DOI: 10.5061/dryad.6m905qg8c
title: Mycorrhizal fungi compromise production of endophytic alkaloids, increasing plant susceptibility to an aphid herbivore

description: Symbiosis plays a critical role in plant biology. Temperate grasses often
 associate with several symbiotic fungi simultaneously,
 including Epichloë endophytes and arbuscular mycorrhizal (AM)
 fungi, in shoots and roots, respectively. These symbionts often modulate
 plant-herbivore interactions by influencing nutritional traits (i.e., AM
 fungi-mediated nutrient uptake) and/or the secondary chemistry (i.e.,
 endophytic alkaloids) of their host plant. Moreover, such grasses also
 accumulate large amounts of silicon (Si) from the soil, which can be
 deposited in tissues to act as a physical antiherbivore defense. Recent
 evidence suggests that both endophytes and AM fungi independently
 facilitate Si uptake. However, the consequences of their interactions for
 piercing-sucking insects (i.e., aphids), or whether Si supply, endophytes
 and AM fungi interact in this regard, are currently unknown. While Si
 deposition may be less effective against aphids than other herbivores
 (i.e., chewing caterpillars), Si supply can also alter plant secondary
 metabolite defenses, which could affect sucking insects. In a factorial
 greenhouse experiment we evaluated whether these components, acting alone
 or in combination, altered 1) foliar primary chemistry, 2) Si and
 symbiont-chemical (endophytic alkaloids) defenses, as well as 3)
 performance of the bird-cherry oat aphid (Rhopalosiphum padi) feeding on
 tall fescue (Festuca arundinacea). Endophytes decreased all aphid
 performance parameters, including population growth and repoduction by
 40%, but their impact was reversed by the presence of AM fungi, leading to
 a 52% increase in aphid performance compared to plants solely hosting
 endophytes. This improvement in performance was associated to reduced
 loline alkaloid levels and higher shoot nitrogen in AM-endophytic plants.
 Endophytes and AM fungi exhibited antagonism, with endophytes reducing AM
 colonization by 34% and AM presence decreasing endophyte loline alkaloids
 by 44%. While both fungi jointly increased Si accumulation by 39% under
 Si-supplied conditions, Si had no noticeable effects on aphids. Moreover,
 although Si supply had no identifiable effects on AM colonisation, it
 reduced endophyte peramine alkaloids by 24%. Synthesis: Our findings
 indicate that symbiotic fungal partnerships and silicon provision may
 benefit plants but could weaken anti-herbivore defenses when combined.
 Revealing the complex interactions among diverse fungal symbionts and
 showcasing their effects on different anti-herbivore defenses (chemical
 and physical) and herbivore performance for the first time.
description: <em>Plants, Insects and Experimental
 procedure</em><em> </em> Two
 hundred tall fescue plants (<em>Festuca arundinacea</em> cv.
 INIA Fortuna) either <em>Epichloë</em>-free (Nil; N = 100) or
 infected with the animal safe AR584 novel <em>Epichloë coenophiala
 </em>(formerly <em>Neotyphodium
 coenophiala</em>)<em> </em>strain (N = 100) were grown
 individually from seed in one-litre pots. Initially, these seeds were
 inoculated with the AR584 endophyte strain by AgResearch NZ, in
 collaboration with INIA under a work cooperation agreement. Following the
 endophyte incorporations, two distinct seed lines were maintained at the
 Margot Forde Forage Germplasm Centre (Palmerston North, NZ). Pots
 contained gamma-irradiated (50 kGy) 1:1 topsoil-sand mix that was
 homogenised with a soil mixer. Sand was used to reduce bioavailable
 phosphorus to &lt;16 mg P kg<sup>-1</sup> and silicon to
 &lt; 11 mg Si kg<sup>-1</sup> (Table S1 Appendix
 S1). The top ¼ of each pot received one of two AM fungi
 treatments: No AM (-AM) or Commercial AM (+AM). +AM was achieved by
 inoculating soil with Start-up Ultra© (Microbe Smart Plty. Ltd., South
 Australia) which contains spores from four isolates of
 <em>Rhizophagus irregularis</em> (formerly<em> Glomus
 intraradices</em>). Before inoculation, spores were extracted and
 separated from the inert substrate (calcined diatomaceous earth) using wet
 sieving and applied at a rate equivalent to the recommended rate of 250 g
 of inoculum per 2.5 L with hydrating water. To generate -AM treatment the
 same steps were performed, except that the inoculant (extracted spores)
 was sterilised by autoclaving twice (121°C) to ensure spores were
 non-viable. To standardise the microbial community within each pot, the
 soil-sand mix in all treatments received a microbial filtrate (300 ml/10
 L) a week after irradiation. This filtrate was created by using the
 extraneous extraction solution (without spores) from both the commercial
 AM fungi after wet sieving and the soil before irradiation following the
 procedure described in Frew et al., (2018). Only the top ¼ of soil in each
 pot was inoculated with the -AM or the +AM solutions.
