DOI: 10.6071/m3cm2f
title: Metabolite, qPCR, and metadata for relationships between Sphaerulina musiva infection and the Populus microbiome and metabolome

description: Pathogenic fungal infections in plants may, in some cases, lead to
 downstream systematic impacts on the plant metabolome and microbiome that
 may either alleviate or exacerbate the effects of the fungal pathogen.
 While Sphaerulina musiva is a well-characterized fungal pathogen which
 infects Populus tree species, an important wood fiber and biofuel
 feedstock, little is known about its systematic effects on the metabolome
 and microbiome of Populus. Here, we investigate the microbiome and
 metabolome of P. trichocarpa and P. deltoides leaves and roots and the
 microbiome of the leaf and root endospheres, phylloplane and rhizosphere
 to understand the systematic impacts of S. musiva abundance and infection
 on Populus species in a common garden field setting. We find that S.
 musiva is indeed present in both P. deltoides and P. trichocarpa, but S.
 musiva abundance was not statistically related to stem canker onset. We
 also find that the leaf and root metabolomes significantly differ between
 the two Populus species and that certain leaf metabolites, particularly
 the phenolic glycosides, salirepin and salireposide, are diminished in
 canker-infected P. trichocarpa trees compared to their uninfected
 counterparts. Furthermore, we found significant associations between the
 metabolome, S. musiva abundance and microbiome composition and
 α-diversity, particularly in P. trichocarpa leaves. This suggests that the
 effects of S. musiva on P. trichocarpa trees are systemic and not confined
 to the site of canker infection.
description: <strong><em>S. musiva
 abundance</em></strong> <em>S.
 musiva</em> abundance was measured by quantitative polymerase chain
 reaction (qPCR) targeting the beta-tubulin gene following Abraham et al.
 (35) (NABtF: 50-CGACCTGAACCACCTTGTCT-30 and NABtR:
 50-CACGGTAACAGCGCGGAACGA-30). Template DNA concentrations were normalized
 to 10 ng ul-1, and PCRs were conducted in a 384 well plate containing
 1 × SYBR Green (iTaq Universal SYBR® Green Supermix), 500 nmol of each
 primer, and 2 ul template DNA for a total volume of 20 ul. To generate a
 standard curve, S. musiva DNA standards were extracted from a cultured
 representative (S. musiva MN14), and diluted 1:10, 1:100, 1:1000 and
 1:10000, quantified using the Qubit dsDNA BR Assay Kit (Invitrogen,
 Waltham, MA). To detect non-specific amplification, negative controls (no
 template control and Marsonnina brun 441) were included in the assays. The
 reactions were carried out using a 7900HT Fast Realtime PCR machine
 (Applied Biosystems, Waltham, MA, USA) under the following conditions:
 initial denaturation at 95 °C for 10 min, followed by 40 cycles of
 amplification at 95 °C for 15 s and 58 °C for 30 s.
 <strong><em>Metabolite extraction and
 processing</em></strong> Leaf and root
 tissue metabolites were processed and analyzed by gas chromatography-mass
 spectrometry (GC-MS). Tissues stored at -80 °C were lyophilized and then
 powdered using a Spex Geno-Grinder (Metuchen, NJ, USA). Approximately 25
 mg of powdered leaf and 45 mg of powdered root material was extracted
 twice with 80% ethanol. Sorbitol (75 µl; 1 mg ml-1) was added to the first
 extract and used as an internal standard. After combining the extracts, a
 1 ml aliquot of the leaf extract and a 250 µl aliquot of the root extract
 was dried under a stream of nitrogen. Both types of samples were then
 derivatized by dissolving aliquots in 500 µl acetonitrile followed by an
 addition of 500 µl of N-Methyl-N-(trimethylsilyl)trifluoroacetamide with
 1% trimethylchlorosilane (MSTFA + 1% TMCS) and heating at 70 °C for 1 h to
 produce trimethylsilyl (TMS) metabolite derivatives. After 2 days, 1 µl
 was injected to the GC-MS and analyzed. Metabolites were identified using
 the Wiley Registry, 10th edition as well as a large, user-created database
 of TMS-derivatized metabolites. Metabolites were quantified relative to
 the internal standard and normalized to the mass extracted, extract volume
 analyzed and injection volume. 
description: The sequence data for this dataset is deposited in the Sequence
 Read Archive under BioProject XXX.