Metabolomics reveal metabolic variation caused by co-culture of Arthrobacter ureafaciens and Trichoderma harzianum and their impacts on wheat germination / La metabolómica revela la variación metabólica causada por el cocultivo de Arthrobacter ureafaciens y Trichoderma harzianum y sus impactos en la germinación del trigo

Arthrobacter ureafaciens DnL1-1 is a bacterium used for atrazine degradation, while Trichoderma harzianum LTR-2 is a widely used biocontrol fungus. In this study, a liquid co-cultivation of these two organisms was initially tested. The significant changes in the metabolome of fermentation liquors were investigated based on cultivation techniques (single-cultured and co-cultured DnL1-1 and LTR-2) using an UPLC-QTOF-MS in an untargeted metabolomic approach. Principle components analysis (PCA) and partial least squares discriminant analysis (PLS-DA) supervised modelling revealed modifications of the metabolic profiles in fermentation liquors as a function of interactions between different strains. Compared with pure-cultivation of DnL1-1, 51 compounds were altered during the cocultivation, with unique and significant differences in the abundance of organic nitrogen compounds (e.g. carnitine, acylcarnitine 4:0, acylcarnitine 5:0, 3-dehydroxycarnitine and O-acetyl-L-carnitine) and trans-zeatin riboside. Nevertheless, compared with pure-cultivation of LTR-2, the abundance of 157 compounds, including amino acids, soluble sugars, organic acids, indoles and derivatives, nucleosides, and others, changed significantly in the cocultivation. Among them, the concentration of tryptophan, which is a precursor to indoleacetic acid, indoleacetic acid, aspartic acid, and L-glutamic acid increased while that of most soluble sugars decreased upon cocultivation. The fermentation filtrates of co-cultivation of LTR-2 and DnL1-1 showed significant promoting effects on germination and radicle length of wheat. A subsequent experiment demonstrated synergistic effects of differential metabolites caused by co-cultivation of DnL1-1 and LTR-2 on wheat germination. Comprehensive metabolic profiling may provide valuable information on the effects of DnL1-1 and LTR-2 on wheat growth.(AU)

Trichoderma harzianum inoculation promotes sweet sorghum growth in the saline soil by modulating rhizosphere available nutrients and bacterial community.

As one of the major abiotic stresses, salinity can affect crop growth and plant productivity worldwide. The inoculation of rhizosphere or endophytic microorganisms can enhance plant tolerance to salt stresses, but the potential mechanism is not clear. In this study, Trichoderma harzianum ST02 was applied on sweet sorghum [Sorghum bicolor (L.) Moench] in a field trial to investigate the effects on microbiome community and physiochemical properties in the rhizosphere soil. Compared with the non-inoculated control, Trichoderma inoculation significantly increased the stem yield, plant height, stem diameter, and total sugar content in stem by 35.52%, 32.68%, 32.09%, and 36.82%, respectively. In addition, Trichoderma inoculation improved the nutrient availability (e.g., N, P, and K) and organic matter in the rhizosphere soil and changed the bacterial community structure and function in both bulk and rhizosphere soil by particularly increasing the relative abundance of Actinobacter and N-cycling genes (nifH, archaeal and bacterial amoA). We proposed that T. harzianum ST02 could promote sweet sorghum growth under saline conditions by regulating available nutrients and the bacterial community in the rhizosphere soil.

Rhizosphere Microbiome and Phenolic Acid Exudation of the Healthy and Diseased American Ginseng Were Modulated by the Cropping History.

The infection of soil-borne diseases has the potential to modify root exudation and the rhizosphere microbiome. However, the extent to which these modifications occur in various monocropping histories remains inadequately explored. This study sampled healthy and diseased American ginseng (Panax quinquefolius L.) plants under 1-4 years of monocropping and analyzed the phenolic acids composition by HPLC, microbiome structure by high-throughput sequencing technique, and the abundance of pathogens by quantitative PCR. First, the fungal pathogens of Fusarium solani and Ilyonectria destructans in the rhizosphere soil were more abundant in the diseased plants than the healthy plants. The healthy American ginseng plants exudated more phenolic acid, especially p-coumaric acid, compared to the diseased plants after 1-2 years of monocropping, while this difference gradually diminished with the increase in monocropping years. The pathogen abundance was influenced by the exudation of phenolic acids, e.g., total phenolic acids (r = -0.455), p-coumaric acid (r = -0.465), and salicylic acid (r = -0.417), and the further in vitro test confirmed that increased concentration of p-coumaric acid inhibited the mycelial growth of the isolated pathogens for root rot. The healthy plants had a higher diversity of rhizosphere bacterial and fungal microbiome than the diseased plants only after a long period of monocropping. Our study has revealed that the cropping history of American ginseng has altered the effect of pathogens infection on rhizosphere microbiota and root exudation.

Metabolomics reveal metabolic variation caused by co-culture of Arthrobacter ureafaciens and Trichoderma harzianum and their impacts on wheat germination.

