Arbuscular mycorrhiza, but not hydrogel, alleviates drought stress of ornamental plants in peat-based substrate

Applied Soil Ecology 146 (2020) 103394

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Arbuscular mycorrhiza, but not hydrogel, alleviates drought stress of ornamental plants in peat-based substrate

T

Jana Rydlová, David Püschel* Department of Mycorrhizal Symbioses, Institute of Botany of the Czech Academy of Sciences, Průhonice, Czech Republic

A R T I C LE I N FO

A B S T R A C T

Keywords: Arbuscular mycorrhizal fungi Flowering Peat substrate Polyacrylamide gel Water deficiency

In a greenhouse study, we examined the application potential of symbiotic arbuscular mycorrhizal fungi (AMF) and water absorbing gel (hydrogel) in planting pot-grown ornamental plants that periodically experience drought stress. We tested the hypotheses that either of these treatments can improve plant growth and flowering under conditions of water deficiency. Three species of ornamental plants – Gazania rigens, Pelargonium peltatum and P. zonale – were grown in pots with peat-based substrate either inoculated or non-inoculated with commercial inoculum of AMF, and either with or without added hydrogel. Generous watering during the first 14 weeks ensured well-grown plants. In the next 15 weeks, plants either were well watered or were periodically exposed to moderate or strong drought stress to an initial wilting point or sufficient to cause strong wilting, respectively, before the substrate was again re-saturated with water. The three tested species differed in their resistance to drought stress, P. peltatum being the most resistant and G. rigens the most sensitive. Strong drought decreased shoot biomass of G. rigens and P. zonale, but it had no effect on shoot biomass of P. peltatum, that even increased flowering. As a response to drought, mycorrhizal colonization of plant roots intensified and AMF significantly increased shoot biomass (and several biometric parameters) of all plant species and promoted flowering of G. rigens and P. zonale under all watering regimes. AMF thus confirmed the hypothesized potential for promoting plant growth under drought. On the other hand, the addition of hydrogel brought no positive growth effect on any plant species. On the contrary, it decreased flowering of all plants and interacted negatively with AMF.

1. Introduction Extended periods without precipitation, especially during summer months, have occurred frequently in recent years. Moreover, these droughts have become common even in the moderate climatic zone of central Europe that in the past had relatively reliable supply of rainwater (Hanel et al., 2018). In addition to seriously threatening agricultural production (Potopová et al., 2016) and creating risk of wildfires that endanger forests (Parente et al., 2019), drought spells can negatively impact such municipal vegetation as flower beds in gardens or parks within cities. Drought is even more difficult to cope with when plants are grown in pots that limit their roots’ space for exploiting water and nutrients. Although municipal ornamental plants can usually benefit from additional watering that is relatively easily provided inside of cities, drought would impact even these plants if watering were restricted due, for example, to temporary prohibition directed to conserving limited water resources.

Pot-grown ornamental plants are planted mainly in peat-based substrates. Although ecological concerns about the use of peat have been raised in recent years, alternative substrates are not yet widespread. For years to come, therefore, peat will arguably remain the key component of horticulture substrates and we should thus aim to improve upon the efficiency of its usage. One possible approach is to inoculate plants with symbiotic arbuscular mycorrhizal fungi (AMF). These fungi constitute a natural component of soils in most terrestrial ecosystems and establish symbiosis with the majority of vascular plant species, providing them with soil nutrients, especially phosphorus (P), in exchange for assimilated carbon (Smith and Read, 2008). Peat-based substrates are, however, well-known for usually lacking beneficial soil microorganisms, including AMF (Koltai, 2010; Linderman and Davis, 2003a; Perner et al., 2007; Püschel et al., 2014). Even subsequent introduction of AMF into peat-based substrates can be difficult due to possible negative effects of some types of peat on such various aspects of AMF symbiosis as germination and early mycelial growth (Ma et al.,

