Crude Metabolites Research Articles

Crude Cell-Free Extract From Deinococcus radiodurans Exhibit Anticancer Activity by Inducing Apoptosis in Triple-Negative Breast Cancer Cells

Extremophilic organisms have the potential to tolerate extremely challenging environments of nature. This property can be accredited to its production of novel secondary metabolites that possess anticancer and other pharmaceutical values. The present study was aimed to investigate the anticancer activity of crude secondary metabolite extract (CSME) obtained from the radiation-tolerant bacterium Deinococcus radiodurans in triple-negative human breast carcinoma (MDA-MB-231) cells. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay showed the antiproliferative potential of CSME in MDA-MB-231 cells (IC50 = 25 μg/ml) and MCF-7 cells (IC50 = 10 μg/ml). Further, the CSME treatment led to the production of intracellular reactive oxygen species (ROS) and nuclear membrane alterations with the formation of apoptotic bodies in MDA-MB-231 cells. Considerable DNA damage and low antioxidant status were observed in CSME-treated MDA-MB-231 cells. The results also showed that the CSME treatment induced apoptotic markers expression in MDA-MB-231 cells. Western blot results illustrated significant upregulation of p53, caspase-3, and caspase-9 proteins expression. Then, we analyzed the presence of secondary metabolites which may be linked with antiproliferative potential of CSME by gas chromatography-mass spectrometry (GC-MS). The results illustrated the presence of 23 bioactive compounds some of which are already reported to possess anticancer properties. The study indicates that the CSME of D. radiodurans possess anticancer properties and exhibit the potential to be used as an anticancer agent.

The Missing Link: Developing a pipeline for accelerated antibiotic discovery from Streptomyces through linking ‘omics data

The genus Streptomyces has proven to be a rich reservoir of specialized metabolites, accounting for 80% of all microbially produced antibiotics including chloramphenicol and nystatin from S. venezuelae and S. noursei respectively. However, the discovery of novel microbial chemistry is still greatly needed to combat antimicrobial resistance. Comparative metabolomics, using platforms such as Global Natural Products Social Molecular Networking (GNPS), as well as tools such as antiSMASH and BiGSCAPE have aided the mining of biosynthetic gene clusters (BGC’s) across datasets but comparing the chemistry to the encoding biosynthetic gene clusters is a significant bottleneck. In this study, ten Streptomyces strains were selected, based on phylogeny and availability of genome sequence. The strains were cultured on 6 types of Actinomycete-specific media to maximise metabolite diversity. Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) was used to obtain spectral data from crude metabolite extracts enabling comparative metabolomics analysis via the GNPS platform. As the genome sequences were publicly available, genome mining of BGC’s was achieved using antiSMASH resulting in 260 BGC’s across the ten strains. This revealed 53 gene cluster families when analysed using BiGSCAPE, the largest encoding for 8 metabolites. In future, both biosynthetic (BGC’s) and chemistry (parent ions) datasets will be computationally linked based on strain presence/absence. The development of standardised datasets that enable cross-‘omics comparison will aid prioritisation of novel antibiotics, especially when combined with bioactivity data.

Evaluate the Effects of Entomopathogenic Fungi Isolates on Wheat Aphid, Schizaphis graminum (Rondani) (Hemiptera: Aphididae)

Four native isolates of entomopathogenic fungi (EPF) Beauveria bassiana (B1and B2), Metarhizium anisopliae (M1 and M2 ) were isolated and evaluated against wheat aphid, Schizaphis graminum (Rondani). Three concentrations of spores suspension from each of the four fungal isolates (1×106, 1×107 and 1×108conidia) were used against adult aphid (one day old). The results showed that (B1) was the most effective according to LC50. Whereas, when testing the toxicity of crude metabolites of fungal isolates in four concentrations for each isolate, it was found that the highest effective toxin was (M1) followed by (B1), (M2) and (B2). Scanning electron microscope photographs (SEM) showed the mode of action of entomopathogenic fungi and its ability to colonize and how it infects aphid.

