Recent advances in the use of Trichoderma-containing multicomponent microbial inoculants for pathogen control and plant growth promotion.

Chemical pesticides and fertilizers are used in agricultural production worldwide to prevent damage from plant pathogenic microorganisms, insects, and nematodes, to minimize crop losses and to preserve crop quality. However, the use of chemical pesticides and fertilizers can severely pollute soil, water, and air, posing risks to the environment and human health. Consequently, developing new, alternative, environment-friendly microbial soil treatment interventions for plant protection and crop yield increase has become indispensable. Members of the filamentous fungal genus Trichoderma (Ascomycota, Sordariomycetes, Hypocreales) have long been known as efficient antagonists of plant pathogenic microorganisms based on various beneficial traits and abilities of these fungi. This minireview aims to discuss the advances in the field of Trichoderma-containing multicomponent microbiological inoculants based on recent experimental updates. Trichoderma strains can be combined with each other, with other fungi and/or with beneficial bacteria. The development and field performance of such inoculants will be addressed, focusing on the complementarity, synergy, and compatibility of their microbial components.

Identifications of Surfactin-Type Biosurfactants Produced by Bacillus Species Isolated from Rhizosphere of Vegetables.

Surfactins are cyclic lipopeptides consisting of a ß-hydroxy fatty acid of variable chain length and a peptide ring of seven amino acids linked together by a lactone bridge, forming the cyclic structure of the peptide chain. These compounds are produced mainly by Bacillus species and are well regarded for their antibacterial, antifungal, and antiviral activities. For their surfactin production profiling, several Bacillus strains isolated from vegetable rhizospheres were identified by their fatty acid methyl ester profiles and were tested against phytopathogen bacteria and fungi. The isolates showed significant inhibition against of E. amylovora, X. campestris, B. cinerea, and F. culmorum and caused moderate effects on P. syringae, E. carotovora, A. tumefaciens, F. graminearum, F. solani, and C. gloeosporioides. Then, an HPLC-HESI-MS/MS method was applied to simultaneously carry out the quantitative and in-depth qualitative characterisations on the extracted ferment broths. More than half of the examined Bacillus strains produced surfactin, and the MS/MS spectra analyses of their sodiated precursor ions revealed a total of 29 surfactin variants and homologues, some of them with an extremely large number of peaks with different retention times, suggesting a large number of variations in the branching of their fatty acid chains.

Response of the mushroom pathogen Cladobotryum mycophilum to prochloraz and metrafenone fungicides and Streptomyces flavovirens actinobacteria.

After an outbreak of cobweb disease of cultivated button mushroom in Serbia in 2003, the isolated fungal pathogen was initially identified as Cladobotryum dendroides (teleomorph Hypomyces rosellus) based on morpho-physiological traits. Molecular analysis indicated re-classification of two strains (isolated in 2004 and 2007) as Cladobotryum mycophilum (teleomorph Hypomyces odoratus). However, subsequent analysis of further five strains (isolated over the period 2003-2010) within the frames of the present study, also confirmed their identification as the exclusive cobweb causal agent C. mycophilum. After artificial inoculation, the symptoms observed on harvested and growing mushrooms were consistent with the appearance of cobweb disease. Pathogen sensitivity to fungicides was estimated by probit analyses. Fungicide susceptibility tests showed that C. mycophilum strains were highly sensitive both to prochloraz (ED50<0.087 µg mL-1) and the newly introduced metrafenone (ED50<0.15 µg mL-1). Furthermore, the growth of all examined strains of C. mycophilum was significantly inhibited by the indigenous actinobacterial strain Streptomyces flavovirens A06. A dual culture assay showed after 72 h that the percentage of radial growth inhibition of the pathogen ranged from 22.38 to 55.73%. Our findings suggest that the antagonistic S. flavovirens A06 might be a potential candidate for controlling the cobweb disease of cultivated button mushroom.

Correction: Tyagi, C., et al. Accelerated Molecular Dynamics Applied to the Peptaibol Folding Problem. International Journal of Molecular Sciences, 2019, 20, 4268.

The authors wish to make the following corrections to this paper [...].

Symbiotic NCR Peptide Fragments Affect the Viability, Morphology and Biofilm Formation of Candida Species.

