Han, Jae Woo
(Center for Eco-friendly New Materials, Korea Research Institute of Chemical Technology)
,
Kim, Bomin
(Center for Eco-friendly New Materials, Korea Research Institute of Chemical Technology)
,
Oh, Mira
(Center for Eco-friendly New Materials, Korea Research Institute of Chemical Technology)
,
Choi, Jaehyuk
(Division of Life Sciences, Incheon National University)
,
Choi, Gyung Ja
(Center for Eco-friendly New Materials, Korea Research Institute of Chemical Technology)
,
Kim, Hun
(Center for Eco-friendly New Materials, Korea Research Institute of Chemical Technology)
Valuable natural compounds produced by a variety of microorganisms can be used as lead molecules for development of new agrochemicals. Furthermore, high-throughput in vitro screening systems with specific modes of action can increase the probability of discovery of new fungicides. In the current stu...
Valuable natural compounds produced by a variety of microorganisms can be used as lead molecules for development of new agrochemicals. Furthermore, high-throughput in vitro screening systems with specific modes of action can increase the probability of discovery of new fungicides. In the current study, a rapid assay tested with various microbes was developed to determine the degree of respiratory inhibition of Saccharomyces cerevisiae in two different liquid media, YG (containing a fermentable carbon source) and NFYG (containing a non-fermentable carbon source). Based on this system, we screened 100 fungal isolates that were classified into basidiomycetes, to find microbial secondary metabolites that act as respiratory inhibitors. Consequently, of the 100 fungal species tested, the culture broth of an IUM04881 isolate inhibited growth of S. cerevisiae in NFYG medium, but not in YG medium. The result is comparable to that from treatment with kresoxim-methyl used as a control, suggesting that the culture broth of IUM04881 isolate might contain active compounds showing the inhibition activity for respiratory chain. Based on the assay developed in this study and spectroscopic analysis, we isolated and identified an antifungal compound (-)-oudemansin A from culture broth of IUM04881 that is identified as Oudemansiella venosolamellata. This is the first report that (-)-oudemansin A is identified from O. venosolamellata in Korea. Taken together, the development of this assay will accelerate efforts to find and identify natural respiratory inhibitors from various microbes.
Valuable natural compounds produced by a variety of microorganisms can be used as lead molecules for development of new agrochemicals. Furthermore, high-throughput in vitro screening systems with specific modes of action can increase the probability of discovery of new fungicides. In the current study, a rapid assay tested with various microbes was developed to determine the degree of respiratory inhibition of Saccharomyces cerevisiae in two different liquid media, YG (containing a fermentable carbon source) and NFYG (containing a non-fermentable carbon source). Based on this system, we screened 100 fungal isolates that were classified into basidiomycetes, to find microbial secondary metabolites that act as respiratory inhibitors. Consequently, of the 100 fungal species tested, the culture broth of an IUM04881 isolate inhibited growth of S. cerevisiae in NFYG medium, but not in YG medium. The result is comparable to that from treatment with kresoxim-methyl used as a control, suggesting that the culture broth of IUM04881 isolate might contain active compounds showing the inhibition activity for respiratory chain. Based on the assay developed in this study and spectroscopic analysis, we isolated and identified an antifungal compound (-)-oudemansin A from culture broth of IUM04881 that is identified as Oudemansiella venosolamellata. This is the first report that (-)-oudemansin A is identified from O. venosolamellata in Korea. Taken together, the development of this assay will accelerate efforts to find and identify natural respiratory inhibitors from various microbes.
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제안 방법
In this study, we developed effective in vitro screening methods for identification of microbial metabolites showing respiratory inhibition, based on the inhibition of growth of Saccharomyces cerevisiae using a non-fermentable carbon source such as glycerol and lactate in a medium [4]. When a QoI fungicide is treated to S.
대상 데이터
To find microbial secondary metabolites that act as respiratory inhibitors, we screened 100 fungal isolates classified into basidiomycetes, which were deposited in the “Culture Collection of Mushrooms” at Incheon National University, Korea.
cerevisiae grown in YG medium. The experiment was conducted twice with three replicates.
The chemical structure of IUM04881E2 was determined using electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. The ESI-MS data of IUM04881E2 was recorded on a single-quadruple mass spectrometer equipped (Acquity QDa; Waters, Manchester, UK). The 1H-NMR spectrum was measured using a Bruker Avance 300 spectrometer (Burker BioSpin, Rheinstetten, Germany) in a chloroform-d (Cambridge Isotope Laboratories, Tewksbury, MA, USA).
For identification of IUM04881 isolate, the ITS region was amplified from gDNA of IUM04881 isolate, and resulting sequences (616 bp of amplicon) were searched using the BLASTn program of NCBI (http://www.ncbi.nlm.nih.gov). Pairwise sequence similarities of the ITS region were determined with the most closely related strains using the BLASTn analysis.
이론/모형
Pairwise sequence similarities of the ITS region were determined with the most closely related strains using the BLASTn analysis. The sequences were aligned using ClustalW implemented in MEGA version 7, and distances were estimated based on the model of Tamura and Nei [8]. A phylogenetic tree was generated using the neighbor-joining method [9] with 1,000 bootstrap analyses (Supplementary Figure 1).
성능/효과
cerevisiae cultures, because metabolically active cells are able to change dye color from blue to red in which resazurin is converted to fluorescent resorufin [6]. Our results showed that the reagent did not change its color in the S. cerevisiae cultures of NFYG medium treated with the IUM04881 culture filtrate, which is comparable to that from treatment with kresoximmethyl. However, all culture filtrate treatment to the S.
5% and 5% culture filtrate treatment compared to the 10% and 20% culture filtrate treatment. Based on the optical density (OD600), the growth inhibition values for the 10% and 20% treatment of IUM04881 culture filtrate against S. cerevisiae grown in NFYG medium were 30% and 76%, respectively, whereas the growth inhibition values for all the other treatments against S. cerevisiae grown in YG medium were less than 18% (Figure 1(C)). Together, these results suggest that the culture broth of IUM04881 isolate might contain active compounds showing the inhibitory activity for respiratory chain.
A phylogenetic tree was generated using the neighbor-joining method [9] with 1,000 bootstrap analyses (Supplementary Figure 1). Phylogenetic analysis showed that the IUM04881 isolate belongs to genus Oudemansiella and mostly relates to Oudemansiella venosolamellata SU200510011 (AB688120) and O. venosolamellata SU20100425 (AB688121) with 99.8% similarity, which resulted in that the IUM04881 was identified as O. venosolamellata.
후속연구
venosolamellata will make this approach accessible for a wide variety of fungicide research. The development of this assay will accelerate efforts to find and identify natural respiratory inhibitors from various microbes.
참고문헌 (11)
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