Isolation, purification and structural elucidation of secondary metabolites from Microcystis aeruginosa bloom from Muttukadu estuary and its in vitro antibacterial, antioxidant and anticancer potency

The biomedical applications of the secondary metabolites produced by the harmful algal blooms were studied over a few decades ago. Only few research studies have been reported on the medicinal properties of the secondary metabolites produced by the cyanobacteria Microcystis aeruginosa. The present study was aimed to screen the Biological activities of GC fractionated Benzeneacetanomide and Norvaline, n-propargyloxycarbonyl mixtures of Microcystis aeruginosa secondary metabolites. The Microcystis aeruginosa samples were collected by plankton net from the blooming area at Muttukadu. The metabolites were extracted by using polar solvent as methanol. The yield percentage of Microcystis aeruginosa methanolic extract showed moderate level of 41% from the dry weight of the pre extracted Microcystis aeruginosa powder. The crude metabolites were purified through column chromatography and GC-MS analysis. The GC-MS results showed the presence of two compounds (2 major peaks), Benzeneacetamide at the retention time (RT) of 7.315 min and l-Norvaline, n-propargyloxycarbonyl (Pyrrol derivaties) at the retention time (RT) of 12.541 min. The GC-MS fractioned compounds were characterized by FT-IR and 1HNMR. The anticancer, antioxidant and antimicrobial activity of the compounds were studied. This study highlights the bioactive potential of Microcystis aeruginosa secondary metabolites that might lead to the development of cancer drug.

Acute Oral Toxicity Studies of the Crude Extract of Endophytic Fungi Isolated from Annona senegalensis Pers

Establishment of safety and toxicity profiles of metabolites of endophytic organisms from known medicinal plants are crucial in their pharmacological and biological applications. The aim of this study was to evaluate the oral acute toxicity (LD50) of crude extract of endophytic fungi isolated from Annona senegalensis Pers. The endophytic fungal metabolite was extracted with ethyl acetate. The LD50 was estimated following the method described by Lorke. Three dose levels (10, 100, and 1000 mg/kg) of the crude extract were administered to three mice each for the first phase using oral gavage needle in a single dose disposable syringe. The animals were observed for possible deaths or other side effects of the test substance in each group within 24 hours of the treatment. In the second phase, which was deduced from the first phase, eight mice were sub-divided into four groups of two mice each and they were treated with doses of 1200, 1600, 2900 and 5000 mg/kg orally. They were also observed within 24 hours and final LD50 value was determined. Results showed that the endophytic fungal extract exhibited no mortality or any histological defect in the liver tissues of the mice. More so, the immunological parameter tested showed significant increase in neutrophils and lymphocytes relative to the control in all the fungal isolates. Additionally, the LD50 for the crude metabolites was > 5000 mg/kg. This study has revealed that crude extract of endophytic fungi isolated from Annona senegalensis Pers did not show oral acute toxicity in mice. Further studies will evaluate long term-toxicity of the crude extract.
 Keywords: Endophytes, LD50, Annona senegalensis, Metabolites, Fungi

Antimicrobial and Antioxidant Effects of a Forest Actinobacterium V002 as New Producer of Spectinabilin, Undecylprodigiosin and Metacycloprodigiosin.

The aim of the study is the research and identification of a Streptomyces strain as a new producer of spectinabilin, undecylprodigiosin and metacycloprodigiosin. Among 54 actinomycete isolates isolated from El-Ogbane forest soils in Algeria, only one isolate, designated V002, was selected for its ability to produce prodigiosins. The selected strain was analysed for its ability to produce three different secondary metabolites as well as their biological activities. V002 belongs to the Streptomyces genus and has significant antimicrobial and antioxidant activities. The taxonomic position of V002 by 16S rRNA sequence analysis showed a similarity of 99.93% with Streptomyces lasiicapitis DSM 103124T and 98.96% with Streptomyces spectabilis DSM 40512T. Fractionation of crude secondary metabolites produced by the strain using HPLC-MS revealed the presence of spectinabilin, undecylprodigiosin and metacycloprodigiosin, which demonstrated significant activity. Strain V002 is considered a new producer of spectinabilin, undecylprodigiosin and metacycloprodigiosin with significant antimicrobial and antioxidant activity.

In Silico and In Vitro Analyses of Glucosamine and Indole Acetaldehyde Inhibit Pathogenic Regulator Gene phcA of Ralstonia solanacearum, a Causative Agent of Bacterial Wilt of Tomato.