 Plants were grown in a single, naturally lit glasshouse chamber
 at 22/18°C (day/night) and 60% relative humidity at the Hawksbury
 Institute for the Environment in Richmond, NSW, Australia. Pots were
 randomly shifted weekly to avoid position bias, and manually irrigated
 three times a week with 50 ml of either a solution with (+Si) or without
 (-Si) silicon (Si). Briefly, the +Si solution contained potassium silicate
 (K<sub>2</sub>SiO<sub>3</sub>; Agsil32, PQ
 Australia) at a concentration of 2 mM, whereas the -Si solution contained
 KCl to balance the addition of potassium in the +Si treatment. Further,
 using HCl, both solutions were adjusted to pH 7 following procedures
 described in Hall et al., (2020). This resulted in a total of eight
 treatments that consisted of combinations of two different endophyte
 treatments (Nil or AR584), two AM fungi treatments (-AM or +AM), and two
 Si supply treatments (-Si or +Si). Each treatment combination contained 25
 pots/replicates (200 plants in total; Fig. 1 Symbiont
 treatments). After eight weeks, 10 plants per treatment
 combination were inoculated and caged with five adult (apterous) bird
 cherry-oat aphid <em>Rhopalosiphum padi </em>(Linnaeus, 1758).
 Transparent cylindrical Perspex cages with meshed air vents were fitted to
 the pots of all plants similar as to those used by figure 1S of
 Cibils-Stewart et al (2021). Cultures of <em>R. padi </em>were
 established from a single parthenogenetic female obtained from a
 laboratory culture at Agriculture Victoria Research (Horsham, VIC,
 Australia) and reared on caged barley (<em>Hordeum
 vulgare</em> cultivar ‘Hindmarsh’). Aphids reared on barley before
 the experiment prevent prior exposure to the plant species, standardize
 the setup, control the aphid population, and reduce variability in
 behaviour and responses. On a weekly basis, ten teneral adult females were
 transferred to new caged barley for 24 h. Adults were then removed,
 leaving only same-age nymphs on plants. This procedure ensured same-aged
 aphids for experiment initiation. Aphid-inoculated
 plants were compared with 10 caged aphid-free plants, selected at random,
 allowing us to determine the impact of aphid infestation on plant
 parameters. Using these plants, treatment effects on aphid population
 performance parameters were determined (section 2.2.1; Fig. 1 Impact on
 aphid population). The remaining five plants per treatment (Table S2) were
 inoculated with three apterous adults <em>R. padi</em> each
 (120 aphids total). Each individual aphid was confined to a clip cage that
 followed Cibils-Stewart et al (2015) design, featuring a 0.5-cm thick foam
 rectangle (6.2 x 3.6 cm outside, 5.1 x 2.5 cm inside) with adhesive on
 tops and bottoms. Pre-applied adhesive secured no-see-um mesh on one side
 for leaf cages, preventing aphid escape while ensuring ventilation for
 aphids. The remaining adhesive was attached to the leaf surface. Following
 Rowe et al., (2020) procedures, each cage was placed on the youngest leaf
 of three different tillers per plant. Using these plants, treatment
 effects on individual aphid performance parameters were determined
 (section 2.2.2) (see Table S2 for treatment combinations; Fig. 1 Impact on
 individual aphid performance). To assess the effect of
 the symbionts and Si supply (alone and in combination) on plant
 physiological performance, net photosynthesis (Anet), stomatal conductance
 (gs), and water-use efficiency (WUE) were measured in aphid-free plants
 with an infrared gas analyser (IRGA, LI6400XT, Li-Cor, Lincoln, NE, USA)
 following Vandegeer et al., (2020) procedures. Additionally, using a
 Minolta chlorophyll SPAD 502 meter chlorophyll content (i.e. greenness)
 was measured in the same leaves, and utilized as a proxy of plant vigour
 (Druille et al., 2013). Immediately after harvest the
 symbiotic status of plants was detected (section 2.3); for this a 1 g
 section of the root was preserved in ethanol to determine AM status
 (section 2.3.2). Roots were carefully washed, and a 1 g (wet weight)
 subsample was taken from the same area from all replicates per treatment
 combination. Subsamples were stored in tissue embedding cassettes in 70%
 ethanol. Remaining shoots and roots were snap frozen in liquid nitrogen,
 freeze-dried, weighed (MS-TA Analytical balances; Mettler Toledo), and
 ball-milled to fine powder (Mixer Mills MM 400; Retsch). Samples were
 stored at -20°C until further chemical analysis (section 2.4). The removed
 root sample was not accounted for in the overall root weight (Fig.
