for 3 and 4. Compound 1, a potent anticancer agent with potency strikingly greater than 5, may potentially be useful in clinic.     

In vitro cytotoxic potential of extracts from Aristolochia foetida Kunth against MCF-7 and bMECs cell lines

The aim of this study was to evaluate the cytotoxic potential of Aristolochia foetida Kunth. Stems and leaves of A. foetida Kunth (Aristolochiaceae) have never been investigated pharmacologically. Recent studies of species of the Aristolochiaceae family found significant cytotoxic activities. Hexane, dichloromethane, ethyl acetate and methanol extracts were analyzed by ¹H NMR and GC–MS to know the metabolites in each extract. In GC–MS analysis, the main compounds were methyl hexadecanoate (3); hexadecanoic acid (4); 2-butoxyethyl dodecanoate (9); ethyl hexadecanoate (20); methyl octadeca-9,12,15-trienoate (28) and (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid (40). The results showed a significant reduction in cell viability of the MCF-7 (breast cancer) cell line caused by organic extracts in a dose-dependent manner. The cytotoxicity activity of the dichloromethane extract from the stems (DSE) showed IC₅₀ values of 45.9 μg/mL and the dichloromethane extract of the leaves (DLE) showed IC₅₀ values of 47.3 μg/mL. DSE and DLE had the highest cytotoxic potential in an in vitro study against the MCF-7 cell line and non-tumor cells obtained from the bovine mammary epithelial (bMECs). DSE and DLE induced a loss in mitochondrial membrane potential (ΔΨm) and can cause cell death by apoptosis through the intrinsic pathway in the MCF-7 cell line. DSE and DLE are cytotoxic in cancer cells and cause late apoptosis. Higher concentrations of DSE and DLE are required to induce a cytotoxic effect in healthy mammary epithelial cells. This is the first report of the dichloromethane extract of A. foetida Kunth that induces late apoptosis in MCF-7 cancer cells and may be a candidate for pharmacological study against breast cancer.     

Evaluation and insights into chitosan antimicrobial activity against anaerobic oral pathogens

The objective of this study was to assess the antimicrobial capability of non-chemically altered chitosan as an alternative to traditional antimicrobials used in the treatment of oral infections. The action mechanism of chitosan was also ascertained. High and low molecular weight chitosan showed antimicrobial activity at low concentrations for all tested bacteria with the MICs varying between 1 and 7 mg/ml with a drop of efficacy relatively to the action of LMW chitosan. In addition chitosan showed also to be an effective bactericidal presenting bactericidal effect within 8 h at the latest. Additionally the evaluation of chitosan's action mechanism showed that both MWs acted upon the bacterial cell wall and were not capable of interacting with the intracellular substances, as showed by the inefficacy obtained in the flocculation assay.     

In vitro antibacterial potential of Eugenia jambolana seed extracts against multidrug-resistant human bacterial pathogens

The present study was carried out to evaluate the possible in vitro antibacterial potential of extracts of Eugenia jambolana seeds against multidrug-resistant human bacterial pathogens. Agar well diffusion and microbroth dilution assay methods were used for antibacterial susceptibility testing. Kill-kinetics study was done to know the rate and extent of bacterial killing. Phytochemical analysis and TLC-bioautography were performed by colour tests to characterize the putative compounds responsible for this antibacterial activity. Cytotoxic potential was evaluated on human erythrocytes by haemolytic assay method and acute oral toxicity study was done in mice. The plant extracts demonstrated varying degrees of strain specific antibacterial activity against all the test isolates. Further, ethyl acetate fraction obtained from fractionation of most active ethanol extract showed maximum antibacterial effect against all the test isolates. Phytochemical analysis and TLC-bioautography of ethyl acetate fraction revealed that phenolics were the major active phytoconstituents. Ethyl acetate fraction also demonstrated no haemolytic activity on human erythrocytes and no gross behavioural changes as well as toxic symptoms were observed in mice at recommended dosage level. The results provide justification for the use of E. jambolana in folk medicine to treat various infectious diseases and may contribute to the development of novel antimicrobial agents for the treatment of infections caused by these drug-resistant bacterial pathogens.     

