Spore Germination Inhibition Assay Research Articles

Activity of fungicide cyclobutrifluram against Fusarium fujikuroi and mechanism of the pathogen resistance associated with point mutations in FfSdhB, FfSdhC2 and FfSdhD

Rice bakanae disease (RBD) is a devastating plant disease caused by Fusarium fujikuroi. This study aimed to evaluate the potential of cyclobutrifluram, a novel succinate dehydrogenase inhibitor (SDHI), to control RBD, and determine the risk and mechanism of resistance to cyclobutrifluram in F. fujikuroi. In vitro experiments showed that cyclobutrifluram significantly inhibited mycelial growth and spore germination, and altered the morphology of mycelia and conidia. Treatment with cyclobutrifluram significantly decreased mycotoxin production and increased cell membrane permeability in F. fujikuroi. The baseline sensitivity of 72 F. fujikuroi isolates to cyclobutrifluram was determined using mycelial growth and spore germination inhibition assays, which revealed EC50 values of 0.0114 – 0.1304 μg mL-1 and 0.0012 – 0.016 μg mL-1, with mean EC50 values of 0.0410 ± 0.0470 μg mL-1 and 0.0038 ± 0.0015 μg mL-1, respectively. Pot experiments demonstrated that the protective effect of cyclobutrifluram against F. fujikuroi was more significant than that of phenamacril and azoxystrobin, indicating that cyclobutrifluram is a promising antifungal agent for the control of RBD. Six cyclobutrifluram-resistant mutants of F. fujikuroi were obtained via fungicide adaptation. Moreover, these mutants exhibited weaker fitness than their parental isolate and positive cross-resistance with other SDHI fungicides, including pydiflumetofen and penflufen; however, no cross-resistance was detected with other classes of fungicides, including phenamacril, fludioxonil, prochloraz, or azoxystrobin. These results indicated that the resistance risk of F. fujikuroi to cyclobutrifluram might be moderate. Sequencing analysis revealed that mutations, including H248D in FfSdhB, A83V in FfSdhC2, and S106F and E166K in FfSdhD, contributed to resistance, which was confirmed by molecular docking and homologous replacement experiments. The results suggest a high potential for cyclobutrifluram to control RBD and a moderate resistance risk of F. fujikuroi to cyclobutrifluram, which are meaningful findings for the scientific application of cyclobutrifluram.

Assessment of the Fungicidal and Nematicidal Potential of Reichardia tingitana (L.) Roth on Phytopathogenic Fungi and Plant Nematode

The primary concern was the removal of numerous soil fumigants and nematicides due to their potential risks to human and environmental safety. Fungal pathogens can cause serious diseases in humans and animals. Among these, root-knot nematodes such as Meloidogyne incognita and Tylenchulus semipenetrans pose a significant threat, leading to substantial damage and yield reduction in various economically important plants. Therefore, this study aimed to assess the fungicidal and nematicidal activities of the ethanol extract (EE) and lupeol (L), the major isolates from the aerial parts of Reichardia tingitana L. Roth (Asteraceae), against Aspergillus flavus and plant-parasitic nematodes. Antifungal actions of EE (10-120 ppm) and L (23.4-281.2 µM) were evaluated through in vitro and in vivo growth assays, spore germination inhibition assays, and the efficacy of inhibiting pod and kernel infection. Nematicidal activity of EE and L was tested by preparing cultures containing egg masses of nematode species M. incognita from infected eggplants and T. semipenetrans from infected citrus roots, using concentrations of 2.5, 5, 10, 20, 40, 80, and 120 ppm. Results showed that R. tingitana (EE) and (L) exhibited nematostatic or nematicidal effects on nematode viability, egg hatch in vitro, and development and reproduction in vivo. Lupeol was particularly effective in inhibiting the colonization of A. flavus in peanuts. EE and L demonstrated high toxicity against nematodes in laboratory exposure and were effective in controlling nematode infestation in eggplant roots for 45 days. Improvement in plant growth parameters, including shoot and root length and weights, varied and was proportional to the doses of EE and L treatments. The antifungal and bio-nematicide effects of the ethanol extract from the aerial parts of R. tingitana were superior to those of lupeol, which could be attributed to the synergistic effect of phytochemicals in the ethanol extract. Both EE and L have potential applications as antifungal and bio-nematicide agents.

