DOI: 10.5061/dryad.jm63xsjm6
title: Data from: Complementary effects of beneficial and non-beneficial mycorrhizal fungi on root phosphatase activity: A mycorrhizal “White Album” effect

description: Mycorrhizal fungi are diverse, with strains differing in the magnitude and
 types of benefits provided to their hosts. In mixture, mycorrhizal strains
 that have complementary functions could provide their hosts with greater
 benefits than any constituent strain in isolation. Conversely, mycorrhizal
 strains could also interfere with one another, competing for host
 resources, providing less benefits than the most beneficial strain. The
 actual, realized effects are likely a composite of these complementary and
 competitive effects. Spatial structure in the root system may
 allow plants to benefit from complementary benefits while reducing
 competition among mycorrhizal strains. I tested the role of spatial
 structure using a species of plant from the family Podocarpaceae, which
 host arbuscular mycorrhizal fungi inside both finely separated nodules and
 longer, contiguous sections of fine root. In root nodules,
 co-inoculation with both growth-promoting and non-growth-promoting
 arbuscular mycorrhizal fungi resulted in complementary increases in
 aggregate root / fungal phosphatase activity. This complementary effect
 was not present in longer, contiguous sections of fine root or in either
 root type with P-fertilization. I dub this interaction a “White
 Album effect”, a reference to the Beatles’ best-selling album, where
 complementary effects of single artists were only revealed when individual
 conflicts were avoided by separating band members and synthesizing their
 contributions in post-production.
description: <em>Growth experiment</em> Seeds
 of <em>Podocarpus gracilior </em>(syn. <em>Afrocarpus
 gracilior; Afrocarpus falcatus gracilior</em>), an east-African
 podocarp commonly grown as a hedge in tropical and Mediterranean climates,
 were purchased from a commercial nursery (The Banana Tree, Oregon House,
 California, USA, banana-tree.com). Seeds were germinated in Metromix soil
 (Hummert International, Earth City, Missouri, USA) that had been
 autoclaved for 8 hours at 121 ºC (in two four treatments on consecutive
 days). After 3 months, seedlings were transferred to
 pots containing a 50:50 (volume:volume) sand:soil mixture. Soil was
 collected from the Kankakee Sands Nature Preserve (Morocoo, Indiana, USA)
 and mixed at a 1:1 ratio with Indiana river sand. The soil mixture was
 autoclaved with the same time/temperature regime as the Metromix used as
 germination media. The background soil mix had a pH of 8.2, with 0.1%
 organic matter, 70 nitrate, 19 P, 33 K, and 102 Mg (all elemental
 quantities in ppm, using Mehlich-3 extraction).
 Experimental seedlings were inoculated either singly or in a
 50:50 mixture of <em>Claroideoglomus candidum</em> and/or
 <em>Gigaspora margarita </em>(deposited as NC172 and NC175, in
 INVAM, respectively). Single-strain inoculum had been maintained at
 Indiana University (Bloomington, IN, USA) in association with
 <em>Sorghum bicolor.</em> In each case, inoculate was prepared
 as follows. Prior to the experimental setup, single species cultures of
 <em>C. candidum</em> and <em>G. margarita</em>
 consisting of spores, mycelium, and fine root segments were air dried,
 chopped, and thoroughly mixed with the potting soil in a 1:5
 (volume:volume) ratio for use as fungal inoculum. The total inoculate
 volume was the same for each treatment, such that in the mixed inoculate
 treatment, each species was present at half the volume as in the
 single-strain treatment. Seedlings were grown over a
 period of 6-months in a greenhouse with daily watering. Photosynthetic
 active radiation was not measured on the greenhouse bench where
 experimental seedlings were grown; however, published figures from an
 experiment conducted during the same period in the same greenhouse give an
 average value of 1339 +/- 71 μmol m<sup>-2</sup>
 s<sup>-1</sup> (Zheng et al., 2015).  Seedlings were
 fertilized twice (2 and 12 weeks after planting) with either a
 no-phosphorus fertilizer (P-,Scott’s 15-0-15) or a complete mixed
 fertilizer (P+, Scott’s 15-15-15). Both fertilizers contained, in units %
 mass, 15% N (from urea) and 15% K (soluble potash), 0.02% Boron, 0.05 %
 Cu, 3% Fe, 0.05% Mn, and 0.05% Zn. The P+ fertilizer also contained 15% P.