The increasing rate of fungal infections causes global problems not only in human healthcare but agriculture as well. To combat fungal pathogens limited numbers of antifungal agents are available therefore alternative drugs are needed. Antimicrobial peptides are potent candidates because of their broad activity spectrum and their diverse mode of actions. The model legume Medicago truncatula produces >700 nodule specific cysteine-rich (NCR) peptides in symbiosis and many of them have in vitro antimicrobial activities without considerable toxicity on human cells. In this work we demonstrate the anticandidal activity of the NCR335 and NCR169 peptide derivatives against five Candida species by using the micro-dilution method, measuring inhibition of biofilm formation with the XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay, and assessing the morphological change of dimorphic Candida species by microscopy. We show that both the N- and C-terminal regions of NCR335 possess anticandidal activity as well as the C-terminal sequence of NCR169. The active peptides inhibit biofilm formation and the yeast-hypha transformation. Combined treatment of C. auris with peptides and fluconazole revealed synergistic interactions and reduced 2-8-fold the minimal inhibitory concentrations. Our results demonstrate that shortening NCR peptides can even enhance and broaden their anticandidal activity and therapeutic potential.

Candida parapsilosis: from Genes to the Bedside.

Patients with suppressed immunity are at the highest risk for hospital-acquired infections. Among these, invasive candidiasis is the most prevalent systemic fungal nosocomial infection. Over recent decades, the combined prevalence of non-albicans Candida species outranked Candida albicans infections in several geographical regions worldwide, highlighting the need to understand their pathobiology in order to develop effective treatment and to prevent future outbreaks. Candida parapsilosis is the second or third most frequently isolated Candida species from patients. Besides being highly prevalent, its biology differs markedly from that of C. albicans, which may be associated with C. parapsilosis' increased incidence. Differences in virulence, regulatory and antifungal drug resistance mechanisms, and the patient groups at risk indicate that conclusions drawn from C. albicans pathobiology cannot be simply extrapolated to C. parapsilosis Such species-specific characteristics may also influence their recognition and elimination by the host and the efficacy of antifungal drugs. Due to the availability of high-throughput, state-of-the-art experimental tools and molecular genetic methods adapted to C. parapsilosis, genome and transcriptome studies are now available that greatly contribute to our understanding of what makes this species a threat. In this review, we summarize 10 years of findings on C. parapsilosis pathogenesis, including the species' genetic properties, transcriptome studies, host responses, and molecular mechanisms of virulence. Antifungal susceptibility studies and clinician perspectives are discussed. We also present regional incidence reports in order to provide an updated worldwide epidemiology summary.

Comprehensive characterization of PAHs profile in Serbian soils for conventional and organic production: potential sources and risk assessment.

This study presents a comprehensive characterization of occurrence and levels of 16 polycyclic aromatic hydrocarbons (PAHs) in arable soils used for conventional and organic production in northern and central part of Serbia as well as cross-border region with Hungary. Furthermore, this study includes a characterization of PAH sources and carcinogenic/non-carcinogenic human health risk for PAHs accumulated in analysed arable soils. The total concentration of 16 PAHs varied between 55 and 4584 µg kg-1 in agricultural soil used for conventional production and between 90 and 523 µg kg-1 in agricultural soil used for organic production. High molecular weight (HMW) PAHs were dominant compounds with similar contribution in both soil types (86% and 80% in conventional and in organic soil, respectively). Principal component analysis and diagnostic ratios of selected PAHs were used for identification of PAH sources in the analysed soils. Additionally, positive matrix factorization was applied for quantitative assessment. The results indicated that the major sources of PAHs were vehicle emissions, biomass and wood combustion, accounting for ~ 93% of PAHs. Exposure of farmers assessed through carcinogenic (TCR) and non-carcinogenic (THQ) risk did not exceed the acceptable threshold (TCR < 10-6 and THQ < 1). Oral ingestion was the main exposure route which accounted for 57% of TCR and 80% of THQ. It was followed by dermal contact. This investigation gives a valuable data insight into the PAHs presence in arable soils and reveals the absence of environmental and health risk. It also acknowledges the importance of comprehensive monitoring of these persistent pollutants.

Comprehensive characterization of stress tolerant bacteria with plant growth-promoting potential isolated from glyphosate-treated environment.

