DOI: 10.5061/dryad.05qfttf13
title: Effects of drought and mycorrhiza on wheat and aphid infestation

description: The impacts of climate change on worldwide crop production become
 increasingly severe. Thus, sustainable enhancements of agricultural
 production are needed. The present study investigated the effects of
 drought and arbuscular mycorrhizal fungi on wheat plants (Triticum
 aestivum) and their interaction with aphids. Considering predicted climate
 change scenarios, wheat plants were exposed to well-watered conditions,
 continuous (CD) or pulsed (PD) drought and plants were grown without (NM)
 or with mycorrhizal (AM) fungi. Ear biomass and harvest index were
 evaluated when grains were produced. Moreover, drought- and
 mycorrhiza-induced changes in the amino acid composition of leaf phloem
 exudates were studied and the population growth and survival of Sitobion
 avenae aphids on those plants measured. Wheat plants responded differently
 towards the irrigation treatments. Under drought stress, ear biomass was
 reduced, while AM resulted in an enhanced harvest index. In phloem
 exudates especially relative concentrations of the osmoprotectant proline
 were modulated by drought. Aphid population size was influenced by the
 interaction of drought and mycorrhiza treatment. This study emphasises the
 pronounced influence of irrigation frequency on plant performance and
 indicates positive contributions of AM that may be relevant for
 agriculture.
description: <b>Plant cultivation and mycorrhiza
 treatments</b> The experiment was carried out in
 a climate chamber at 22.7 ± 1.6 °C (mean ± SD) with a relative humidity
 (r.h.) of 61 ± 5% and 16 h light:8 h dark. Untreated grains of spring
 wheat cultivar Tybalt were provided from Borries-Eckendorf (Leopoldshöhe,
 Germany). Plants were grown in 2 L pots (11.3 ×11.3 × 23 cm) with a 2:1
 mixture of steamed (4 h, at 120 °C) sand and soil (Fruhstorfer Pikiererde,
 HawitaGroup, Vechta, Germany). This mixture was used to be able to harvest
 and clean the roots at the end of the experiment to determine the total
 root length colonisation (TRLC). The pots had holes at their base to
 enable draining of surplus water and were placed on holders to prohibit
 water loss. To pots of the arbuscular mycorrhizal treatment (AM; n = 30)
 150 mL of fungal inoculum (sand spore mixture, pH 7; Inoq GmbH, Schnega,
 Germany) were added, containing the generalist fungus <i>Rhizoglomus
 irregulare</i><b> </b>(Błaszk., Wubet, Renker &
 Buscot) Sieverd., G. A. Silva & Oehl (Glomerales, Glomeromycota).
 This AMF species was used because it is commercially available for
 potential use in agriculture, colonises wheat roots and is known to affect
 growth and physiological responses of spring wheat (Campos et al., 2019;
 Zhang et al., 2018). The inoculum was mixed with the upper third of the
 substrate in each pot. To provide comparable microbial conditions in the
 non-mycorrhized control treatment (NM; n = 30), 150 mL sterilised (3 h, at
 120 °C) inoculum were mixed with the upper third of the substrate of each
 pot and 45 mL of a microbial solution obtained from the inoculum before
 sterilisation by filtrating a washing of the inoculum through a 20 µm
 sieve were added. AM pots received 45 mL water instead. To simulate field
 conditions with regard to plant density and nutrient competition each pot
 contained two plants in opposite corners, at a distance of about 10 cm.
 Sufficient germination was guaranteed by putting three seeds in the pots.
 Surplus seedlings were removed six days post sowing (dps). Plants were
 fertilised 18 (1 g / plant) and 35 dps (0.6 g / plant) with a solid
 long-term, phosphate-free mineral fertiliser (Floranid N-P-K 14-0-19,
 containing 3% Mg, 11% S and traces of B, Cu, Fe, Mn and Zn; Manna,
 Düsseldorf, Germany).  
 <b>Establishment of irrigation
 treatments</b> Initially, all pots were kept
 well-watered near field capacity [~ 18% soil water content (SWC)
 determined in preliminary experiment] until 24 dps to gain robust plants.
 The water requirement was determined gravimetrically. Therefore, every
 other day 15 representative pots were chosen, weighed and weights
 averaged. Pots then received the calculated amount of water to re-gain the
 requested soil moisture. Within each mycorrhiza treatment (NM/AM), pots
 were randomly attributed at 24 dps to one of three irrigation treatments,
 a control (CTR), continuous drought stress (CD) or pulsed drought stress
 (PD) (n = 10 per mycorrhiza and irrigation treatment). CTR pots were
 continued to be weighed as described above and watered to 18% SWC. CD and
 PD pots were left unwatered until a SWC of ~8% was reached. Then, CD pots
 received 40% of the water amount that was added to the CTR pots. A
 reduction of precipitation to 40% may occur in certain hot and dry
 summers, especially in certain regions under ongoing climate change.
 Furthermore, this amount of water had been shown to result in a
 significantly reduced aboveground biomass of wheat in a previous study
 under similar growth conditions, without killing the plants (Stallmann,
 Schweiger & Müller, 2018). CTR and CD plants were watered every
 other day. Pots of the PD treatment were irrigated only every eight days
 with the cumulated amount the CD pots had received in that time period
 (Fig. 1). All pots were randomly distributed and their position was
 changed weekly. The number of replicates was reduced by three pots (2 CD
 AM, 1 PD AM) due to a local fungal infection and by a further pot (CD NM)
 due to a failure during phloem exudate sampling. If not stated otherwise,
 in the following “treatment” is referred to as a combination of
 mycorrhizal treatment and irrigation treatment, e.g. AM CTR.