Inhibitory Effect and Control Efficacy of Picoxystrobin against Neopestalotiopsis clavispora, Causing Vine Tea Leaf Blight

Vine tea (Ampelopsis grossedentata) is a traditional herb widely consumed in southwestern China that possesses paramount potential for human health. In 2021, the outbreak of typical leaf blight disease was observed in almost all vine tea plantations in Zhangjiajie of Hunan province, resulting in significant economic losses of vine tea production. In this study, we identified Neopestalotiopsis clavispora as the causal agent of vine tea leaf blight via its morphological characteristics and molecular identification. The sensitivity distribution of N. clavispora isolates to picoxystrobin were determined based on mycelial growth and spore germination inhibition assays. The EC50 values for mycelial growth ranged from 0.0062 to 0.0658 µg/mL, with a mean of 0.0282 ± 0.0148 µg/mL. The EC50 values for spore germination ranged from 0.0014 to 0.0099 µg/mL, and the mean value was 0.0048 ± 0.0022 µg/mL. Picoxystrobin increased fungal cell membrane permeability, but inhibited fungal ATP biosynthesis. Moreover, picoxystrobin exhibited good in planta control efficacy on vine tea leaves. Three picoxystrobin-resistant mutants were obtained in the current study, but no mutations were detected in the N. clavispora Cytb gene. Competitive ability assays showed that the conidium production and pathogenicity of all picoxystrobin-resistant mutants decreased as compared to their progenitors, indicating that picoxystrobin-resistant mutants suffer fitness penalty. These findings provide important evidence for picoxystrobin in vine tea leaf blight management and increase understanding of the resistance mechanism of picoxystrobin against N. clavispora.

Green chemistry principles for the synthesis of anti fungal active gum acacia-gold nanocomposite - natamycin (GA-AuNC–NT) against food spoilage fungal strain Aspergillus ochraceopealiformis and its marked Congo red dye adsorption efficacy

In this present study, a highly stable gum acacia -gold nanocomposite fabricated with food preservative agent natamycin (GA-AuNC–NT) was prepared via green science principles under in vitro conditions. Various characterisation techniques reveal highly stable structural, functional properties of the synthesised nanocomposite with marked antifungal activity and adsorption efficacy against congo red dye. The antifungal activity was investigated against the fungal strain Aspergillus ochraceopealiformis isolated from spoiled, expired bread. The well diffusion assay, fungal hyphae fragmentation assay and spore germination inhibition assay were used to determine the antifungal activity of the synthesised nanocomposite. Potential antifungal activity of the synthesised nanocomposite was confirmed by recording zone of inhibition, high rate of hyphae fragmentation and marked spore germination inhibition against the tested fungal strain. The molecular mechanism of antifungal activity was studied by measuring oxidative stress marker genes like catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) induction adopting quantitative real-time polymerase chain reaction (q RT-PCR). Among the various treatment, a notable reduction in all the tested marker genes expression was recorded in the nanocomposite treated fungal strain. Release profile studies using different solvents reveals sustained or controlled release of natamycin at the increasing periods. The synthesised nanocomposite's high safety or biocompatibility was evaluated with the Wistar animal model by determining notable changes in behavioural, biochemical, haematological and histopathological parameters. The synthesised nanocomposite did not exhibit any undesirable changes in all the tested parameters confirming the marked biosafety or biocompatibility. The nanocomposite was coated on the bread packaging material. The effect of packaging on the proximate composition, antioxidative enzymes status, and fungal growth of bread samples incubated under the incubation period were studied. Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) studies reveal that the nanocomposite was effectively coated on the packaging material without changing size, shape, and functional groups. No changes in the proximate composition and antioxidative enzymes of the packaged bread samples incubated under different incubation periods reveal the nanocomposite's marked safety. The complete absence of the fungal growth also indicates the uniqueness of the nanocomposite. Further, the sorption studies revealed the utilisation of Langmuir mechanism and pseudo II order model successfully The present finding implies that the synthesised nanocomposite can be used as an effective, safe food preservative agent and adsorbent of toxic chemicals.