Arthrobacter ureafaciens DnL1-1 is a bacterium used for atrazine degradation, while Trichoderma harzianum LTR-2 is a widely used biocontrol fungus. In this study, a liquid co-cultivation of these two organisms was initially tested. The significant changes in the metabolome of fermentation liquors were investigated based on cultivation techniques (single-cultured and co-cultured DnL1-1 and LTR-2) using an UPLC-QTOF-MS in an untargeted metabolomic approach. Principle components analysis (PCA) and partial least squares discriminant analysis (PLS-DA) supervised modelling revealed modifications of the metabolic profiles in fermentation liquors as a function of interactions between different strains. Compared with pure-cultivation of DnL1-1, 51 compounds were altered during the cocultivation, with unique and significant differences in the abundance of organic nitrogen compounds (e.g. carnitine, acylcarnitine 4:0, acylcarnitine 5:0, 3-dehydroxycarnitine and O-acetyl-L-carnitine) and trans-zeatin riboside. Nevertheless, compared with pure-cultivation of LTR-2, the abundance of 157 compounds, including amino acids, soluble sugars, organic acids, indoles and derivatives, nucleosides, and others, changed significantly in the cocultivation. Among them, the concentration of tryptophan, which is a precursor to indoleacetic acid, indoleacetic acid, aspartic acid, and L-glutamic acid increased while that of most soluble sugars decreased upon cocultivation. The fermentation filtrates of co-cultivation of LTR-2 and DnL1-1 showed significant promoting effects on germination and radicle length of wheat. A subsequent experiment demonstrated synergistic effects of differential metabolites caused by co-cultivation of DnL1-1 and LTR-2 on wheat germination. Comprehensive metabolic profiling may provide valuable information on the effects of DnL1-1 and LTR-2 on wheat growth.

Antagonistic effects of Talaromyces muroii TM28 against Fusarium crown rot of wheat caused by Fusarium pseudograminearum.

Fusarium crown rot (FCR) caused by Fusarium pseudograminearum is a serious threat to wheat production worldwide. This study aimed to assess the effects of Talaromyces muroii strain TM28 isolated from root of Panax quinquefolius against F. pseudograminearum. The strain of TM28 inhibited mycelial growth of F. pseudograminearum by 87.8% at 72 h, its cell free fermentation filtrate had a strong antagonistic effect on mycelial growth and conidial germination of F. pseudograminearum by destroying the integrity of the cell membrane. In the greenhouse, TM28 significantly increased wheat fresh weight and height in the presence of pathogen Fp, it enhanced the antioxidant defense activity and ameliorated the negative effects of F. pseudograminearum, including disease severity and pathogen abundance in the rhizosphere soil, root and stem base of wheat. RNA-seq of F. pseudograminearum under TM28 antagonistic revealed 2,823 differentially expressed genes (DEGs). Most DEGs related to cell wall and cell membrane synthesis were significantly downregulated, the culture filtrate of TM28 affected the pathways of fatty acid synthesis, steroid synthesis, glycolysis, and the citrate acid cycle. T. muroii TM28 appears to have significant potential in controlling wheat Fusarium crown rot caused by F. pseudograminearum.

Chromosome doubling influences the morphological, physiological, biochemical and genetic traits related to essential oil biosynthesis of peppermint (Mentha piperita) under salinity stress.

Peppermint (Mentha piperita L.) is an important medicinal aromatic plant. In this study, the morphology, physiology, biochemistry and gene expression of chromosomes doubling peppermint (D1 lines) were analyzed. The analysis showed that D1 lines had larger, thicker and darker leaves, and stronger roots when planted in the pots, but delayed growth in the field condition. Under NaCl stress, the D1 lines increased cell oxidative defense through more active antioxidant enzymes and decreased the oxidative damages of cell membrane, leading to a significantly greater survival rate and photosynthesis intensity than WT lines. The size and density of glandular trichomes of D1 lines was larger, which contributed to its higher essential oil yield. In addition, chromosome doubling reduced the inhibition of NaCl stress on essential oil yield and quality, through changing the expression of genes in the oil biosynthesis pathway. The traits of chromosome doubling peppermint provide new technical and theoretical evidence for peppermint germplasm improvement.

An Intronless ß-amyrin Synthase Gene is More Efficient in Oleanolic Acid Accumulation than its Paralog in Gentiana straminea.