In this study, medicinal plant (Solanum surattense)-associated bacteria were isolated and their extracellular secondary metabolites were extracted. Dual-plate application of crude secondary metabolites proved that SSL2I and SSL5 had a good inhibitory activity against Ralstonia solanacearum. These biocontrol bacteria were identified as Bacillus subtilis and Bacillus velezensis by 16S rRNA gene sequencing analysis. The crude extracts of secondary metabolites were identified based on high-resolution liquid chromatography/mass spectrometry (HR-LCMS) analysis. On the basis of HR-LCMS analysis, we selected the compounds such as glucosamine and indole acetaldehyde for in silico analysis and inhibition of pathogenic gene of phcA from R. solanacearum. The specificity of identified pathogenic gene of R. solanacearum and its cytoplasmic localization were identified by BLASTP and PSORTB bioinformatics tools. The protein-protein interaction between the identified secondary metabolites and pathogenic gene revealed that the compound had antagonistic potential against pathogenic gene of phcA. Furthermore, the synthetic forms of the above metabolites showed that both the compounds had the ability to inhibit R. solanacearum under in vitro condition. On the basis of in silico and in vitro analyses, it was concluded that medicinal plant-associated Bacillus spp. could be used as a biocontrol agent in managing wilt disease caused by R. solanacearum.

Metabolites of the rust mycoparasite, Sphaerellopsis paraphysata for management of rust in pearl millet

The mycoparasite, Sphaerellopsis paraphysata was isolated from the uredospores of pearl millet rust pathogen Puccinia substriata with promising natural parasitic association. The culture had definite margin, sometimes submerged in media and sporulation occurred on the third week of incubation. The mycelium was hyaline and septate. The secondary metabolites were explored to contain the uredospore production thereby reduce the rust incidence. The solvent extraction method was used to extract the secondary metabolites produced from crude extracts of S. paraphysata . The fractions of the secondary metabolites were analyzed using GC-MS. The major components present were Phenol, Decane, 3-Butoxy-1,1,1,5,5,5, hexamethyl-3-(trimethylsiloxy) trisiloxane, Butanoic acid, 3-methyl-3-methyl butyl ester, which were reported to have anti-microbial, anti-fungal and anti-oxidant properties. The crude metabolite of S. paraphysata in the dilution of 1:5 recorded the maximum lysis of the uredospores of P. substriata (92.67%). Further, SEM study showed severely lysed broken cell wall which led to leakage of cellular contents. The spraying of secondary metabolites on rust infected pearl millet crop reduced the incidence of rust. The present study paved the way for the development of new biologically originated molecule for the management of pearl millet rust disease.

Biocontrol of chocolate spot disease (Botrytis cinerea) in faba bean using endophytic actinomycetes Streptomyces: a field study to compare application techniques.

Sustainable agriculture is needing economic applications for disease control. One possibility is offered by local medical plants. Endophytes of medical plants, such as actinomycetes Streptomyces sp. have previously shown antagonistic activities against fungal phytopathogens. In the present field experiment, we aimed to verify the efficiency of endophytic Streptomyces against one of the common pathogens, Botrytis cinerea, causing chocolate spot disease for faba bean (Vicia fabae L.). We tested two strains of Streptomyces (MG788011, MG788012) and three techniques to apply the biocontrol agent: (1) coating the seeds with spores, (2) spraying mycelia and (3) spraying the crude metabolites over the plants. The technique using the crude metabolites was the most efficient to prevent the disease symptoms. Both of the endophytic strains diminished the disease symptoms and improved the plant growth. The study offers a potential biological control technique to prevent chocolate spot disease and, at the same time, increase the yields of faba bean in sustainable agriculture.

In Vitro Antimicrobial Activity of Piper retrofractum Fruit Extracts against Microbial Pathogens Causing Infections in Human and Animals.