  Each mycorrhizal x fertilizer treatment had 8 replicates, for a total of
 64 plants.  <em>Plant performance
 measurements</em> Plants were harvested by gently
 removing them from their pots and washing adhering soil particles from
 roots under water. Root segments for enzyme assays, and root length
 colonized by mycorrhizal fungi measurements were excised and weighed. The
 rest of the plant was dried at 80°C for one week and leaves, stems, and
 roots weighed separately. Dried leaf samples were sent to the Cornell
 Nutrient Analysis Laboratory (cnal.cals.cornell.edu) and foliar P
 concentration analysed via dry ash elemental analysis using inductively
 coupled plasma atomic emission spectrometry (ICP-AES, Spectro, Analytical
 Instruments). Additionally, dry combustion at 1100°C was used to measure
 %C and %N of foliar dry mass using non dispersive infrared and thermal
 conductivity detection analysis, respectively (SNC-100 Carbon and Nitrogen
 Analyzer, Skalar Primacs). <em>Mycorrhizal
 colonization</em> The % root length colonized was
 measured using the standard clearing and staining protocol of (McGonigle
 et al., 1990). Eight 2 cm nodulated root segments (technical replicates)
 were cleared in KOH and stained with Trypan blue. Mycorrhizal colonization
 was scored as “present” if it appeared in at least one technical
 replicate, based on 6 complete passes along the root axis for the cortex
 of both the fine roots and the adhering nodules.
 <em>Phosphatase enzyme assays</em>
 Immediately following plant harvest, four root tips that
 contained both root nodules and long, un-nodulated sections of fine root
 were excised from each experimental plant for use in phosphomono- and
 diesterase enzyme assays. Root samples contained either a 2 cm section of
 roots with all nodules removed (fine roots) or 10 individual root nodules.
 For each replicate plant and root structure, potential enzyme activity was
 assayed in 8 subsamples (technical replicates). Statistical analyses were
 conducted on the average value of up to 8-technical replicates, with
 exclusions made for outliers &gt; 2 standard deviations from the mean
 of each experimental plant x root-type. Root samples
 were added to fluorescence assay well plates along with 200 μL of acetate
 buffer (pH 5.0). Potential P-mono and -diesterase activity was measured
 fluorometrically by adding MUB-phosphate substrates to root samples.
 Additionally, blank wells (buffer), sample controls (buffer and root
 sample), reference standards (MUB standard and buffer), quench standard
 wells (MUB standard and root sample), and negative control wells
 (substrate and buffer) were run to control for quenching by the substrate
 and buffer alone. Samples were incubated in the dark at 23°C for 2 hrs. 
 At the end of incubation, 20 μL of 1 M NaOH was added to each well,
 raising the pH to improve fluorescence (German et al., 2011). Fluorescence
 was measured on a plate reader using 365 nm excitation and 450 nm emission
 filters. Potential enzyme activity rates for each sample were calculated
 as μmol MUB g<sup>−1</sup> dry root
 h<sup>−1</sup>. Dry root mass was determined by multiplying
 fresh root mass to the ratio of dry to fresh root mass of one additional
 replicate sample that was not assayed for enzymes.
 <em>Statistical Analyses </em> For
 all dependent variables (plant growth, biomass allocation, foliar nutrient
 concentrations, and nodule and fine root P-mono and di-esterase activity),
 ANOVA was used to partition variance among the main effects of mycorrhizal
 treatment (df=3), fertilization (df=1) and the interaction between
 mycorrhiza x fertilization (df=3) (table 1). Post-hoc
 tests were used to assess differences in treatment means among mycorrhizal
 treatments within each fertilization treatments (least significant
 difference with Bonferroni correction for multiple contrasts, using the
 ‘agricolae’ package in R).  Chi-square tests were used
 to assess the probability that mycorrhizal colonization frequency was
 distributed according to chance between different groups (rejecting the
 null-hypothesis when p&lt;0.05). Differences in the frequency of
 mycorrhizal fungal colonization were tested between different root types
 (nodules vs. fine-roots, df=1) and mycorrhizal and fertilization
 treatments (df=9).   Linear regression was used to
 assess the amount of variation in nodule enzyme activity that could be
 explained by fine root enzyme activity and, within a root group, the
 amount of P-diesterase activity that could be explained by variability in
 P-monoesterase activity. Additionally, principal component analyses were
 used to assess the relationship between enzyme activities, growth, and
 foliar nutrient concentrations.