Paralogous members of the oxidosqualene cyclase (OSC) family encode a diversity of enzymes that are important in triterpenoid biosynthesis. This report describes the isolation of the Gentiana straminea gene GsAS2 that encodes a ß-amyrin synthase (ßAS) enzyme. Unlike its previously isolated paralog GsAS1, GsAS2 lacks introns. Its predicted protein product was is a 759 residue polypeptide that shares high homology with other known ß-amyrin synthases (ßASs). Heterologously expressed GsAS2 generates more ß-amyrin in yeast than does GsAS1. Constitutive over-expression of GsAS2 resulted in a 5.7 fold increase in oleanolic acid accumulation, while over-expression of GsAS1 led to a 3 fold increase. Additionally, RNAi-directed suppression of GsAS2 and GsAS1 in G. straminea decreased oleonolic acid levels by 65.9% and 21% respectively, indicating that GsAS2 plays a more important role than GsAS1 in oleanolic acid biosynthesis in G. straminea. We uses a docking model to explore the catalytic mechanism of GsAS1/2 and predicted that GsAS2, with its Y560, have higher efficiency than GsAS1 and mutated versions of GsAS2 in ß-amyrin produce. When the key residue in GsAS2 was mutagenized, it produced about 41.29% and 71.15% less ß-amyrin than native, while the key residue in GsAS1 was mutagenized to that in GsAS2, the mutant produced 38.02% more ß-amyrin than native GsAS1.

MAPK-mediated regulation of growth and essential oil composition in a salt-tolerant peppermint (Mentha piperita L.) under NaCl stress.

Peppermint (Mentha × piperita L.) is an important and commonly used flavoring agent worldwide, and salinity is a major stress that limits plant growth and reduces crop productivity. This work demonstrated the metabolic responses of essential oil production including the yield and component composition, gene expression, enzyme activity, and protein activation in a salt-tolerant peppermint Keyuan-1 with respect to NaCl stress. Our results showed that Keyuan-1 maintained normal growth and kept higher yield and content of essential oils under NaCl stress than wild-type (WT) peppermint.Gas chromatography-mass spectrometry (GC-MS) and qPCR results showed that compared to WT seedlings, a 150-mM NaCl stress exerted no obvious changes in essential oil composition, transcriptional level of enzymes related to essential oil metabolism, and activity of pulegone reductase (Pr) in Keyuan-1 peppermint which preserved the higher amount of menthol and menthone as well as the lower content of menthofuran upon the 150-mM NaCl stress. Furthermore, it was noticed that a mitogen-activated protein kinase (MAPK) protein exhibited a time-dependent activation in the Keyuan-1 peppermint and primarily involved in the modulation of the essential oil metabolism in the transcript and enzyme levels during the 12-day treatment of 150 mM NaCl. In all, our data elucidated the effect of NaCl on metabolic responses of essential oil production, and demonstrated the MAPK-dependent regulation mechanism of essential oil biosynthesis in the salt-tolerant peppermint, providing scientific basis for the economic and ecological utilization of peppermint in saline land.

Proper regeneration from in vitro cultured Arabidopsis thaliana requires the microRNA-directed action of an auxin response factor.

MicroRNAs (miRNAs) are important for the regulation of gene expression, and are involved in many developmental processes. A set of miRNAs which were differentially expressed between cells of totipotent (C1) and non-totipotent (C2) Arabidopsis thaliana calli was identified, some of which were affected during callus formation or shoot regeneration. One of those down-regulated after 10 days' incubation in shoot induction medium (SIM) was MIR160a, for which transcript abundance was lower in C1 than in C2. Over-expression of MIR160 compromised shoot regeneration from in vitro cultured A. thaliana cells, while the transgenic expression of a miR160-resistant form of ARF10 was associated with a high level of shoot regeneration. The latter transgenic line also showed an elevated expression level of shoot meristem-specific genes CLAVATA3, CUP-SHAPEDCOTYLEDON1 and -2, and WUSCHEL. ARF10 expression was concentrated at the initiation sites of shoots or leaves, while during the early phase of shoot regeneration, the accumulation of the ARF10 mRNA was lower in the wild type than in the mARF10 transgenics, in contrast to the pattern of miR160 expression. Thus, miR160 and ARF10 both appear to be components of the regulation of shoot regeneration in vitro.

Cloning and Functional Analysis of a beta-amyrin synthase gene associated with oleanolic acid biosynthesis in Gentiana straminea MAXIM.

Phytosterols and triterpenes are synthesized by oxidosqualene cyclases (OSCs) via the isoprenoid pathway. Here, GsAS1--a full-length beta-amyrin synthase cDNA isolated from Gentiana straminea MAXIM.--was characterized. Its open reading frame consists of 2268 bp, predicted to encode a 756 residue protein containing four QW and one Asp-Cys-Thr-Ala-Glu (DCTAE) motifs, which are both well conserved among known triterpene synthases. The deduced GsAS1 peptide sequence shares 76.2% homology with that of Panax ginseng beta-amyrin synthase. A phylogenetic analysis showed that GsAS1 is closely related to other plant OSCs, and particularly to the beta-amyrin synthases. When the GsAS1 sequence was heterologously expressed in Escherichia coli, an 88 kDa gene product was produced, and this reacted with the appropriate antibody. The sequence was also heterologously expressed in the Pichia pastoris yeast. GsAS1 is expressed in a tissue-specific manner, with its expression in the leaf being ca. 4.5-fold than that in the root, and nearly three-fold than that in the stem. GsAS1 expression was up-regulated by treatment with methyl jasmonate (MeJA) over a period from 6 h to 10 d post treatment. The accumulation oleanolic acid increased after induction by MeJA.