Sesquiterpenes of the ectomycorrhizal fungus Pisolithus microcarpus alter root growth and promote host colonization.

Trees form symbioses with ectomycorrhizal (ECM) fungi, maintained in part through mutual benefit to both organisms. Our understanding of the signaling events leading to the successful interaction between the two partners requires further study. This is especially true for understanding the role of volatile signals produced by ECM fungi. Terpenoids are a predominant class of volatiles produced by ECM fungi. While several ECM genomes are enriched in the enzymes responsible for the production of these volatiles (i.e., terpene synthases (TPSs)) when compared to other fungi, we have limited understanding of the biochemical products associated with each enzyme and the physiological impact of specific terpenes on plant growth. Using a combination of phylogenetic analyses, RNA sequencing, and functional characterization of five TPSs from two distantly related ECM fungi (Laccaria bicolor and Pisolithus microcarpus), we investigated the role of these secondary metabolites during the establishment of symbiosis. We found that despite phylogenetic divergence, these TPSs produced very similar terpene profiles. We focused on the role of P. microcarpus terpenes and found that the fungus expressed a diverse array of mono-, di-, and sesquiterpenes prior to contact with the host. However, these metabolites were repressed following physical contact with the host Eucalyptus grandis. Exposure of E. grandis to heterologously produced terpenes (enriched primarily in γ -cadinene) led to a reduction in the root growth rate and an increase in P. microcarpus-colonized root tips. These results support a very early putative role of fungal-produced terpenes in the establishment of symbiosis between mycorrhizal fungi and their hosts.

Fungal metabolism and free amino acid content may predict nitrogen transfer to the host plant in the ectomycorrhizal relationship between Pisolithus spp. and Eucalyptus grandis.

Ectomycorrhizal (ECM) fungi are crucial for tree nitrogen (N) nutrition; however, mechanisms governing N transfer from fungal tissues to the host plant are not well understood. ECM fungal isolates, even from the same species, vary considerably in their ability to support tree N nutrition, resulting in a range of often unpredictable symbiotic outcomes. In this study, we used isotopic labelling to quantify the transfer of N to the plant host by isolates from the ECM genus Pisolithus, known to have significant variability in colonisation and transfer of nutrients to a host. We considered the metabolic fate of N acquired by the fungi and found that the percentage of plant N acquired through symbiosis significantly correlated to the concentration of free amino acids in ECM extra-radical mycelium. Transcriptomic analyses complemented these findings with isolates having high amino acid content and N transfer showing increased expression of genes related to amino acid transport and catabolic pathways. These results suggest that fungal N metabolism impacts N transfer to the host plant in this interaction and that relative N transfer may be possible to predict through basic biochemical analyses.

Speciation Underpinned by Unexpected Molecular Diversity in the Mycorrhizal Fungal Genus Pisolithus.

The mutualistic ectomycorrhizal (ECM) fungal genus Pisolithus comprises 19 species defined to date which colonize the roots of >50 hosts worldwide suggesting that substantial genomic and functional evolution occurred during speciation. To better understand this intra-genus variation, we undertook a comparative multi-omic study of nine Pisolithus species sampled from North America, South America, Asia, and Australasia. We found that there was a small core set of genes common to all species (13%), and that these genes were more likely to be significantly regulated during symbiosis with a host than accessory or species-specific genes. Thus, the genetic "toolbox" foundational to the symbiotic lifestyle in this genus is small. Transposable elements were located significantly closer to gene classes including effector-like small secreted proteins (SSPs). Poorly conserved SSPs were more likely to be induced by symbiosis, suggesting that they may be a class of protein that tune host specificity. The Pisolithus gene repertoire is characterized by divergent CAZyme profiles when compared with other fungi, both symbiotic and saprotrophic. This was driven by differences in enzymes associated with symbiotic sugar processing, although metabolomic analysis suggest that neither copy number nor expression of these genes is sufficient to predict sugar capture from a host plant or its metabolism in fungal hyphae. Our results demonstrate that intra-genus genomic and functional diversity within ECM fungi is greater than previously thought, underlining the importance of continued comparative studies within the fungal tree of life to refine our focus on pathways and evolutionary processes foundational to this symbiotic lifestyle.

Purification and use of carotenoid standards to quantify cis-trans geometrical carotenoid isomers in plant tissues.

Reverse-phase high-performance liquid chromatography (HPLC) is a preferred method used to identify and quantify carotenoids. Here, we describe a straightforward, reliable, and cost-effective protocol to purify and develop individual carotenoid standards for absolute quantification of carotenoids, including selected cis-trans (geometric) isomers. Analytical techniques to extract, purify and collect individual carotenoids using an HPLC system equipped with a Diode Array Detector (DAD) and fraction collector are described. Carotenoids were separated and identified by their characteristic ultraviolet-visible (UV-Vis) absorption spectra and individually isolated based on their retention times using a C30 column. This chapter outlines how to prepare standard calibration curves using known quantities of purified and/or commercially available carotenoids. A series of molar extinction and slope coefficients for phytoene, phytofluene, ζ-carotene, neurosporene, pro-lycopene, all trans-lycopene, lutein, ß-carotene, zeaxanthin, antheraxanthin, violaxanthin, neoxanthin, capsanthin, capsorubin and ß-cryptoxanthin are defined to enable absolute quantification of their abundance in plant, animal, and bacterial tissues. Different approaches for reporting carotenoid abundance by absolute concentration, relative composition, and/or using ratios of different pigments are provided as a convenient resource for carotenoid researchers.

Toxic pollutants emitted from thermal decomposition of phthalimide compounds.

Phthalimide (PI) and tetrahydrophthalimide (THPI) are two structurally similar compounds extensively used as intermediates for the synthesis of variety of industrial chemicals. This paper investigates the thermal decomposition of PI and THPI under oxygen rich to oxygen lean conditions, quantifying the production of toxicants and explaining their formation pathways. The experiments involved a plug flow reactor followed by silica cartridges, activated charcoal trap and a condenser, with the decomposition products identified and quantified by Fourier transform infrared spectroscopy (FTIR), gas chromatography-mass spectrometry (GC-MS) and micro gas chromatography (µGC). The density functional theory (DFT) calculations served to obtain dissociation energies and reaction pathways, to elucidate the reaction mechanism. The oxidation of PI and THPI produced several toxic nitrogen-containing gases and volatile organic compounds, including hydrogen cyanide, isocyanic acid, nitrogen oxides, benzonitrile, maleimide and tentatively identified benzenemethanimine. The detection of dibenzo-p-dioxin (DD) and dibenzofuran (DF) suggests potential formation of the toxic persistent organic pollutants (POPs) in fires involving PI and THPI, in presence of a chlorine source. The oxidation of THPI produced 2-cyclohexen-1-one, a toxic unsaturated ketone. The results of the present study provide the data for quantitative risk assessments of emissions of toxicants in combustion processes involving PI and THPI.