Inhibitory effect of cinnamon ( Cinnamomum tamala (Buch.-Ham.) T.Nees & Eberm.) essential oil and its aldehyde constituents on growth and spore germination of phytopathogenic fungi

Plant pathogens lead to severe damage of the agricultural crops, worldwide. The disease management is currently being done by the synthetic chemicals. In the present report, Cinnamomum tamala (Buch.-Ham.) T. Nees & Eberm. oil was analyzed by gas chromatography-FID and GC/MS, which revealed (E)-cinnamaldehyde (40.6%), (E)-cinnamyl acetate (19.6%), linalool (10.2%) and benzaldehyde (3.6%) as marker constituents. Contact, volatile and spore germination inhibition assays were performed to determine the antifungal activities of Cinnamomum tamala (Buch.-Ham.) T.Nees & Eberm. against Rhizoctonia solani AG 4, Choanephora cucurbitarum and Bipolaris australiensis. C. tamala oil 500-560 ppm showed complete fungal growth inhibition in poison food technique over concentration range of. Solid-phase microextraction analysis of drop diffusion assay revealed the presence of benzaldehyde in headspace of Petri-plate, and found to be one of the strongest antifungal compounds against tested phytopathogens. Similarly, an oil concentration of 500 ppm inhibits spore germination. Therefore, products containing such compositions can be used for the plant diseases management.

Activity of the Novel Succinate Dehydrogenase Inhibitor Fungicide Pydiflumetofen Against SDHI-Sensitive and SDHI-Resistant Isolates of Botrytis cinerea and Efficacy Against Gray Mold.

Succinate dehydrogenase inhibitor (SDHI) fungicides are currently the most frequently used fungicides for controlling gray mold. However, isolates of Botrytis cinerea resistant to SDHI fungicides have emerged in the field. Pydiflumetofen is a new SDHI fungicide that can control a variety of fungal diseases, but its efficacy against gray mold and whether the activity of pydiflumetofen is affected by the current SDHI-resistant isolates is currently unknown. The sensitivity of 291 single-spore B. cinerea isolates collected from 2017 to 2019 to pydiflumetofen was determined by spore germination inhibition assays. The mean EC50 value (fungicide concentration resulting in a 50% inhibition compared with that of the control) of pydiflumetofen was 0.06 ± 0.01, 0.07 ± 0.02, and 0.05 ± 0.02 mg/liter in 2017, 2018, and 2019, respectively. There was no significant difference in the sensitivity of B. cinerea to pydiflumetofen among the 3 years. Furthermore, pydiflumetofen at 300 mg/liter effectively controlled gray mold on cucumber leaves (80.9%), and its efficacy was superior to that of boscalid at 400 mg/liter (42.7%). The isolates carrying P225F, N230I, H272Y, and H272R mutations in the SdhB subunit were associated with the less sensitivity of B. cinerea to SDHI fungicides. After establishing the baseline sensitivity of B. cinerea to pydiflumetofen (EC50 of 0.03 ± 0.003 mg/liter), we found that the P225F and H272Y mutant isolates showed low to moderate levels of resistance to pydiflumetofen, and the H272R and N230I mutant isolates showed low levels of resistance. The reduced sensitivity to pydiflumetofen resulted from the positive correlation of pydiflumetofen with the other four SDHI fungicides (i.e., boscalid, fluopyram, isopyrazam, and benzovindiflupyr). These results suggest that pydiflumetofen provides effective control for the management of gray mold but must be used with caution.

IN VITRO ANTIMYCOTIC EVALUATION AND PRELIMINARY COMBINATORIAL STUDIES OF IBUPROFEN WITH STANDARD ANTIFUNGAL DRUGS AGAINST ASPERGILLUS SPP.