Long pepper (Piper retrofractum Vahl) is a Thai medicinal herb which has been used as one of the common ingredients in variety of Thai foods. Here, we investigated antimicrobial activities of crude bioactive metabolites extracted from fruits of P. retrofractum against 10 pathogenic organisms (bacteria and yeast) causing opportunistic infections in human or animals including Bacillus subtilis ATCC6633, Staphylococcus aureus ATCC25923, Enterococcus faecalis ATCC2921, Escherichia coli ATCC25922, Klebsiella pneumonia TISTR1843, Pseudomonas aeruginosa ATCC741, Salmonella typhi (clinical isolate), Vibrio parahaemolyticus (XN98 and 5HP), and Candida albicans ATCC90020. The results of disk diffusion test showed that the extract from methanol solvent exhibited greater antibacterial activity than other solvents with inhibition zones ranging from 0.5 to 8.0 mm, respectively. Subsequently, minimal inhibition concentration (MIC) determined by the colorimetric assay confirmed that methanol extracts showed consistent results with disk diffusion method. In summary, in vitro assays suggest that methanol is the best solvent for extraction of bioactive metabolites from P. retrofractum fruits. This crude extract can inhibit the majority of human and animal pathogens. This opens up a potential use of pepper fruits in prevention of food-contaminating microorganisms.

AntiMycobacterial activity of endophytic actinobacteria from selected medicinal plants

Background: Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), poses a serious threat to human life. In addition, certain nontuberculous mycobacterial (NTM) species also cause infections in immune suppressed and even in immunocompetent individuals. With this view, the present study investigated the antimycobacterial properties of endophytic actinobacteria using Mycobacterium smegmatis as a surrogate model. Methods: Endophytic actinobacterial cultures were isolated from seven medicinal plants and their antimicrobial activity against two Gram-positive bacteria, three Gram-negative bacteria, fungi, and M. smegmatis was studied. Crude bioactive metabolites from one potential culture EAM4 were tested against Staphylococcus aureus, Escherichia coli, and M. smegmatis by disc-diffusion method, whereas activity against MTB H37Rv was tested by luciferase reporter phage (LRP) assay. The potential actinobacterial culture EAM4 was identified based on their 16S rRNA analysis. Results: Thirty actinobacterial cultures were isolated and were belonged to the genus Streptomyces. Maximum number of actinobacterial strains was isolated from Selaginella stellata (36.6%) and Pilea microphylla (36.6%). Gram-positive pathogens are inhibited by more number of actinobacterial cultures (>90%) than Gram-negative pathogens (50%). Twenty-eight of thirty actinobacterial cultures showed inhibitory activity against M. smegmatis. Bioactive metabolites from the potential culture EAM4 showed better inhibition against the S. aureus, E. coli, and M. smegmatis. In the LRP assay, 79% inhibition was exhibited against the MTB H37Rv. The potential actinobacterial strain EAM4 was identified as Streptomyces sp. based on their 16S rRNA analysis. Conclusion: Findings of the present study revealed that the endophytic cultures of Streptomyces investigated in this study are the promising source for isolating antimycobacterial metabolites effective against TB and nontuberculous mycobacterial pathogens.

Identification and Characterization of Antagonism Band of Secondary Metabolite from T. asperellum MK045610 against F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici based on HPTLC and GC-MS

The present investigation was carried out on identification and characterization of an antagonism band of extract secondary metabolites from Trichoderma asperellum MK045610 against Fusarium oxysporum f. sp. ciceri and Fusarium oxysporum f. sp. lycopersici. In this experiment, analysis was performed by High Performance Thin Layer Chromatography (HPTLC), paper disc assay, gas chromatography-Mass spectrometry (GC-MS). Firstly, extract crude secondary metabolites were used for partial purification based on HPTLC. Secondly, the paper disc assay method was used for the determination of antifungal property on PDA plate from the partial purified compound. Thirdly, GC-MS was used for identification of partial purified compound based on peaks. Identified compounds are named as Phenol, 3, 5-bis (1,1-dimethylethyl), Pentadecanoic Acid, 14-methyl, methyl ester, Benzenepropanoic acid, 3,5,-bis (1,1-dimethyl ethyl)-4-hydroxy-methyl ester and represented to antifungal property . Conclusively, Secondary metabolite of Trichoderma asperellum MK045610 has a significant role in radial growth inhibition of Fusarium oxysporum f. sp. ciceri and Fusarium oxysporum f. sp. lycopersici.