The application of plant growth-promoting bacteria in agricultural systems is an efficient and environment-friendly strategy to improve crop yields and maintain soil quality. However, as different soils have diverse and specific ecological characteristics and may represent adverse abiotic conditions, in vivo application requires the careful selection of the desired beneficial microorganisms. In this study we report Ensifer adhaerens SZMC 25856 and Pseudomonas resinovorans SZMC 25875 isolates recovered from glyphosate-treated soil to possess yet undiscovered plant growth-enhancing potential. The strains were found to promote the growth of tomato seedlings significantly, to have the ability of synthesizing indole-3-acetic acid and siderophores, to tolerate pH in the range of 6.59-7.96, salinity up to 12.5 g L-1 NaCl and drought up to 125 g L-1 polyethylene glycol 6000, as well as to survive in the presence of various pesticides including glyphosate, diuron, chlorotoluron, carbendazim and thiabendazole, and heavy metals such as Al, Fe, Mn, Zn, Pb and Cu. The plant growth-promoting traits of the examined E. adhaerens and P. resinovorans isolates and their tolerance to numerous abiotic stress factors make them promising candidates for application in different agricultural environments, including soils polluted with glyphosate.

Sensitivity of Trichoderma strains from edible mushrooms to the fungicides prochloraz and metrafenone.

Twenty-two strains of Trichoderma spp. (T. harzianum species complex [THSC], Trichoderma aggressivum f. europaeum, Trichoderma pleuroti, and Trichoderma pleuroticola) causing green mold disease on edible mushrooms (button mushroom, shiitake and oyster mushroom), collected during 2004-2018 from four countries (Serbia, North Macedonia, Croatia, and Hungary) were examined. Based on their ITS (internal transcribed spacer) sequences, strains from shiitake mushroom in Serbia were identified as members of the THSC, while in samples obtained from Serbian and North-Macedonian oyster mushroom farms THSC, T. pleuroti and T. pleuroticola were detected, which represent the first findings in the region. In fungicide susceptibility tests, all examined Trichoderma strains were found to be highly sensitive to prochloraz (ED50<0.4 µg mL-1) and considerably susceptible to metrafenone (ED50 < 4 µg mL-1). The most sensitive taxon to both fungicides was THSC from oyster mushroom. The toxicity of metrafenone was satisfying and strains from oyster mushroom showed the highest sensitivity (ED50 < 1.43 µg mL-1), while strains originating from button mushroom and shiitake displayed similar susceptibilities (ED50 < 3.64 µg mL-1). After additional in vivo trials, metrafenone might also be recommended for the control of green mold disease in mushroom farms.

Size-dependent activity of silver nanoparticles on the morphological switch and biofilm formation of opportunistic pathogenic yeasts.

BACKGROUND: Dimorphism and biofilm formation are important virulence factors of some opportunistic human pathogenic yeasts. Such species commensally colonize skin or mucosal surfaces generally in yeast form, but under particular circumstances, convert into virulent hyphae and disseminate internal organs or cause mucocutaneous infections. The yeast-to-hypha shape-conversion promotes the development of a biofilm, a thick extracellular matrix with sessile cells within. The biofilm is capable to prevent the penetration of antifungal drugs, rendering the surviving biofilm-resident cells intrinsic sources of recurrent infections. The aim of this study was to evaluate the ability of silver nanoparticles (AgNPs) to attenuate the morphological switch and biofilm formation of several opportunistic pathogenic yeasts and to determine whether this feature depends on the nanoparticle size. RESULTS: AgNPs in three different sizes were prepared by chemical reduction approach and characterized by transmission electron microscopy, ultraviolet-visible spectroscopy and dynamic light scattering. The antifungal activity was evaluated by the microdilution method, the inhibitory capacity on biofilm formation and the biofilm degradation ability of differently sized AgNPs was assessed by viability assay. The morphological state of opportunistic pathogenic yeast cells in monoculture and in co-culture with human keratinocytes in the presence of AgNPs was examined by flow cytometry and scanning electron microscopy. All the three AgNPs inhibited the growth of the examined opportunistic pathogenic yeasts, nevertheless, AgNPs with the smallest diameter exhibited the most prominent toxic activities. AgNPs attenuated the biofilm formation in a nanoparticle size-dependent manner; however, their biofilm destruction capacity was negligible. AgNPs with the smallest size exerted the most significant effect on suppressing the morphological change of pathogens in monoculture as well as in a co-culture with keratinocytes. CONCLUSIONS: Our results confirm that AgNPs are capable to hinder yeast-to-hypha morphological conversion and biofilm formation of opportunistic pathogens and this biological effect of AgNPs is size-dependent.