Objective: The prevalence of invasive mycoses is increased in the immunocompromised patients with an increase in resistance developed againstcurrent antifungal drugs. This has led to the need for discovering novel combinations of the antifungal drugs to combat against resistant pathogenic spp.This study mainly targets to evaluate the antifungal activity of ibuprofen (IBU) alone and in combination with the standard antifungal drugs (polyenesand azoles) against eight isolates of Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger.Methods: The study was performed using the disc diffusion assay (DDA), microbroth dilution assay and spore germination inhibition assay. Moreover,cytotoxicity was checked by heamolytic assay.Results: Minimum inhibitory concentration (MIC) of IBU against A. fumigatus and A. flavus using DDA is found to be in the range of 250-275 μg/disc while for A. niger isolates, the range was 500-575 μg/disc. Likewise, by broth microdilution assay and spore germination inhibitory assay, MICdetermined, were in the range of 500-750 μg/ml against A. fumigatus and A. flavus while for A. niger, it was 1000-1500 μg/ml.Conclusion: IBU demonstrated its antimycotic potential against all the eight isolates of Aspergillus spp. Moreover, preliminary combinatorialevaluation of IBU with the standard antifungal drugs reported by DDA revealed an increase in zone of inhibition as compared to the drugs alone.Further research regarding the confirmation of synergistic interaction between the selected drugs is in progress.

Growth Inhibition of Grain Spoilage Fungi by Selected Herbs and Spices Essential Oils

Natural plant extracts are promising alternatives for chemical food additives and synthetic pesticides. In this study, essential oils of selected herbs and spices were tested for their antimicrobial activities against Aspergillus flavus and Aspergillus niger, two of the most common food spoilage microorganisms. Agar disk diffusion assay was used for screening of the most effective essential oils, agar dilution assay was used to determine Minimum Inhibitory Concentration (MIC) of the essential oils and broth dilution assay was employed to the spore germination inhibition assay. Tests were also conducted to examine the effects of the essential oils for sorghum kernel protection against the tested fungi, and the optimal protective dosages on the sorghum grains were also determined. From the preliminary tests, essential oils of Cinnamomum zeylanicum (Cinnamon) and Thymus schimperi (thymus) were found to be the most effective. However piper nigrum (black pepper) had no effect on the test organisms. In MIC, spore germination inhibition and grain protection assay, cinnamon essential oil was found to be superior where its MIC on the isolates was found to be 0.0156% and its optimum protective dosage on the sorghum grain was 5%. It inhibited spore germination at a concentration of 3iL/mL. The effect of thymus oil was also very much comparable to these results (no significant difference at P>0.05). Finally, it was concluded that essential oil extracts of cinnamon and thymus can be a useful source of antifungal agents for protection of grain spoilage by fungi.

Antifungal activity of fungicides and plant extracts against yellow sigatoka disease causing Mycosphaerella musicola

Mycosphaerella musicola causes yellow sigatoka disease in banana plantations, and affects overall yield and quality of the fruit. Synthetic fungicides are used to control disease. An integrated approach by using plant based extracts and synthetic fungicides can control the disease efficiently by reducing the usage of fungicides. In vitro studies were carried out to test methanolic extracts of ten plants belonging to seven different families having previous reports on antimicrobial activity against Mycosphaerella musicola, methanolic extracts of two plants showed significant antifungal activity as well as significant inhibition of spore germination in spore germination inhibition assay. All eight fungicides exhibited inhibitory action on M. musicola in poison food technique, where as MIC range of each fungicide varied significantly. The results revealed that integrated disease management by using efficient plant extracts and effective fungicides can control the disease and the pathogen in fields efficiently.

English

A Streptomyces sp. strain named 5K10, isolated from soil samples of Kaziranga National Park, Assam, India was found to secrete an antimicrobial proteinaceous compound, showing in vitro antagonistic effect against some pathogenic bacteria, yeasts, dermatophytes and filamentous fungi. Capacity to produce extracellular antimicrobial substances by the strain 5K10 was assessed by conventional spot inoculation method in agar media against test microorganisms as well as fungal spore germination inhibition assay by microscopic observations. The fermented culture broth with ammonium sulfate precipitation revealed a biologically active proteinaceous compound and molecular weight estimated to be about 14.3 kDa on SDS-PAGE gel. Using Candida albicans MTCC 227 as the indicator strain, antagonistic activity of the proteinaceous compound in the region of the SDS-PAGE gel was confirmed. Bioactivity of the extracellular protein was sensitive to proteinases and partially susceptible to prolonged heat treatment. The results suggest that Streptomyces sp. 5K10 might be an important bioresource of lead molecules for developing antimicrobial agents to control bacterial and fungal infections.   Key words: Antifungal activity, extracellular protein, dermatophytes, Streptomyces.