LC–MS and docking profiling reveals potential difference between the pure and crude fucoidan metabolites

Fucoidan, a macromolecule, a type of polysaccharide and contains high percentage of fucose and sulfate ester groups and other compounds in low percentage. In the last decades, a number of interesting biological activities have been studied so fucoidan has been major focus of interest for exploring new drugs against the leading human diseases. It is known in relation to biological pathways and function that it activates the pathways which mediate apoptosis. Using LC–MS and interdisciplincary approach, we developed our goal to elucidate the potential difference between the crude and pure fucoidan and the interaction of fucoidan with potential signaling molecules STAT4 and IL12RB2. Based on our analysis, we conclude that there is major difference between crude and pure fucoidan in terms of identified metabolites and their functional relevance. It leads to the conclusion that there are 18 functions which are common between crude and pure fucoidans, 47 functions pure fucoidan specific, and 84 functions crude fucoidan specific which leads to the conclusion that even the fucoidans obtained from the same organism behave functionally completely different between the processing states (with/without processing) while docking study with STAT4 and IL12RB2 display very strong binding between fucoidan and both these signaling molecules.

SCREENING OF ANTIMICROBIAL POTENTIAL OF NATURALLY OCCURRING FRESHWATER FILAMENTOUS GREEN ALGAE CLADOPHORA GLOMERATA COLLECTED FROM BIRBHUM DISTRICT, WEST BENGAL, INDIA.

Microalgae have been recognized in the last several years as source of novel anti-viral, anti-algal, antibacterial and antifungal metabolites. Cladophora glomerata is a branched filamentous green algae commonly found in freshwater bodies. The aim of the present work is to study the antimicrobial screening of the cell extracts of naturally occurring freshwater green algae Cladophora glomerata against clinically significant microorganisms both Gram positive and Gram negative bacteria and pathogenic fungi. In this study four test pathogens were considered, these test bacterial strains were Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus and fungal strain was Candida krusei. Crude metabolites were extracted using four different solvents namely chloroform, methanol, butanol and petroleum benzene. The antimicrobial activities of crude extracts were evaluated using agar cup diffusion methods. Among the solvents highest antimicrobial activity was detected in methanol extracts against both Gram positive and Gram negative bacteria, i.e. Bacillus subtilis (19.66±0.57 mm) followed by Pseudomonas aeruginosa (15.66±0.57 mm) and Staphylococcus aureus (15.33±0.57 mm) respectively. Moderate activity were detected in chloroform extract against Bacillus subtilis (14.00±0.57 mm) and Pseudomonas aeruginosa (13.33±1.00 mm) respectively. The less activity was detected in petroleum benzene extract against Staphylococcus aureus (10.66±1.00 mm) and no activity was found against fungal pathogen Candida krusei. The present finding that may serve as leads for the development of new pharmaceuticals addressing the novel therapeutic needs of mankind. It is concluded that the branched filamentous green algae Cladophora glomerata occurring in natural freshwater bodies represents a new source of antimicrobial formulation with stable and biologically active compounds. Keywords: Cladophora glomerata, Freshwater bodies, Solvent extracts, Antimicrobial potential.

Chromatographic Detection of Antibacterial Antibiotics from Pseudomonas fluorescens (TNAU PF1) and Their Effect on Bacterial Wilt Pathogen Ralstonia solanacearum (KK2)

Bacterial wilt of tomato is a devastating disease incited by Ralstonia solanacearum (KK2) causing heavy yield loss in India. The antagonist Pseudomonas fluorescens (TNAU PF1) was characterized for antibacterial ability against the bacterial wilt pathogen. Thin-layer chromatography (TLC) identified six distinct antibiotics in crude metabolite of TNAU PF1 strain. The crude antibiotics and the antibiotics 2,4-diacetylphloroglucinol and Phenazine of TNAU PF1 showed best in vitro antibacterial activity against the bacterial wilt pathogen R. solanacearum. The ability of crude antibiotics in inhibiting Ralstonia colonies was higher than the individual antibiotics (2,4-diacetylphloroglucinol and Phenazine) under in vitro. Further, GC-MS analysis of the semi-purified crude extract of Pseudomonas fluorescens (TNAU PF1) identified six antibacterial compounds viz., Phloroglucinol dimethyl ether, Patulin, Hemipyocyanin, Phthalic acid, butyl 2-pentyl ester, Phenazine and 1-Phenazinecarboxylic acid that would be synergistically inhibited the colony multiplication of the pathogen. The study emphasized the antagonistic activity of the Pseudomonas fluorescens (TNAU PF1) for the management of bacterial wilt disease.