CRISPR-Cas9-Based Mutagenesis of the Mucormycosis-Causing Fungus Lichtheimia corymbifera.

Lichtheimia corymbifera is considered as one of the most frequent agents of mucormycosis. The lack of efficient genetic manipulation tools hampers the characterization of the pathomechanisms and virulence factors of this opportunistic pathogenic fungus. Although such techniques have been described for certain species, the performance of targeted mutagenesis and the construction of stable transformants have remained a great challenge in Mucorales fungi. In the present study, a plasmid-free CRISPR-Cas9 system was applied to carry out a targeted gene disruption in L. corymbifera. The described method is based on the non-homologous end-joining repair of the double-strand break caused by the Cas9 enzyme. Using this method, short, one-to-five nucleotide long-targeted deletions could be induced in the orotidine 5'-phosphate decarboxylase gene (pyrG) and, as a result, uracil auxotrophic strains were constructed. These strains are applicable as recipient strains in future gene manipulation studies. As we know, this is the first genetic modification of this clinically relevant fungus.

CRISPR-Cas9-mediated disruption of the HMG-CoA reductase genes of Mucor circinelloides and subcellular localization of the encoded enzymes.

Terpenoid compounds, such as sterols, carotenoids or the prenyl groups of various proteins are synthesized via the mevalonate pathway. A rate-limiting step of this pathway is the conversion of 3-methylglutaryl-CoA (HMG-CoA) to mevalonic acid catalyzed by the HMG-CoA reductase. Activity of this enzyme may affect several biological processes, from the synthesis of terpenoid metabolites to the adaptation to various environmental conditions. In this study, the three HMG-CoA reductase genes (i.e. hmgR1, hmgR2 and hmgR3) of the ß-carotene producing filamentous fungus, Mucor circinelloides were disrupted individually and simultaneously by a recently developed in vitro plasmid-free CRISPR-Cas9 method. Examination of the mutants revealed that the function of hmgR2 and hmgR3 are partially overlapping and involved in the general terpenoid biosynthesis. Moreover, hmgR2 seemed to have a special role in the ergosterol biosynthesis. Disruption of all three genes affected the germination ability of the spores and the sensitivity to hydrogen peroxide. Disruption of the hmgR1 gene had no effect on the ergosterol production and the sensitivity to statins but caused a reduced growth at lower temperatures. By confocal fluorescence microscopy using strains expressing GFP-tagged HmgR proteins, all three HMG-CoA reductases were localized in the endoplasmic reticulum.

Accelerated Molecular Dynamics Applied to the Peptaibol Folding Problem.

The use of enhanced sampling molecular dynamics simulations to facilitate the folding of proteins is a relatively new approach which has quickly gained momentum in recent years. Accelerated molecular dynamics (aMD) can elucidate the dynamic path from the unfolded state to the near-native state, "flattened" by introducing a non-negative boost to the potential. Alamethicin F30/3 (Alm F30/3), chosen in this study, belongs to the class of peptaibols that are 7-20 residue long, non-ribosomally synthesized, amphipathic molecules that show interesting membrane perturbing activity. The recent studies undertaken on the Alm molecules and their transmembrane channels have been reviewed. Three consecutive simulations of ~900 ns each were carried out where N-terminal folding could be observed within the first 100 ns, while C-terminal folding could only be achieved almost after 800 ns. It took ~1 µs to attain the near-native conformation with stronger potential boost which may take several µs worth of classical MD to produce the same results. The Alm F30/3 hexamer channel was also simulated in an E. coli mimicking membrane under an external electric field that correlates with previous experiments. It can be concluded that aMD simulation techniques are suited to elucidate peptaibol structures and to understand their folding dynamics.

Tripleurin XIIc: Peptide Folding Dynamics in Aqueous and Hydrophobic Environment Mimic Using Accelerated Molecular Dynamics.