In vitro radiosensitization by eribulin in human cancer cell lines

BackgroundThe objective was to to determine the radiosensitizing properties of eribulin and the potential mechanisms of radiosensitization in cervical (HeLa) and pharyngeal (FaDu) cancer cell lines.Materials and methodsCytotoxicity was evaluated by the crystal violet method. The 10% and 50% inhibitory concentration (IC10, IC50) for 24-hour drug exposure were determined. The surviving fraction at 2 Gy (SF2) and the sensitizer enhancement ratio (SER) were calculated from radiation cell survival curves in the presence or absence of eribulin. Combination index (CI) was calculated to determine if there is a true synergistic interaction between eribulin and irradiation. Cell cycle changes were assessed by propidium iodide staining and flow cytometry. Apoptotic cells were detected by annexin V and TUNEL-assay.ResultsMean IC50s and IC10s were 1.58 nM and 0.7 nM and 0.7 nM and 0.27 nM for HeLa and FaDu cells, respectively. Radiosensitization was observed in both lines with a SER up to 2.71 and 2.32 for HeLa and FaDu cells, respectively. A true synergistic effect was showed with a CI of 0.82 and 0.76 for HeLa and FaDu cells, respectively. Eribulin induced significant G2/M cell arrest and marked apoptosis. Irradiation combined with 3 nM eribulin increased the apoptotic response to radiation in Hela cells.ConclusionEribulin shows a true in vitro radiosensitizing effect in HeLa and FaDu cells by inducing significant G2/M phase arrest. In HeLa, the enhancement radiation-induced apoptosis could be an additional mechanism of radiosensitization. Further studies are needed to evaluate the clinical benefits of concurrent eribulin and radiotherapy as a novel therapeutic strategy for cancer.     

Novel Aza-resveratrol analogs: Synthesis,characterization and anticancer activity against breast cancer cell lines

Novel Aza-resveratrol analogs were synthesized, structurally characterized and evaluated for cytotoxic activity against MDA-MB-231 and T47D breast cancer cell lines, which exhibited superior inhibitory activity than parent resveratrol compound. The binding mechanism of these compounds with estrogen receptor-α was rationalized by molecular docking studies which indicated additional hydrogen binding interactions and tight binding in the protein cavity. Induction of Beclin-1 protein expression in breast cancer cell lines after treatment with newly synthesized resveratrol analogs indicated inhibition of growth of these cell lines through autophagy. The study highlighted the advantage of introducing the imino-linkage in resveratrol motif in enhancing the anticancer potential of resveratrol suggesting that these analogs can serve as better therapeutic agents against breast cancer and can provide starting point for building more potent analogs in future.     

In vitro antiproliferative activity against human colon cancer cell lines of representative 4-thiazolidinones. Part I

The characterization of two cyclooxygenase isoforms (COX), the rate-limiting enzyme for the synthesis of prostaglandins (PGs) from arachidonic acid, has allowed the development of COX-2 selective inhibitors as non-steroidal anti-inflammatory drugs (NSAIDs) with significant gastric tolerability. However, PGs are also important in cancer pathogenesis. Thus, there is an increasing interest in studying COX-2 inhibitors as potential drugs aimed at the prevention and treatment of cancer, especially colorectal cancer. The purpose of this study was to determine the inhibitory effects of some representative 4-thiazolidinones, already widely investigated as potential NSAIDs, on the growth of five human colon carcinoma cell lines with a different COX-2 expression, and to correlate them with COX-2 inhibitory properties. Our results preliminarily revealed that 2-phenylimino derivative 3 and 2,4-thiazolidindione 4 were the most active compounds. In particular, 3 mainly inhibited the HT29 cell line characterized by a high COX-2 expression, whereas 4 showed antiproliferative properties on all tested cell lines, suggesting molecular targets other than COX-2 inhibition.     