   <b>Phloem exudate collection and plant
 harvest</b> To examine the influence
 of the different treatments on the phloem sap composition of developing
 wheat plants, phloem exudates were collected when ears were fully emerged
 and plants started flowering (52 dps, T1; Fig. 1). At this time point, the
 aphid bioassays were started as well (see below). Phloem exudates were
 collected from half of the replicates (group A; n = 4-5 per treatment)
 from one plant per pot, following a method modified after Kos et al.
 (2011) and Schweiger, Heise, Persicke & Müller (2014c). To
 guarantee a sufficient turgor of the phloem sap the phloem exudate
 collections took place 24 h after watering all plants. The three youngest
 leaf blades of the main shoot were cut at their base and placed into a 50
 mL Falcon tube with 1 mL of an 8 mM ethylenediaminetetraacetic acid
 solution (EDTA; 99%, AppliChem GmbH, Darmstadt, Germany; pH = 7, adjusted
 with NaOH) in the dark for 2 h (20 °C, 60% r.h.). After this first
 incubation, leaves were washed in Millipore water (MicroPure Water
 Purification System, Thermo Fisher Scientific, Niederelbert, Germany),
 transferred to a new 50 mL Falcon tube with 1 mL Millipore water and
 incubated for another 2 h in the dark. These second collections were used
 for subsequent chemical analyses. Blanks were prepared by keeping
 Millipore water without plant material in Falcon tubes for the same
 duration. For amino acid analysis, 300 µL of the exudates and blanks were
 frozen in liquid nitrogen and stored at -80 °C. Leaf blades used for
 phloem exudate sampling were dried for 48 h at 40 °C and
 weighed. When flowering was
 completed and grains were watery ripe (68 dps, T2), the total (remaining)
 aboveground plant biomass of both plants per pot was harvested for groups
 A and B. Biomass was separated into vegetative and generative (ears) parts
 and dried for 96 h at 40 °C to determine the dry biomass. The HI was
 calculated by dividing the dry ear biomass by the dry total aboveground
 biomass.  
 <b>Amino acid analysis of phloem
 exudates</b> To analyse the amino acid
 composition, phloem exudates and blanks were lyophilised and redissolved
 in 80% methanol with norvaline and sarcosine (Agilent Technologies,
 Waldbronn, Germany) as internal standards for primary and secondary amino
 acids, respectively. Samples were analysed <i>via</i>
 high-performance liquid chromatography coupled with fluorescence detection
 (HPLC-FLD; 1260/1290 Infinity HPLC and FLD, Agilent Technologies, Santa
 Clara, CA, USA) following the protocol of Jakobs & Müller (2018).
 Identification of amino acids was performed <i>via</i>
 comparison of retention times with those of reference standards measured
 within the same worklist. Amino acids were quantified by integrating the
 corresponding peaks, using OpenLab ChemStation C.01.07 (Agilent
 Technologies). Data were normalised by dividing the peak areas by the
 areas of the corresponding internal standards. Peak areas were related to
 the dry weight of the leaf blades from which the phloem exudates had been
 collected. Amino acids were only included into further analysis when they
 were found in at least three of the five replicates (or two of four
 replicates) of one treatment and not in the blanks. To compare the
 relative composition of amino acids in dependence of the plant treatment,
 for each amino acid the mean percentage (i.e., its mean peak area divided
 by the mean total peak area of all amino acids) was determined for each
 treatment group. The amino acid data were only compared visually, because
 the sample sizes (n = 4-5) were too small for proper statistical
 analyses.   <b>Root sampling
 and quantification of AMF colonisation</b> To
 determine the TRLC with AMF two subsamples of roots were taken per pot at
 T2 by punching a cork borer (2.8 cm i.d.) twice vertically into the
 substrate for about 22 cm. Subsequently roots were washed and
 representative subsamples were bleached (10% KOH; 20 min, 95 °C), dyed
 with an acetic solution of ink (royal blue, Ink 4001, Pelikan Group GmbH,
 Berlin, Germany) (1:1:8 ink:acetic acid:water; 15 min, 90 °C) and
 conserved (4:2:1 90% lactic acid:89% glycerine:water, 4 °C in the dark).
 The TRLC was determined using the grid-line intersect method (Giovanetti
 & Mosse, 1980) by separately counting different AMF structures (i.
 e., hyphae, vesicles, arbuscules) in 200 intersects per sample.
   <b>Bioassays with
 aphids</b> Aphids of <i>S. avenae</i>
 were obtained from Koppert Biological Systems (Suffolk, UK) and kept in
 tents (58 ´ 58 ´ 58 cm) on wheat plants (cultivar: Tybalt) for several
 generations at 16 h light:8 h dark. To examine the influences of the plant
 treatments on aphid performance we investigated the growth of aphid
 populations as well as the survival of single nymphs on leaves of the
 experimental plants over 16 days (from T1 to T2). To record the population
 growth clip cages (2 cm i.d.) with three eight days-old apterous aphids
 were attached to the second youngest leaf of the main shoot of one intact
 plant per pot (groups A and B, n = 8-10 / treatment). Under the used
 conditions, aphids of <i>S. avenae</i> turn into adults at
 about 9 days and live for about 20 - 60 days. For group A, the plant in
 each pot was used that had not been used for phloem exudate collections
 (see above). Numbers of living adult aphids and their offspring were
 counted after 16 days on the plants (T2). To record the survival of single
 aphids, a one-day-old nymph was fixed in a clip cage on the flag leaf of
 the same shoot (n = 8-10 / treatment). Survival of these nymphs was
 checked every day and offspring counted and removed.