Peptaibols are a special class of fungal peptides with an acetylated N-terminus and a C-terminal 1,2-amino alcohol along with non-standard amino acid residues. New peptaibols named tripleurins were recently identified from a strain of the filamentous fungal species Trichoderma pleuroti, which is known to cause green mould disease on cultivated oyster mushrooms. To understand the mode of action of these peptaibols, the three-dimensional structure of tripleurin (TPN) XIIc, an 18-mer peptide, was elucidated using an enhanced sampling method, accelerated MD, in water and chloroform solvents. Non-standard residues were parameterized by the Restrained Electrostatic Potential (RESP) charge fitting method. The dihedral distribution indicated towards a right-handed helical formation for TPN XIIc in both solvents. Dihedral angle based principal component analysis revealed a propensity for a slightly bent, helical folded conformation in water solvent, while two distinct conformations were revealed in chloroform: One that folds into highly bent helical structure that resembles a beta-hairpin and another with an almost straight peptide backbone appearing as a rare energy barrier crossing event. The hinge-like movement of the terminals was also observed and is speculated to be functionally relevant. The convergence and efficient sampling is addressed using Cartesian PCA and Kullback-Leibler divergence methods.

Genome analysis of a Bacillus subtilis strain reveals genetic mutations determining biocontrol properties.

Several Bacillus strains are used as biocontrol agents, as they frequently have strong antagonistic effects against microbial plant pathogens. Bacillus strain SZMC 6179J, isolated from tomato rhizosphere, was previously shown to have excellent in vitro antagonistic properties against the most important fungal pathogens of tomato (Alternaria solani, Botrytis cinerea, Phytophthora infestans and Sclerotinia sclerotiorum) as well as several Fusarium species. Taxonomic investigations revealed that it is a member of the B. subtilis subsp. subtilis group and very closely related with the reference type strain B. subtilis subsp. subtilis 168. The sequenced genome of strain SZMC 6179J contains the genes responsible for the synthesis of the extracellular antibiotics surfactin, fengycin and bacilysin. Compared to strain 168, a prophage-like region is missing from the genome of SZMC 6179J, while there are 106 single nucleotide polymorphisms and 23 deletion-insertion polymorphisms. The high biocontrol potential of strain SZMC 6179J may results from a single base deletion in the sfp gene encoding the transcription factor of the surfactin and fengycin operons. Hypermutated regions reflecting short-time evolutionary processes could be detected in SZMC 6179J. The deletion-insertion polymorphism in the sfp gene and the detected hypermutations can be suggested as genetic determinants of biocontrol features in B. subtilis.

Exophiala dermatitidis Endophthalmitis: Case Report and Literature Review.

We report a case of a 59-year-old male patient with a postoperative fungal infection of the left eye. A dark-pigmented yeast, Exophiala dermatitidis (previously known as Wangiella dermatitidis), was identified from the culture of the biopsy taken from the posterior capsule. The infection was successfully eradicated by a combination of surgical and medical (i.e., voriconazole and fluconazole) treatment. This is the first report of successfully treated E. dermatitidis endophthalmitis, which demonstrates that a prompt and aggressive antifungal therapy combined with surgical intervention is necessary to prevent vision loss in cases of endophthalmitis due to Exophiala species. Beside the case description, we also aim to provide a literature review of previously reported eye infections caused by Exophiala species in order to help the future diagnosis and management of the disease.

Sterigmatocystin production is restricted to hyphae located in the proximity of hülle cells.

Aspergillus nidulans produces sterigmatocystin, a secondary metabolite mycotoxin, for the protection of its reproductive structures. Previous studies on grazing behavior of fungivore arthropods, regulation of sexual development, and secondary metabolite biosynthesis have revealed the association of sterigmatocystin biosynthesis with sexual reproduction, but the spatial distribution of sterigmatocystin producing hyphae within the colony has never been investigated. In this work, we aimed to locate the site of sterigmatocystin production within the colony by employing a yCFP reporter system. We demonstrated that the stcO promoter is active only in vegetative hyphae that surround groups of hülle cells and the activity decreases and eventually ceases as the distance between the hypha and the hülle cells increases. This phenomenon indicates that the vegetative mycelium might consist of morphologically uniform, but functionally different hyphae.

An Organic Solvent-Tolerant Lipase with Both Hydrolytic and Synthetic Activities from the Oleaginous Fungus Mortierella echinosphaera.

