Biochemical and molecular characterisation of the bacterial endophytes from native sugarcane varieties of Himalayan region

Seven endophytic bacterial isolates were finally recovered from native sugarcane varieties at hilly areas namely Berinag, Champawat and Didihat of Uttarakhand state in northern Himalayan region. New isolates and two standard cultures—Azospirillum brasilense and Gluconacetobacter diazotrophicus, were evaluated for their morphological, biochemical and molecular characteristics. Morphologically all were rod shaped, Gram-negative bacteria. Their plant growth promotory properties were also assessed which proved isolates RtBn and StBn as IAA producing. Except isolate StBn, all were phosphate solubilising and except RtBn all produced siderophores. Molecular characterisation of the isolates was performed using amplified 16S r-DNA restriction analysis. Similarity index in unweighted pair group method with arithmetic mean programme clustered the isolates according to their geographical distribution. Native isolates showed insignificant similarity with South American strains used as standards. nifH amplification was observed with all the isolates used in the study which again establish them as potential N-fixers

Volatiles Mediated Interactions Between Aspergillus oryzae Strains Modulate Morphological Transition and Exometabolomes

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 (S1VMI: 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 S1VMI was delayed relative to its non-conditioned control (S1) cultivated without partner strain (S2) in twin plate. Selectively evaluating A. oryzae RIB 40 (S1VMI vs. S1) for altered phenotypes concomitant to SD, we observed a marked disparity for corresponding growth rates (S1VMI < S1)7days, media pH (S1VMI > S1)7days, and biochemical characteristics viz., protease (S1VMI > S1)7days, amylase (S1VMI > nS1)3–7days, and antioxidants (S1VMI > 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 S1VMI 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 S1VMI. 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 S1VMI 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 (S1VMI: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

The antileukaemic cell cycle regulatory activities of swainsonine purified from <i>Metarhizium anisopliae</i> fermentation broth

<div><p>Swainsonine is a <i>Metarhizium</i> secondary metabolite known differentially for its specific mannosidase inhibitory, toxic and therapeutic activities. Here, the standard and purified swainsonine from <i>Metarhizium anisopliae</i> fermentation broth were comparatively evaluated for their <i>in situ</i> antileukaemic activities in human promyelocytic cell line, HL-60. Both the standard (IC₅₀ = 6.96 μM) and purified (IC₅₀ = 9.50 μM) compounds inhibited the leukaemic cell proliferation without inflicting cell membrane disruption at 48 h of post-treatment incubation. The DNA cell cycle analysis showed approximately 48.81% and 60.72% of the treated cells arrested in the synthetic phase (S-phase) at 36 and 48 h, respectively, upon treatment with IC₅₀ concentration of the purified swainsonine. However, only 29.62% of cells were arrested in S-phase with standard swainsonine at 48 h, suggesting the comprehensive action of certain other metabolites sharing the similar paradigm of antiproliferative properties in <i>Metarhizium</i> broth extract.</p></div

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

Perplexing metabolomes in fungal-insect trophic interactions: A terra incognita of mycobiocontrol mechanisms

The trophic interactions of entomopathogenic fungi in different ecological niches viz., soil, plants, or insect themselves are effectively regulated by their maneuvered metabolomes and the plethora of metabotypes. In this article, we discuss a holistic framework of co-evolutionary metabolomes and metabotypes to model the interactions of biocontrol fungi especially with mycosed insects. Conventionally, the studies involving fungal biocontrol mechanisms are reported in the context of much aggrandized fungal entomotoxins while the adaptive response mechanisms of host insects are relatively overlooked. The present review asserts that the selective pressure exerted among the competing or interacting species drives alterations in their overall metabolomes which ultimately implicates in corresponding metabotypes. Quintessentially, metabolomics offers a most generic and tractable model to assess the fungal-insect antagonism in terms of interaction biomarkers, biosynthetic pathway plasticity, and their co-evolutionary defense. The fungi chiefly rely on a battery of entomotoxins viz., secondary metabolites falling in the categories of NRP's (non-ribosomal peptides), PK's (polyketides), lysine derive alkaloids, and terpenoids. On the contrary, insects overcome mycosis through employing different layers of immunity manifested as altered metabotypes (phenoloxidase activity) and overall metabolomes viz., carbohydrates, lipids, fatty acids, amino acids, and eicosanoids. Here, we discuss the recent findings within conventional premise of fungal entomotoxicity and the evolution of truculent immune response among host insect. The metabolomic frameworks for fungal-insect interaction can potentially transmogrify our current comprehensions of biocontrol mechanisms to develop the hypervirulent biocontrol strains with least environmental concerns. Moreover, the interaction metabolomics (interactome) in complementation with other -omics cascades could further be applied to address the fundamental bottlenecks of adaptive co-evolution among biological species