 1). <em>Aphid parameters</em>
 <em>Aphid population </em> Three
 weeks after aphid inoculation, aphid populations were categorized
 (apterous, adults and nymphs) and counted. The finite rate of population
 change was then calculated for the entire 21-day trial
 (λ<sub>21</sub>) as a ratio of change in aphid densities at
 the start (N<sub>0</sub>) and end (N<sub>21</sub>)
 of assay, where λ<sub>21</sub> =
 N<sub>21</sub>/N<sub>0</sub>.
 <em>Individual aphid performance</em>
 After 24 h, the original aphid and all but the youngest nymph
 (founder nymphs) was removed from cage. Nymphal development (Pr;
 pre-reproductive period), adult fecundity (F; number of born nymphs
 daily), and longevity (L) of the founder nymphs were recorded daily for
 the entire lifespan of each aphid. Newly born nymphs were removed daily
 from clip cages after daily adult fecundity recordings. Using the above
 parameters, the intrinsic rate of increase (r<sub>m</sub>) and
 the generation time (GT) were calculated as r<sub>m</sub> =
 0.74 (ln F)/L) and GT=4Pr/3, respectively (Wyatt & White,
 1977). <em>Symbiont
 detection</em> <em>Endophyte in
 shoots</em> The fresh cut end of one tiller of
 each plant was pressed onto a nitrocellulose membrane (tissue-print
 immunoblotting) to confirm absence (Nil) or presence (AR584) of
 <em>Epichloë</em> in planta following di Menna, et al., (2012)
 procedures; this was further confirmed with histological staining
 following Cibils-Stewart et al., (2020) procedures. No colonization was
 detected in the endophyte-free (Nil) plants.
 <em>AM fungi in roots</em>
 Mycorrhizal colonization was scored  following Frew et al (2018)
 procedures with minor modifications. Briefly, samples within cassettes
 were rinsed with cold water and cleared with 10% KOH at room temperature
 for five consecutive days. Samples were then water-rinsed and stained with
 5% ink-vinegar in a 60°C water bath for 10 mins (Vierheilig et al., 2005).
 Finally, samples were rinsed until water ran clear and were submerged in
 lacto glycerol (de-staining solution) overnight. Ten 1 cm segments per
 sample were mounted on glass slides with glycerine under a cover slip and
 a minimum of 25 intersects were scored for presence of AM fungi using the
 intersect method (McGonigle et al., 1990). Only hyphae for which there was
 a visible connection to AM fungal structures (arbuscules, vesicles,
 spores) were counted. No colonization was detected in the -AM
 plants. <em>Chemical
 analyses</em><em> </em>
 <em>Shoot silicon, carbon and nitrogen</em>
 Eighty milligrams of ground leaf material were analysed to
 measure Si concentration using an X-ray fluorescence spectrometer (Epsilon
 3 ×; PANalytical, EA Almelo, The Netherlands) following the method of
 Reidinger et al., (2012) and using certified plant material of known Si
 concentrations (see Hiltpold et al., 2016 for full details). A subsample
 of 6-7 mg of ground tissue of aphid-free plants used for Si analysis was
 further utilized to measure carbon and nitrogen concentrations with an
 elemental analyser (FLASH EA 1112 Series CHN analyser, ThermoFinnigan,
 Waltham, MA, USA). Epichloë
 <em>alkaloid</em><em>
 concentrations</em> Shoot peramine production was
 analysed according to Berry et al., (2019) using a Thermo LTQxl linear
 ion-trap mass spectrometer equipped with an Accela 1250 HPLC in
 endophytic-plants only. Chromatography was achieved using a SeQuant
 ZIC-HILIC column (150 × 2.1mm, 5 µ, Merck KGaA, Darmstadt, Germany).
 Samples were extracted with 500 µL of 50% methanol (containing 1.7 µg/mL
 homoperamine as an internal standard) for 1 h in the dark. Samples (20 mg)
 were then centrifuged (5000 g, 5 min) and the supernatant transferred to 2
 mL amber HPLC vials via a 0.45 µm syringe filter (PVDF). Peak integration
 was conducted using LCQuan 2.7 (Thermo Fisher Scientific Inc., San Jose,
 CA, USA); alkaloid concentrations were determined from peak areas and
 calculated standard curves. Production of the three loline derivatives
 <em>N</em>-acetylloline (NAL),
 <em>N</em>-acetylnorloline (NANL), and
 <em>N</em>-formylloline (NFL) (added to afford the total
 lolines) weas quantified following Bastías et al. (2018).