Antifungal Activity of Methanol-Extracted Secondary Metabolites of Rhizobacteria Isolated from Rhizosphere of Oil Palm Trees Against Ganoderma Boninense Pat.

Several rhizobacteria were isolated from the rhizosphere of healthy oil palm trees showed prospective ability as biocontrol agent against pathogenic fungi Ganoderma boninense Pat. The fungus is the causal agent of basal stem rot (BSR) disease can cause yield reduction up to 80%. Therefore, searching for effective compound to control the disease is a continuous process. Rhizosphere bacteria isolates were grown on ISP2 agar media and incubated for 14 days at room temperature. The media with fully grown cultures were cut into cubes and added with methanol. It was shaken and the bacterial cells were then removed through centrifugation and series of filtration. The final filtration was conducted using 0.2 µm to sterilize the filtrate. The filtrates were subjected for antifungal activity against G. boninense using agar well diffusion. The result showed that the crude metabolites produced by the rhizosphere bacteria demonstrated the ability to inhibit the growth of G. boninense in the agar diffusion method. The highest inhibition reached 51.63%. Furthermore, G. boninense mycelia were also experienced malformation by the presence of the secondary metabolites. The isolated rhizosphere bacteria showed promising ability to produce antifungal compounds which were able to inhibit the growth of G. boninense as well as causing the morphological changes of its mycelia.

Prevalence of antibiotic resistant mastitis pathogens in dairy cows in Egypt and potential biological control agents produced from plant endophytic actinobacteria.

Dairy production is threatened by antibiotic resistant pathogens worldwide, and alternative solutions to treat mastitis are not available. The prevalence of antibiotic resistant strains is not well known in less developed countries. The prevalence of pathogenic bacteria and their resistance to 21 commercial antibiotics were studied in milk samples taken from 122 dairy cows suffering from the symptoms of mastitis in Egypt. The bacterial species were identified with molecular methods, and antibiotic resistance was studied with disc diffusion method. The prevalence of Streptococcus aureus, Escherichia coli and Pseudomonas aeruginosa were 30%, 17% and 3.5%, respectively. Most (90%) of the S. aureus strains showed resistance to penicillin whereas only 10% of the strains were resistant to oxacillin. Nearly half (40%) of E. coli strains showed resistance to streptomycin. Six P. aeruginosa strains showed resistance to several antibiotics, including ceftriaxone, enrofloxacin and levofloxacin. This points out that despite P. aeruginosa was not common, it should be followed up carefully. Potential biocontrol agents against antibiotic resistant mastitis bacteria were searched among 30 endophytic actinobacterial strains derived from wild medicinal plants. Three plants, namely Mentha longifolia, Malva parviflora and Pulicaria undulata were chosen for a more detailed study; their endophytic actinobacteria were used to prepare metabolic extracts. The crude metabolites of the actinobacteria were extracted with ethyl acetate. All metabolic extracts inhibited the growth of S. aureus, methicillin-resistant Staphylococcus aureus (MRSA), E. coli and P. aeruginosa in vitro. The 16S rRNA sequence analysis revealed that the most efficient actinobacterial strains were two Micromonospora sp. and one Actinobacteria bacterium. We conclude that the combination of the metabolites of several endophytic actinobacteria derived from several medicinal plants would be the most efficient against pathogens. Different metabolite cocktails should be studied further in order to develop novel biocontrol agents to treat antibiotic resistant mastitis bacteria in dairy cows.