In vitro antimicrobial activity against Pseudomonas aeruginosa and acute oral toxicity of marine algae Gracilaria changii

Methanol extract of the Gracilaria changii has been screened for antimicrobial activity against Pseudomonas aeruginosa. Antimicrobial activities were carried out using disc diffusion assay and broth dilution method against P. aeruginosa. The methanol extract of G. changii showed a good antimicrobial activity against P. aeruginosa with MIC (Minimum Inhibitory Concentration) value of 6.25 mg/ml. Exposure of P. aeruginosa cells to 6.25 mg/ml of methanol extract of G. changii resulted in complete inhibition of the bacterial cells. The main abnormalities noted via SEM and TEM studies were the alterations in morphology and cytology of the bacterial cells. The main reason for this deterioration was discussed. The effect of the methanol extract on the growth profile for the bacteria was also done and confirmed the bactericidal effect of the G. changii methanol extract on P. aeruginosa by changing the normal growth profile of P. aeruginosa. In an acute toxicity study using mice, the median lethal dose (LD₅₀) of the extract was greater than 2000 mg/kg, and we found no pathological changes in macroscopic examination by necropsy of mice treated with extract. We conclude that G. changii might be safely used as an antimicrobial agent.     

Potential anticancer activities of Rhus coriaria (sumac) extract against human cancer cell lines

Therapeutic strategies of plant origin are a better choice as both dietary plant products or its isolated active constituents against the development and progression of cancer. The present study aims to evaluate the anticancer activity of sumac (Rhus coriaria) against different human cancer MCF-7, PC-3, and SKOV3 cell lines. In addition, the study tries to explore a prospective mechanism of action, assessment of in vitro enzyme-inhibitory capacity of sumac extract against hCA I, II, IX, and XII. In the present study, the potential antitumor effects of sumac (Rhus coriaria) were explored in the human cancer cell lines; MCF-7, PC-3, and SKOV3 using in vitro assays. Apoptotic, cell survival, ELISA immunoassays were also conducted to reveal the inhibitory effects of sumac extract against hCA I, II, IX, and XII. In addition, both Clioquinol and Acetazolamide (AZM) were used as standards to explore the in vitro enzyme-inhibitory capacity of sumac extract against hCA I, II, IX, and XII. The hydro-alcoholic extract of R. coriaria (Sumac) was subjected to phytochemical analysis using GC/MS assays. Sumac at non-cytotoxic doses of 50 and 100 µM significantly modulates the growth of the MCF-7, PC-3, and SKOV3 cancer cells with a higher inhibitory effect and selectivity to carbonic anhydrase (CA) isoforms; hCA I, II, hCA IX, and XII. The data showed that sumac at doses of 50 and 100 µM significantly inhibited the growth, proliferation, and viability of cancer cells by activating the apoptotic process via caspase-3 overexpression and the regulation of Bcl-2 anti-apoptotic protein.     

比较五种中草药牙膏对口腔常见菌的抗菌潜力

目的评价5种国产中草药牙膏对口腔常见菌的抗菌潜力。方法运用扩散法来评价5种国产中草药牙膏———美加净复合中草药牙膏(MAX)、洁银中药牙膏(JY)、田七特效中药牙膏(TQ)、中华中草药牙膏(ZH)、高露洁草本美白牙膏(Colgate H)对口腔常见菌:变形链球菌(S.mutants)、血链球菌(S.sanguis)、粘放线菌(A.viscosus)和白色念珠菌(C.albicans)的抗菌潜力。结果阴性对照组未见产生抗菌区域,而阳性对照组对4种口腔常见菌产生明显的抗菌区域。TQ对除了白色念珠菌(C.albicans)外的其他3种口腔常见菌都有明显的抗菌作用,其余4种牙膏在膏状和稀释状态下对所有4种常见菌均显示了明显和持续的抗菌作用。膏状状态下的抗菌区域比稀释状态下有显著的增大(P<0.05)。结论除TQ对C.albicans显示无效外,所有5种国产中草药牙膏对口腔内的常见菌均有明显的抗菌作用。     

