DataSheet1.DOCX

<p>Notwithstanding its mitosporic nature, an improbable morpho-transformation state i. e., sclerotial development (SD), is vaguely known in Aspergillus oryzae. Nevertheless an intriguing phenomenon governing mold's development and stress response, the effects of exogenous factors engendering SD, especially the volatile organic compounds (VOCs) mediated interactions (VMI) pervasive in microbial niches have largely remained unexplored. Herein, we examined the effects of intra-species VMI on SD in A. oryzae RIB 40, followed by comprehensive analyses of associated growth rates, pH alterations, biochemical phenotypes, and exometabolomes. We cultivated A. oryzae RIB 40 (S1_VMI: KACC 44967) opposite a non-SD partner strain, A. oryzae (S2: KCCM 60345), conditioning VMI in a specially designed “twin plate assembly.” Notably, SD in S1_VMI was delayed relative to its non-conditioned control (S1) cultivated without partner strain (S2) in twin plate. Selectively evaluating A. oryzae RIB 40 (S1_VMI vs. S1) for altered phenotypes concomitant to SD, we observed a marked disparity for corresponding growth rates (S1_VMI < S1)_7days, media pH (S1_VMI > S1)_7days, and biochemical characteristics viz., protease (S1_VMI > S1)_7days, amylase (S1_VMI > nS1)_3–7days, and antioxidants (S1_VMI > S1)_7days levels. The partial least squares—discriminant analysis (PLS-DA) of gas chromatography—time of flight—mass spectrometry (GC-TOF-MS) datasets for primary metabolites exhibited a clustered pattern (PLS1, 22.04%; PLS2, 11.36%), with 7 days incubated S1_VMI extracts showed higher abundance of amino acids, sugars, and sugar alcohols with lower organic acids and fatty acids levels, relative to S1. Intriguingly, the higher amino acid and sugar alcohol levels were positively correlated with antioxidant activity, likely impeding SD in S1_VMI. Further, the PLS-DA (PLS1, 18.11%; PLS2, 15.02%) based on liquid chromatography—mass spectrometry (LC-MS) datasets exhibited a notable disparity for post-SD (9–11 days) sample extracts with higher oxylipins and 13-desoxypaxilline levels in S1_VMI relative to S1, intertwining Aspergillus morphogenesis and secondary metabolism. The analysis of VOCs for the 7 days incubated samples displayed considerably higher accumulation of C-8 compounds in the headspace of twin-plate experimental sets (S1_VMI:S2) compared to those in non-conditioned controls (S1 and S2—without respective partner strains), potentially triggering altered morpho-transformation and concurring biochemical as well as metabolic states in molds.</p

Presentation_1_Comprehensive Secondary Metabolite Profiling Toward Delineating the Solid and Submerged-State Fermentation of Aspergillus oryzae KCCM 12698.pdf

<p>Aspergillus oryzae has been commonly used to make koji, meju, and soy sauce in traditional food fermentation industries. However, the metabolic behaviors of A. oryzae during fermentation in various culture environments are largely uncharacterized. Thus, we performed time resolved (0, 4, 8, 12, 16 day) secondary metabolite profiling for A. oryzae KCCM 12698 cultivated on malt extract agar and broth (MEA and MEB) under solid-state fermentation (SSF) and submerged fermentation (SmF) conditions using the ultrahigh performance liquid chromatography-linear trap quadrupole-ion trap-mass spectrometry (UHPLC-LTQ-IT-MS/MS) followed by multivariate analyses. We observed the relatively higher proportions of coumarins and oxylipins in SSF, whereas the terpenoids were abundant in SmF. Moreover, we investigated the antimicrobial efficacy of metabolites that were extracted from SSF and SmF. The SSF extracts showed higher antimicrobial activities as compared to SmF, with higher production rates of bioactive secondary metabolites viz., ketone-citreoisocoumarin, pentahydroxy-anthraquinone, hexylitaconic acid, oxylipins, and saturated fatty acids. The current study provides the underpinnings of a metabolomic framework regarding the growth and bioactive compound production for A. oryzae under the primarily employed industrial cultivation states. Furthermore, the study holds the potentials for rapid screening and MS-characterization of metabolites helpful in determining the consumer safety implications of fermented foods involving Koji mold.</p

Data_Sheet_1_A Growth-Promoting Bacteria, Paenibacillus yonginensis DCY84T Enhanced Salt Stress Tolerance by Activating Defense-Related Systems in Panax ginseng.docx

<p>Panax ginseng (C.A. Mayer) is a well-known medicinal plant used in traditional medicine in Korea that experiences serious salinity stress related to weather changes or incorrect fertilizer application. In ginseng, the use of Paenibacillus yonginensis DCY84^T to improve salt stress tolerance has not been thoroughly explored. Therefore, we studied the role of P. yonginensis DCY84^T under short-term and long-term salinity stress conditions in a controlled environment. In vitro testing of DCY84^T revealed high indole acetic acid (IAA) production, siderophore formation, phosphate solubilization and anti-bacterial activity. We determined that 10-min dip in 10¹⁰ CFU/ml DCY84^T was sufficient to protect ginseng against short-term salinity stress (osmotic stress) upon exposure to 300 mM NaCl treatment by enhancing nutrient availability, synthesizing hydrolyzing enzymes and inducing osmolyte production. Upon exposure to salinity stress (oxidative and ionic stress), strain DCY84^T-primed ginseng seedlings were protected by the induction of defense-related systems such as ion transport, ROS scavenging enzymes, proline content, total sugars, and ABA biosynthetic genes, as well as genes involved in root hair formation. Additionally, ginseng primed with DCY84^T and exposed to 300 mM NaCl showed the same metabolite profile as control ginseng plants, suggesting that DCY84^T effectively reduced salt stress. These results indicated that DCY84^T can be widely used as a microbial inoculant to protect ginseng plants against salinity stress conditions.</p

Data_Sheet_2_A Growth-Promoting Bacteria, Paenibacillus yonginensis DCY84T Enhanced Salt Stress Tolerance by Activating Defense-Related Systems in Panax ginseng.xlsx

<p>Panax ginseng (C.A. Mayer) is a well-known medicinal plant used in traditional medicine in Korea that experiences serious salinity stress related to weather changes or incorrect fertilizer application. In ginseng, the use of Paenibacillus yonginensis DCY84^T to improve salt stress tolerance has not been thoroughly explored. Therefore, we studied the role of P. yonginensis DCY84^T under short-term and long-term salinity stress conditions in a controlled environment. In vitro testing of DCY84^T revealed high indole acetic acid (IAA) production, siderophore formation, phosphate solubilization and anti-bacterial activity. We determined that 10-min dip in 10¹⁰ CFU/ml DCY84^T was sufficient to protect ginseng against short-term salinity stress (osmotic stress) upon exposure to 300 mM NaCl treatment by enhancing nutrient availability, synthesizing hydrolyzing enzymes and inducing osmolyte production. Upon exposure to salinity stress (oxidative and ionic stress), strain DCY84^T-primed ginseng seedlings were protected by the induction of defense-related systems such as ion transport, ROS scavenging enzymes, proline content, total sugars, and ABA biosynthetic genes, as well as genes involved in root hair formation. Additionally, ginseng primed with DCY84^T and exposed to 300 mM NaCl showed the same metabolite profile as control ginseng plants, suggesting that DCY84^T effectively reduced salt stress. These results indicated that DCY84^T can be widely used as a microbial inoculant to protect ginseng plants against salinity stress conditions.</p