Mosquito control potential of secondary metabolites isolated from Aspergillus flavus and Aspergillus fumigatus

Repeated use of same or different types of chemical pesticide causes environmental pollution and results in resistance in mosquitoes. Currently vector control programs need alternative tools from biological source. Plant and fungi crude metabolites maybe jointly or independently prove its efficacy on mosquitoes. In the present study, we tested the Aspergillus flavus and Aspergillus fumigatus crude metabolites on larvae of Anopheles stephensi, Culex quinquefasciatus, and Aedes aegypti, after 24 h and 48 h exposure in laboratory condition. Crude metabolites were characterized using GC-MS analysis. Our results clearly, shows that lower LC50 and LC90 value were observed in A. flavus on A. aegypti was 18.018 mg/l, 31.053 mg/l for 24 h exposure whereas, A. aegypti had 39.687 mg/l, 82.263 mg/l 48 h exposure respectively. Also lower LC50 and LC90 values in A. fumigatus on C. quinquefasciatus were, 11.436 mg/l and 28.309 mg/l, for 24 h exposure, A. fumigatus crude metabolites are highly toxic to mosquito larvae than A. flavus crude metabolites. Fungi cultures were confirmed as A. flavus and A. fumigatus by using 18S rDNA sequencing. Present study concluded that fungi derived secondary metabolites is eco-friendly in nature, cheaper, effective, target-specific and lower toxicity to non-target organisms.

Identification and Characterization of Antagonism Band of Secondary Metabolite from T. asperellum MK045610 against F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici based on HPTLC and GC-MS

The present investigation was carried out on identification and characterization of an antagonism band of extract secondary metabolites from Trichoderma asperellum MK045610 against Fusarium oxysporum f. sp. ciceri and Fusarium oxysporum f. sp. lycopersici. In this experiment, analysis was performed by High Performance Thin Layer Chromatography (HPTLC), paper disc assay, gas chromatography-Mass spectrometry (GC-MS). Firstly, extract crude secondary metabolites were used for partial purification based on HPTLC. Secondly, the paper disc assay method was used for the determination of antifungal property on PDA plate from the partial purified compound. Thirdly, GC-MS was used for identification of partial purified compound based on peaks. Identified compounds are named as Phenol, 3, 5-bis (1,1-dimethylethyl), Pentadecanoic Acid, 14-methyl, methyl ester, Benzenepropanoic acid, 3,5,-bis (1,1-dimethyl ethyl)-4-hydroxy-methyl ester and represented to antifungal property. Conclusively, Secondary metabolite of Trichoderma asperellum MK045610 has a significant role in radial growth inhibition of Fusarium oxysporum f. sp. ciceri and Fusarium oxysporum f. sp. lycopersici.

Characterization of secondary metabolites produced during interaction of Pseudomonas fluorescens with Fusarium oxysporum

Characterization of the Secondary Metabolites (SM) produced by Pseudomonas fluorescens (NAIMCC-B-00361) while growing in mixed culture conditions with fungal pathogen Fusarium oxysporum (NAIMCC-F-00811) was done to better mimic antagonism and interaction in a natural environment. Nutrient media, viz. King’s B Broth (KBB), Potato Dextrose Broth (PDB), Pigment Production Medium (PPM), Czapek Dox broth Medium (CDB) and half minimal media (50% KBB and 50% PDB) were assessed and KBB was found to be the best medium for the production of these metabolites. Antifungal assay of crude metabolite extract was done using poison food technique and the results showed that the crude chloroform extract of metabolites (mixed culture of P. fluorescens and F. oxysporum) grown in KBB medium showed 50.47% inhibition of mycelial growth of F. oxysporum followed by the chloroform extract of solo culture of P. fluorescens which showed 45.38% inhibition of mycelial growth of Bipolaris oryzae. Antibacterial assay of crude metabolite extract was done using Agar well diffusion technique and the results revealed that the crude chloroform extract of mixed culture of P. fluorescens and F. oxysporum grown in KBB medium showed 25.67 mm zone of inhibition against Bacillus subtilis, whereas the extract of solo culture of P. fluorescens showed 18.67 mm zone of inhibition against Klebsella pneumoniae. The qualitative confirmation of antibiotic production by solo culture of P. fluorescens and mixed culture using TLC revealed the presence of antibiotics, i.e. 2, 4-DAPG, pyrrolnitrin and phenazine. HPLC analysis of crude chloroform extract of SM produced by solo culture of P. fluorescens and mixed culture showed the presence of 2,4-DAPG at retention time 20.707 and 20.698 respectively.