Safety and Efficacy of Green Foliage in the Treatment of Fungal Infections and Associated Clinical Conditions

Forty five crude extracts of nine selected medicinal plants, based on their use in respiratory and other disorders in traditional systems of medicine were analyzed for their potential activity against three pathogenic species of Aspergillus. The presence of phenols, tannins, flavanoids, terpenoid, steroids, alkaloids and saponins in the different extracts was established. The crude extracts were examined for antifungal activity in concentration ranging from 5000.0 to 19.53 µg/ml using microbroth dilution assay in which twenty two extracts exhibited the anti-Aspergilli activity. The petroleum ether extract of Justicia adhatoda and water extract of Commelina bengalensis exhibited the maximum activity against Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. Their in vitro minimum inhibitory concentrations (MICs) were found to be 156.0-312.0 µg/ml by microbroth dilution and spore germination inhibition assays. In disc diffusion assay, at concentration of 10 µg/disc of some crude extracts showed significant activity against Aspergilli. The toxicity (in vitro and in vivo) of bio-active fractions was evaluated, in which the extracts isolated from J. adhatoda were found to be non-toxic. Gas chromatography-mass spectrometry (GCMS) studies were performed for various extracts (petroleum ether, chloroform and acetone) of J. adhatoda which resulted in the identification of several bioactive compounds. The antifungal activity along with acute toxicity, cyto-toxicity as well as genotoxicity of extract fractions from J. adhatoda justifies the use of such screening in the expedition for new drugs.

Inhibition of conidiophore development in Aspergillus fumigatus by an Escherichia coli DH5α strain, a promising antifungal candidate against aspergillosis

The opportunistic human pathogen Aspergillus fumigatus produces a massive number of asexual spores (conidia) as the primary means of dispersal, survival, genome protection and infection of hosts. In this report, we investigated secretory and cytosolic proteins of non-pathogenic bacterial species (mostly belonging to human microbiome) for antifungal potential against A. fumigatus, A. flavus and A. niger. Our preliminary results revealed that cytosolic proteins of E. coli DH5α were most active and the less toxic against various pathogenic isolates of A. fumigatus (the major pathogenic species), depicting a minimum inhibitory concentration (MIC) of 62.50 μg/mL, 62.50 μg/mL and 12.50 μg/disc using microbroth dilution assay (MDA), percentage spore germination inhibition assay (PSGI) and disc diffusion assay (DDA), respectively. E. coli protein was non-toxic against human erythrocytes at doses up to 1000 μg/mL as compared to standard drug, amphotericin B which lysed 100% of erythrocytes at a concentration of 37.50 μg/mL. Time kill analysis proved it to be fungicidal in a concentration and time-dependent manner. Scanning electron microscopic studies (SEM) were carried out to prevail what kind of damage it causes to A. fumigatus. SEM results reported that conidiophore (structures forming conidia) development was halted as a major consequence, reducing the number of conidiophores to insignificant values as well as alteration in their morphological attributes. This feature may contribute to the development of new prevention strategies against Aspergillus infections. Hyphal atrophy was also observed, evidenced by shrinking and flattening of hyphal walls and reduced, abrupt hyphal branching. Such actions may effectively reduce the invasive ability of Aspergillus as well as it can sterilize the fungal burden by obstructing the conidiation pathway of A. fumigatus. Hence, E. coli DH5α, being a commensal species, can lead to the development of antifungal molecule with novel targets in fungal metabolism, which will help in combating the antifungal resistance and toxicity associated with current therapy.