In vitro activity of novel in silico‐developed antimicrobial peptides against a panel of bacterial pathogens

Antimicrobial‐peptide‐based therapies could represent a reliable alternative to overcome antibiotic resistance, as they offer potential advantages such as rapid microbicidal activity and multiple activities against a broad spectrum of bacterial pathogens. Three synthetic antimicrobial peptides (AMPs), AMP72, AMP126, and also AMP2041, designed by using ad hoc screening software developed in house, were synthesized and tested against nine reference strains. The peptides showed a partial β‐sheet structure in 10‐mM phosphate buffer. Low cytolytic activity towards both human cell lines (epithelial, endothelial, and fibroblast) and sheep erythrocytes was observed for all peptides. The antimicrobial activity was dose dependent with a minimum bactericidal concentration (MBC) ranging from 0.17 to 10.12 μM (0.4–18.5 µg/ml) for Gram‐negative and 0.94 to 20.65 μM (1.72‐46.5 µg/ml) for Gram‐positive bacteria. Interestingly, in high‐salt environment, the antibacterial activity was generally maintained for Gram‐negative bacteria. All peptides achieved complete bacterial killing in 20 min or less against Gram‐negative bacteria. A linear time‐dependent membrane permeabilization was observed for the tested peptides at 12.5 µg/ml. In a medium containing Mg²⁺ and Ca²⁺, the peptide combination with EDTA restores the antimicrobial activity particularly for AMP2041. Moreover, in combination with anti‐infective agents (quinolones or aminoglycosides) known to bind divalent cation, AMP126 and AMP2041 showed additive activity in comparison with colistin. Our results suggest the following: (i) there is excellent activity against Gram‐negative bacteria, (ii) there is low cytolytic activity, (iii) the presence of a chelating agent restores the antimicrobial activity in a medium containing Mg²⁺ and Ca²⁺, and (iv) the MBC value of the combination AMPs–conventional antibiotics was lower than the MBC of single agents alone. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

耐碳青霉烯类肠杆菌科细菌体外抗菌活性分析

目的了解临床分离的59株碳青霉烯酶类抗生素耐药的肠杆菌科细菌(CRE)体外药敏情况。方法采用法国生物梅里埃公司VITEK-2Compact全自动细菌分析鉴定系统进行细菌鉴定及药敏,MALDI-TOF MS确认。琼脂稀释法测定临床常用抗生素的最低抑菌浓度。结果分离的菌株以肺炎克雷伯菌为主。59株CRE除对亚胺培南和美罗培南耐药率100.0%外,对哌拉西林/他巴唑坦和阿莫西林/克拉维酸同样完全耐药。头孢他啶、头孢噻肟、氨曲南耐药率分别为91.5%、98.5%和94.8%。阿米卡星有较好的抗菌活性,耐药率为44.8%。替加环素耐药率最低,为22.0%。结论 CRE耐药率较高,头孢类、青霉素类及其复合制剂单独用药不适合作为本地区CRE菌株抗菌药物,需联合用药。替加环素目前可作为CRE理想用药,阿米卡星也可作为次选。     

In vitro biological evaluation of novel 7-O-dialkylaminoalkyl cytotoxic pectolinarigenin derivatives against a panel of human cancer cell lines

The effect of novel pectolinarigenin derivatives bearing a dialkylaminoalkyl substituent at O-7 on cell proliferation was evaluated in vitro in a panel of seven human cancer cell lines including renal adenocarcinoma ACHN, amelanotic melanoma C32, colorectal adenocarcinoma Caco-2, lung large cell carcinoma COR-L23, malignant melanoma A375, lung carcinoma A549 and hepatocellular carcinoma Huh-7D12 cell lines. Pectolinarigenin (2), obtained by hydrolysis of rutinose unit of the pectolinarin (1) isolated from Linaria reflexa, exhibited cytotoxic activity against Caco-2, A549 and A375 cell lines with IC(50) values of 5.3-8.2 microM. The most active pectolinarigenin derivative was 3 characterized by a dimethylamino-propoxy group in O-7 with IC(50) values of 7.2 and 7.4 microM against COR-L23 and A549 cell lines, respectively. A structure-activity relationship analysis of synthesized compounds was performed. None of the tested compounds affected the proliferation of skin fibroblasts 142BR suggesting a selective activity against tumor cells.     