description: # Complementary effects of beneficial and non-beneficial mycorrhizal fungi
 on root phosphatase activity: a mycorrhizal “White Album” Effect
 [https://doi.org/10.5061/dryad.jm63xsjm6](https://doi.org/10.5061/dryad.jm63xsjm6) ## Description of the data and file structure This is a ReadMe file for data deposited for Steidinger 2024 (Complementary effects of beneficial and non-beneficial mycorrhizal fungi on root phosphatase activity: a mycorrhizal "White Album" Effect). ### Files and variables #### File: Steidinger\_2024\_FE2024\_00721.zip **Description:** A zip file containing .Rmd and .csv files needed for recreating analysis.  The archive contains 6 files (including this ReadMe). **Steidinger2024_FE.Rmd** R code required to replicate all analysis presented in the manuscript (and supplement). The code is annotated for clarity throughout. It requires both R and RStudio. Additionally, there are package dependencies listed inside, which may have to be installed. The code works by accessing four .csv files (included in the archive), which are described below. I begin with some definitions and synonyms. The same mycorrhizal treatments are described using different character strings. **Definitions & Synonyms** \[Myc, Trt] = Mycorrhizal treatment \[Control] = No mycorrhizal fungi \[Glomus white, Glom] = single strain innoculum of Claroideoglomus candidum (Glomus white is an out of date synonym) \[Gigaspora, Gigaspora margarita] = single strain innoc of Gigaspora margarita \[Glomus + Gigaspora, GigGlo, Mix] = 50:50 mixture (by volume) of Claroideoglomus candidum and Gigaspora margarita \[Fert, fert] = Fertilization treatment \[NoP, No P] = No phosphorus added to experimental plants \[P, P+] = phosphorus added to experimental plants **file: Podocarp_drymass.csv** [64 rows by 11 columns] \[1] Myc= mycorrhizal treatment (Glomus white, Gigaspora margarita, Glomus + Gigaspora, Control) \[2] Fert = fertilization treatment (NoP, P) \[3] Rep# = replication number (plant) \[4] Leaf.mass = mass of dry leaves (in mg) per plant \[5] Stem.mass = mass of dry stems (in mg) per plant \[6] Root.mass = mass of dry roots (in mg) per plant \[7] Total.Mass = total dry mass of plant \[8] LMF = leaf mass fraction (Leaf.mass/Total.Mass) \[9] SMF= stem mass fraction (Stem.mass/Total.Mass) \[10] RMF = root mass fraction (Root.mass/Total.Mass) \[11] AG/BG = ratio of above ground to below ground mass (Leaf.mass + Stem.mass)/Root.mass **file: PodocarpEnzyme_IU.csv** [64 rows by 7 columns] All enzyme data [4-7] is in units of micromol per hour per gram of dry tissue \[1] Myc = mycorrhizal treatment (Glow, Gig, GigGlo, Control) \[2] fert = fertilisation treatment (NoP, P) \[3] rep = replication (plant) \[4] nod_ap = nodule phosphomonoesterase activity \[5] fr_ap = fine root phosphomonoesterase activity \[6] nod_pd = nodule phosphodiesterase activity \[7] fr_pd = fine root phosphodiesterase activity **file: PodocarpCN8June2016.csv** [64 rows by 6 columns] In this file, mycorrhizal treatment is Trt pN and pC are in % of foliar dry mass \[1] Trt = mycorrhizal treatment (Control, Glomus, Gigaspora, Mix) \[2] Fert = fertilisation treatment (No P, P+) \[3] Rep = replication (plant) \[4] Samp = sample number (for analyser, numbered sequentially 1-64) \[5] pN = % N \[6] pC = % C **file: 27May2016_PodocarpFoliarNutrients.csv** [64 rows by 17 columns] \[1] Myc = mycorrhizal treatment (Control, Glomus, Gigaspora, Mix) \[2] Fert = fertilisation treatment (No P, P+) \[3] Rep = replication (plant) \[4] Sample # = sample number (for analyser) \[5] CNAL Sample = Cornell Nutrient Analysis Lab Sample (concatenation of 26369-Sample #) \[6] Al = foliar Aluminum concentration (ppm) \[7] B = foliar Boron concentration (ppm) \[8] Ca = foliar Calcium concentration (ppm) \[9] Cu = foliar Copper concentration (ppm) \[10] Fe = foliar Iron concentration (ppm) \[11] K = foliar Potassium concentration (ppm) \[12] Mg = foliar Magnesium concentration (ppm) \[13] Mn = foliar Manganese concentration (ppm) \[14] Mo = foliar Molybdenum concentration (ppm) \[15] Na = foliar Sodium concentration (ppm) \[16] P = foliar Phosphorus concentration (ppm) \[17] Zn = foliar Zinc concentration (ppm) ## Code/software R Studio used for analysis