Lipase enzymes of the oleaginous fungal group Mortierella are rarely studied. However, considering that most commercial lipases are derived from filamentous fungal sources, their investigation can contribute to the cost-effective development of new biotechnological processes. Here, an extracellular lipase with a molecular mass of 30 kDa was isolated from Mortierella echinosphaera CBS 575.75 and characterized. The purified lipase exhibited an optimal p-nitrophenyl palmitate (pNPP)-hydrolyzing activity at 25 °C and pH 6.6-7.0 and proved to be highly stable at temperatures up to 40 °C and under broad pH conditions. The enzyme was active under low temperatures, retaining 32.5% of its activity at 10 °C, and was significantly stable in polar and non-polar organic solvents. The Km, Vmax, and kcat for pNPP were 0.336 mM, 30.4 µM/min, and 45.7 1/min for pNPP and 0.333 mM, 36.9 µM/min, and 55.6 1/min for pNP-decanoate, respectively. The pNPP hydrolysis was inhibited by Hg2+, N-bromosuccinimide, and sodium dodecyl sulfate, while ethylenediaminetetraacetic acid and metal ions, such as Ca2+, Mg2+, Na⁺, and K⁺ enhanced the activity. The purified lipase had non-regioselective activity and wide substrate specificity, showing a clear preference for medium-chained p-nitrophenyl esters. Besides its good transesterification activity, the enzyme appeared as a suitable biocatalyst to operate selective esterification reactions to long-chained alkyl esters. Adsorption to Accurel MP1000 improved the storage stability of the enzyme at 5 °C. The immobilized lipase displayed tolerance to a non-aqueous environment and was reusable for up to five cycles without significant loss in its synthetic and hydrolytic activities. These findings confirm the applicability of both the free and the immobilized enzyme preparations in future research.

Effects of Different Cultivation Parameters on the Production of Surfactin Variants by a Bacillus subtilis Strain.

Surfactins are lipopeptide-type biosurfactants produced mainly by Bacillus species, consisting of a peptide loop of seven amino acids and a hydrophobic fatty acid chain (C12⁻C16). These molecules have been proven to exhibit various biological activities; thus, their therapeutic and environmental applications are considered. Within the surfactin lipopeptide family, there is a wide spectrum of different homologues and isomers; to date, more than 30 variants have been described. Since the newest members of these lipopeptides were described recently, there is no information that is available on their characteristic features, e.g., the dependence of their production from different cultivation parameters. This study examined the effects of both the different carbon sources and various metal ions on the surfactin production of a selected B. subtilis strain. Among the applied carbon sources, fructose and xylose had the highest impacts on the ratio of the different variants, regarding both the peptide sequences and the lengths of the fatty acids. Furthermore, the application of metal ions Mn2+, Cu2+ and Ni2+ in the media completely changed the surfactin variant compositions of the fermenting broths leading to the appearance of methyl esterified surfactin forms, and resulted in the appearance of novel surfactin variants with fatty acid chains containing no more than 11 carbon atoms.

High-Frequency Occurrence of Surfactin Monomethyl Isoforms in the Ferment Broth of a Bacillus subtilis Strain Revealed by Ion Trap Mass Spectrometry.

Surfactins are cyclic lipopeptides consisting of a ß-hydroxy fatty acid of various chain length and a peptide ring of seven amino acids linked together by a lactone bridge, forming the cyclic structure of the peptide chain. These compounds are produced mainly by Bacillus species and possess numerous biological effects such as antimicrobial (antiviral, antibacterial, and antifungal) activities. A mixture of surfactins extracted from Bacillus subtilis strain SZMC 6179J was examined by HPLC-ESI-IT-MS technique, enhancing their separation to reveal novel lipopeptide varieties with higher masses and to characterize their structures. During the MS² spectra analyses of their sodiated molecular ions [M + Na]⁺, a previously rarely encountered group of surfactins was detected and two novel types of the group were discovered containing methyl esterified aspartic acid residue in their fifth amino acid position. The relative amounts of these monomethyl isoforms exceeded 35% of the produced surfactin in total. In the m/z value of 1114, all the detected isoforms possessed aspartic acid 4-methyl ester residue in their fifth amino acid position (C17-[Lxx4, AME5], C18-[AME5]), offering an opportunity to separate a pure fraction of the compound and to study its biological activities in the future.