Screening the antimicrobial potential of twelve medicinal plants against venereal diseases causing pathogens

The antimicrobial potential of selected ethnomedicinal plants in traditional healers of Silent valley, Palakkad district of Kerala, India against venereal diseases causing pathogens. Twelve medicinal plants and their parts were collected from the various places of Silent Valley, Kerala, India. These plant parts were dried and mixed with different kinds of solvents respectively hexane, chloroform, methanol and water. In this study, six microbial strains were selected, in which five bacterial and a fungal strain. Among the bacterial strains, two strains were gram positive and three strains for gram negative bacteria. Totally, twelve medicinal plant parts mixed with various solvents were treated against the selected pathogenic organisms. Among them, methanolic extract of A. occidentate, C. indica, H. rosa-sinensis and M. oleifera exhibited excellent antibacterial activity than other parts of plants and standard drugs. As well as, methanolic extract of H. rosa-sinensis showed good antifungal activity against C. albicans. While,the least inhibition was noted with aqueous extract of C. indica against S. aureus. The MIC ranges from 0.78μg/ml to 50μg/ml and MBC/MFC 1.52μg/ml to 50μg/ml. The methanolic flower extract of H. rosa-sinensis has showed effective zone of inhibition against all the pathogens, particularly N. gonorrhoeae (30 mm) and C. albicans (26mm), than other extracts and standard drugs. Therefore, we concluded that, flower extract had potential therapeutic activity against venereal diseases. This research will be helpful to discover the new therapeutic drug molecule in Pharmaceutical sectors.     

In vitro activity of amphibian peptides alone and in combination with antimicrobial agents against multidrug-resistant pathogens isolated from surgical wound infection

The in vitro activities of three amphibian peptides magainin II amide, citropin 1.1 and temporin A alone and in combination with eight clinically used antimicrobial agents (imipenem, ceftazidime, clarithromycin, vancomycin, amikacin, polymyxin E, ciprofloxacin and linezolid) were investigated against several multidrug-resistant Pseudomonas aeruginosa and Staphylococcus aureus strains isolated from surgical wound infections. Antimicrobial activities were measured by MIC, MBC and time-kill studies. P. aeruginosa strains were more susceptible to magainin II amide and less susceptible to temporin A. S. aureus isolates were highly susceptible to temporin A and citropin 1.1. The combination studies showed synergy between citropin 1.1 and clarithromycin. Magainin II amide and temporin A showed synergism with imipenem and ceftazidime. Finally, all peptides showed synergistic effects with polymyxin E. These results provide evidence for the potential use of these antimicrobial peptides in the topical or systemic treatment of surgical wound infections.     

In vitro activity of glucosinolate-derived isothiocyanates against postharvest fruit pathogens*

The authors assayed the ability of some reaction products, essentially isothiocyanates, derived from the myrosinase-catalysed hydrolysis (neutral pH) of six natural glucosinolates to inhibit germination and mycelial growth of Botrytis cinerea, Rhizopus stolonifer, Monilinia laxa, Mucor piriformis and Penicillium expansum, the leading postharvest fungal pathogens of fruit and vegetable crops. All of the tested products showed antifungal activity, although they proved less effective against mycelial growth than in germination control. The isothiocyanates produced by enzymatic hydrolysis of glucoraphenin, sinalbin and sinigrin were particularly effective because they completely inhibited conidic germination of all five pathogens considered. The sinigrin-derived isothiocyanate exhibited a wide pathogen-control spectrum, either inhibiting conidia germination altogether or delaying by 3–6 days the onset of mycelial growth compared to the control.