  <em>Statistical analysis</em><em>
 </em> R was utilized for all statistical analyses
 (R Core Team, 2015) and all figures were produced using the ‘ggplot2’
 package (Wickham, 2016). Assumptions of normality for residuals for all
 models were verified according to the inspection of quantile-quantile
 plots, and variables log-transformed in case of lack of normality (aphid
 λ). Shoot and root biomass of freeze-dried tissue,
 along with foliar Si concentration were analysed with a four-way analysis
 of variance (ANOVA) using Si supply, AM fungi, endophyte and herbivory as
 fixed effects. AM fungi colonisation, alternatively, was analysed with a
 three-way ANOVA using Si supply, endophyte, and herbivory as fixed
 effects; only +AM plants were utilized for this analysis.
 Aphid parameters were analysed using a three-way ANOVA, with Si
 supply, AM fungi and endophyte as fixed effects using aphid-inoculated
 plants. Likewise, plant physiological and nutritional traits were analysed
 using a three-way ANOVA, with Si supply, AM fungi and endophyte as fixed
 effects, aphid-free plants only. For all models described above,
 differences between treatment means were determined by Tukey’s HSD test
 using the ‘emmeans’ package (Lenth et al., 2018). A multivariate analysis
 of variance (manova) using Si supply, AM fungi and herbivory as fixed
 effect factors was utilized to determine differences in overall alkaloid
 profiles (sum of all alkaloids produced by the AR584-strain) and to assess
 treatment effects on individual alkaloids at the strain-specific level.
 Finally linear regressions were used to determine the relationship between
 aphid parameters and specific alkaloid expressions.
description: # Mycorrhizal fungi compromise production of endophytic alkaloids,
 increasing plant susceptibility to an aphid herbivore
 [https://doi.org/10.5061/dryad.6m905qg8c](https://doi.org/10.5061/dryad.6m905qg8c) ## Description of the data and file structure In a factorial greenhouse experiment we evaluated whether these components, acting alone or in combination, altered 1) foliar primary chemistry (PYTO cvs), 2) Si and symbiont-chemical (endophytic alkaloids) defenses(POP cvs), as well as 3) performance of the bird-cherry oat aphid (*Rhopalosiphum padi*) feeding on tall fescue (*Festuca arundinacea*) (IND cvs). ### Files and variables #### File: IND.csv **Description:**  ##### Variables * BLOCK: BLOCK DESIGN * PL: PLANT NUMBER * VAR: VARIETY * AMF: WITH OR WITHOUT AMF * Si: WITH OR WITHOUT Si supply * CAGE : TYPE OF CAGE * C#: CAGE NUMBER * DTA: DAYS TO ADULT * DaA: DAYS TO ADULT * Tfec(7): TOTAL FECUNDITY * Fec.d: DAILY FECUNDITY * lnfec: ENDO COLONIZATION * long: LONGEVITY IN DAYS * r: DAILY INTRINISIC RATE OF INCREASE * gt: GENERATION TIME #### File: PHYTO.csv **Description:**  ##### Variables * BLOCK: BLOCK DESIGN * PL: PLANT NUMBER * APHIDS: WITH OR WITHOUT APHID * SI: WITH OR WITHOUT SI SUPPLY * VAR: VARIETY * AMF: WITH OR WITHOUT AMF * SDM : SHOOT DRY MASS in mg * REGROWTH : NEW GROWTH in mg #### File: POP.csv **Description:**  ##### Variables * BLOCK: BLOCK DESIGN * PL: PLANT NUMBER * APHID: WITH OR WITHOUT APHID * Si: WITH OR WITHOUT Si supply * AMF: WITH OR WITHOUT AMF * VAR: VARIETY * SIAMFVAR: COMBINED SI SUPPLY, AMF AND VARIETY TRTS * AMFVAR: COMBINES AMF AND VARIERTY TRTS  * ALL: ALL TRT COMBINATIONS * AP: TOTAL NUMBER OF APTEROUS APHIDS * AL: TOTAL NUMBER OF ALATE APHIDS * NY: TOTAL NUMBER OF NYMPHS APHIDS * r: INTRINSIC RATE OF GENERATION CHANGE * l: lLONGEVITY IN DAYS * e: EXTRINSIC RATE OF GENERATION CHANGE * T#: TOTAL NUMBER OF APHIDS * SDM: SHOOT DRY WEIGHT IN MG * RDM: ROOT DRY WEIGHT IN MG * SI%: PERCENT: PERCENT ROOT COLONIZED  * %ARB: PERCENT AMF ARBUSCLES * %VES: PERCENT AMF VESICLES  * R.Peramine: ROOT PERAMINES  * R.TotalLolines: ROOT TOTAL LOLINES * R.NAL: ROOT NAL (LOLINE PRECUSOR) * BLOCK: BLOCK * S. Peramine: SHOOT PERAMINES  * S.TotalLolines: SHOOT TOTAL LOLINES * S. NAL: SHOOT NAL (LOLINE PRECUSOR) ## CODE/SOFTWARE R was used, code provided upon request.  ## Access information The information is not published elsewhere.