Advancement in Infection Control of Opportunistic Pathogen (Aspergillus spp.): Adjunctive Agents

There is continuous emergence of resistant strains which leads to urgent need to discover new antifungal agents. The investigation of adjunctive agents for antifungal activity might help to optimize the therapy for Invasive Aspergillosis (IA). The chelating agents Ethylenediamine Tetraacetic Acid (EDTA) & Disodium salt of EDTA (DiEDTA) as adjunct to antifungal drugs have been investigated against 8 pathogenic isolates of Aspergillus spp. The MIC (Minimum Inhibitory Concentration) found by DDA (Disc Diffusion Assay) is 7.50-15.0 μg/disc; by MDA (Microbroth Dilution Assay) is 30.0-49.13 μg/ml & SGIA (Spore germination Inhibition Assay) is 30.0-49.13 μg/ml. Moreover, these agents did not show any toxicity up to a concentration of 312.5 μg/ml. The antifungal activity is also confirmed by another method i.e time kill curve analysis. While these agents were ten times less active than gold standard drug (Amphotericin B; AmpB) but eight times less toxic than AmpB. This leads to preliminary investigation of in vitro combination of chelating agents with antifungal drugs (Polyenes & Azoles) by DDA. These combinations showed a significant increase in zone of inhibition in contrast to single drug used. This preliminary work with chelating agents suggest that EDTA as an enhancing agent with antifungal properties in combination with antifungal drugs can be used in pharmaceutical preparations. Further investigation is in progress.

A novel antifungal anthraquinone from seeds of Aegle marmelos Correa (family Rutaceae)

A new anthraquinone, 1-methyl-2-(3′-methyl-but-2′-enyloxy)-anthraquinone ( 1) has been isolated from seeds of Aegle marmelos Correa and was characterized on the basis of spectral analysis (UV, IR, 1H NMR, 13C NMR, 2D NMR and mass spectroscopy). The compound exhibited significant antifungal activity against pathogenic strains of Aspergillus species and Candida albicans in disc diffusion assay (MIC value of 6.25 μg/disc), microbroth dilution and percent spore germination inhibition assays (MIC value of 31.25–62.5 μg/ml).

Antifungal Properties of Some Mexican Medicinal Plants

The antifungal properties of some extracts from Artemisia ludoviciana Nutt., Heliopsis longipes 'A. Gray' Blake., Satureja macrostema Benth. and Tagetes lucida Cav. were analyzed, using the agar disc diffusion method. After 72 h incubation, the plant extracts inhibited the growth of fungi, but the ethyl acetate and methanol-chloroform extracts from A. ludoviciana, H. longipes and T. lucida inhibited all the fungi assayed: Candida albicans, Colletotrichum linde- muthianum, Mucor circinelloides, Saccharomyces cerevisiae, and Sporothrix schenckii. The spore germination inhibition assay suggested that methanol-chloroform extract from A. ludoviciana was the most active against the fungi tested. The methanol-chloroform and ethyl acetate extracts from A. ludoviciana and T. lucida had pronounced antifungal activity against fungi tested. Compounds found in the leaves methanol-chloroform extracts from A. ludoviciana were analyzed by using GC/MS; the major compounds were: 1-8-cineole, camphor, borneol, cis-verbenol, myrtenol, eugenol, cariophyllene, � -farnesene, spathulenol, derivatives of caryophyllene and derivatives of spathulenol.

보리 흰가루병 곰팡이 Fusarium graminearum에 대한 산초 추출물의 항진균 활성

The powdery mildew, a fungal plant disease found in varieties of plant cultures, is occurred by attack with Fusarium sp., Sphaerotheca sp., Leveilluna sp., and Eryshipe sp.. In this study we investigated the control of Fusarium graminearum, a barley powdery mildew fungus, by natural plant extracts. Among the 900 plant extracts tested, Zanthoxylum schinifolium, Ligusticum acutilobum, Bidens frondosa L., Dictamnus dasycarpus, Evodia officinalis, Disporum sessile, Scopolia japonica Max., Styrax japonica S. et Z., Dictamnus dasycarpus Turcz., Sinomenium acutum Rehder et Wils., Eugenia aromaticum, Rubus parvifolius L., Reynoutria elliptica, Coptis chinensis, Paeonia lactiflora Pall., Rheum undalatum, Paeonia suffruticosa, Oenothera odorata Jacq., Euphorbia pekinensis Rupr., and Nepeta cataria were selected based on spore germination inhibition assay. Further mycelial growth inhibition assay with economical and safety considerations led us to finally select Z. schinifolium (sancho) for control of F. graminearum. To produce antifungal sancho extract, methanol was suitable for extraction and subsequent fractionations of the extract showed that the water residue mainly had antifungal activity. The sancho extract and its fractions showed minor antibacterial activity against different pathogenic or food spoilage bacteria, but they did not show any harmful effects against young tomato plant by treatment of 1,000 ㎍/ml in green chamber test. These results suggested that the extract of sancho has high potentials on control of a powdery mildew fungus, F. graminearum.