Ultrahigh Pressure Processing Produces Alterations in the Metabolite Profiles of Panax ginseng

Ultrahigh pressure (UHP) treatments are non-thermal processing methods that have customarily been employed to enhance the quality and productivity of plant consumables. We aimed to evaluate the effects of UHP treatments on ginseng samples (white ginseng: WG; UHP-treated WG: UWG; red ginseng: RG; UHP-treated RG: URG; ginseng berries: GB; and UHP-treated GB: UGB) using metabolite profiling based on ultrahigh performance liquid chromatography-linear trap quadrupole-ion trap-tandem mass spectrometry (UHPLC-LTQ-IT-MS/MS) and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). Multivariate data analyses revealed a clear demarcation among the GB and UGB samples, and the phenotypic evaluations correlated the highest antioxidant activities and the total phenolic and flavonoid compositions with the UGB samples. Overall, eight amino acids, seven organic acids, seven sugars and sugar derivatives, two fatty acids, three notoginsenosides, three malonylginsenosides, and three ginsenosides, were identified as significantly discriminant metabolites between the GB and UGB samples, with relatively higher proportions in the latter. Ideally, these metabolites can be used as quality biomarkers for the assessment of ginseng products and our results indicate that UHP treatment likely led to an elevation in the proportions of total extractable metabolites in ginseng samples

Metabolomics Reveal Optimal Grain Preprocessing (Milling) toward Rice <i>Koji</i> Fermentation

A time-correlated mass spectrometry (MS)-based metabolic profiling was performed for rice <i>koji</i> made using the substrates with varying degrees of milling (DOM). Overall, 67 primary and secondary metabolites were observed as significantly discriminant among different samples. Notably, a higher abundance of carbohydrate (sugars, sugar alcohols, organic acids, and phenolic acids) and lipid (fatty acids and lysophospholipids) derived metabolites with enhanced hydrolytic enzyme activities were observed for <i>koji</i> made with DOM of 5–7 substrates at 36 h. The antioxidant secondary metabolites (flavonoids and phenolic acid) were relatively higher in <i>koji</i> with DOM of 0 substrates, followed by DOM of 5 > DOM of 7 > DOM of 9 and 11 at 96 h. Hence, we conjecture that the rice substrate preprocessing between DOM of 5 and 7 was potentially optimal toward <i>koji</i> fermentation, with the end product being rich in distinctive organoleptic, nutritional, and functional metabolites. The study rationalizes the substrate preprocessing steps vital for commercial <i>koji</i> making

Integrated Metabolomics and Volatolomics for Comparative Evaluation of Fermented Soy Products

Though varying metabolomes are believed to influence distinctive characteristics of different soy foods, an in-depth, comprehensive analysis of both soluble and volatile metabolites is largely unreported. The metabolite profiles of different soy products, including cheonggukjang, meju, doenjang, and raw soybean, were characterized using LC-MS (liquid chromatography–mass spectrometry), GC-MS (gas chromatography–mass spectrometry), and headspace solid-phase microextraction (HS-SPME) GC-MS. Principal component analysis (PCA) showed that the datasets for the cheonggukjang, meju, and doenjang extracts were distinguished from the non-fermented soybean across PC1, while those for cheonggukjang and doenjang were separated across PC2. Volatile organic compound (VOC) profiles were clearly distinct among doenjang and soybean, cheonggukjang, and meju samples. Notably, the relative contents of the isoflavone glycosides and DDMP (2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one) conjugated soyasaponins were higher in soybean and cheonggukjang, compared to doenjang, while the isoflavone aglycones, non-DDMP conjugated soyasaponins, and amino acids were significantly higher in doenjang. Most VOCs, including the sulfur containing compounds aldehydes, esters, and furans, were relatively abundant in doenjang. However, pyrazines, 3-methylbutanoic acid, maltol, and methoxyphenol were higher in cheonggukjang, which contributed to the characteristic aroma of soy foods. We believe that this study provides the fundamental insights on soy food metabolomes, which determine their nutritional, functional, organoleptic, and aroma characteristics