Microwave-assisted synthesis of antimicrobial dihydropyridines and tetrahydropyrimidin-2-ones: Novel compounds against aspergillosis

Ten 4-aryl-1,4-dihydropyridine and three 4-aryl-1,2,3,4-tetrahydropyrimidin-2-one derivatives have been synthesized and examined for their activity against pathogenic strains of Aspergillus fumigatus and Candida albicans. Although none of the three compounds belonging to pyrimidin-2-one series showed any activity against two pathogens, two of the compounds of the dihydropyridine series, that is, diethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate and dimethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate, exhibited significant activity against A. fumigatus in disc diffusion, microbroth dilution and percent spore germination inhibition assays. The most active diethyl dihydropyridine derivative exhibited a MIC value of 2.92 μg/disc in disc diffusion and 15.62 μg/ml in microbroth dilution assays. The MIC 90 value of the most active compound by percent germination inhibition assay was found to be 15.62 μg/ml. The diethyl dicarboxylate derivative of dihydropyridine also exhibited appreciable activity against C. albicans. The in vitro toxicity of the most active diethyl dihydropyridine derivative was evaluated using haemolytic assay, in which the compound was found to be non-toxic to human erythrocytes even at a concentration of 625 μg/ml. The standard drug amphotericin B exhibited 100% lysis of erythrocytes at a concentration almost 16 times less than the safer concentration of the most active dihydropyridine derivative.

Investigations on anti-Aspergillus properties of bacterial products

To investigate the anti-Aspergillus properties of bacterial products. In the present study, 12 bacterial strains were screened for antifungal activity against Aspergilli. The culture supernatant and lysates of Pseudomonas aeruginosa, Bacillus cereus, Escherichia coli (BL21, DH5alpha, HB101, XL Blue), Klebsiella pneumoniae, Streptomyces thermonitrificans, Streptococcus pneumoniae, Enterobacter aerogenes, Staphylococcus aureus and Salmonella typhi were examined for antifungal activity in protein concentration ranging from 1000.0 to 7.8 microg ml-1 using microbroth dilution assay. The lysate of Salm. typhi and E. coli BL21 exhibited the maximum activity against Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. Their in vitro minimum inhibitory concentrations (MICs) were found to be 15.6-31.2 microg ml-1 by microbroth dilution and spore germination inhibition assays. In disc diffusion assay, a concentration of 3.1 microg disc-1 of Salm. typhi lysate showed significant activity against Aspergilli. Escherichia coli BL21 exhibited similar activity at 6.2 microg disc-1. The work on identification of molecule endowed with antimycotic properties is in progress. The products of Salm. typhi and E. coli demonstrated significant activity against Aspergillus species. This is the first time that E. coli has been reported for anti-Aspergillus activity. It could be an important source of biologically active compounds useful for developing better new antifungal drugs/or probiotics.

Studies on antimycotic properties of Datura metel

The hexane, chloroform, acetone and methanolic fractions of Datura metel L. were investigated for antifungal properties using pathogenic species of Aspergillus. The chloroform fraction was found to be endowed with antifungal activity. The minimum inhibitory concentration (MIC) of chloroform fraction of D. metel L. was 625.0 μg ml −1 against all the three species of Aspergillus, i.e. A. fumigatus, A. flavus and A. niger, by microbroth dilution and percent spore germination inhibition assays . The MIC by disc diffusion assay was observed to be 12.5 μg disc −1. The chloroform fraction of D. metel, when investigated for potency, turned to be 9.2 times less active than amphotericin B. However, it was important to note that the cytotoxicity of chloroform fraction in vitro was 117.8 times less than amphotericin B.