⁎ Corresponding author at: Department of Mycorrhizal Symbioses, Institute of Botany of the Czech Academy of Sciences, Zámek 1, 252 43 Průhonice, Czech Republic. E-mail address: [email protected] (D. Püschel).

https://doi.org/10.1016/j.apsoil.2019.103394 Received 21 June 2019; Received in revised form 14 October 2019; Accepted 16 October 2019 Available online 30 October 2019 0929-1393/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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J. Rydlová and D. Püschel

Gazania rigens (L.) Gaertn. (var. Daybreak Red Stripe), Pelargonium peltatum (L.) L'Hér. (var. Tornado), and P. zonale (L.) L'Hér. (var. Gizela). Plants were well watered during the first 14 weeks. For the next 15 weeks, a specific watering regime was established that included both plenty of water and periodic exposure of plants to moderate or strong drought that caused initial or strong wilting, respectively. The effects of AMF, hydrogel and watering on plant growth and flowering were tested in a full factorial design with ten replicates per treatment, thus resulting in 360 pots.

2006), establishing colonization (Calvet et al., 1992), development of intraradical colonization (Linderman and Davis, 2003b; Ma et al., 2007), and effectiveness of the symbiosis itself (Vestberg et al., 2005). On the other hand, if AMF are successfully introduced to the peat substrate, these symbionts can become highly beneficial for growth and flowering of certain species of ornamental plants (Püschel et al., 2014). While most of the mycorrhizal benefits usually stem from increased nutrient acquisition by the colonized plants (Hodge and Fitter, 2010; Jansa et al., 2003; Smith et al., 2011), a less-explored and less-understood, yet important phenomenon is the role of AMF in water relations between plants and soil (Augé, 2001, 2004; Augé et al., 2003). It is assumed that arbuscular mycorrhizal symbiosis can increase water uptake by the host plants under conditions of water deficiency due to penetration of hyphae into very small pores inaccessible to roots (Allen, 2007) and that AMF mycelium can also contribute indirectly to water redistribution through the soil (Allen, 2007; Bitterlich et al., 2018a, b). In a previous experiment with Pelargonium zonale (Püschel et al., 2014), we experienced that the clearly positive effects of mycorrhizal inoculation were maintained even under conditions of partial water deficiency, to which ornamental plants are often subjected. Although water deficiency negatively affected most traits related to plant growth, mycorrhizal drought-stressed plants were distinctly more vital than were their well-watered, but non-mycorrhizal counterparts (Püschel et al., 2014). When pot-grown ornamental plants cultivated in peat-based substrates are watered, retention of water received, especially from short and intensive watering, might not be sufficient. Significant amounts of water can leach from the pots immediately or soon evaporate from the substrate. For these reasons, another possible way to improve the water regime of pot-grown ornamental plants might be to add water-absorbing gel (hydrogel). Hydrogels can improve physical soil properties, mainly water retention (Bres and Weston, 1993; Jobin et al., 2004; Martinez et al., 2001), and thus positively affect plant growth (Ochoa et al., 2009; Su et al., 2017; Suresh et al., 2018). Hydrogel absorbs mainly gravitation water, which under natural conditions rapidly permeates the soil profile and becomes unavailable for plants (Leciejewski, 2009). Therefore, some manufacturers of commercial mycorrhizal inocula add hydrogel to their products. Theoretically, such synergistic application of AMF and hydrogel could be particularly effective for pot-grown ornamental plants, the watering of which can be erratic, quick, yet excessive. Moreover, the small volume of substrate in their pots and its water-holding capacity limit the later accessibility of water. We tested in a greenhouse study how the application of AMF and addition of hydrogel to the cultivation substrate bear out as means presumably facilitating growth of ornamental plants exposed to moderate or strong drought causing symptoms of wilting. We hypothesized that each of these steps would provide the plants with an advantage under drought that should result in increased growth and flowering compared to the respective controls, and that the combination of AMF and hydrogel would act synergistically giving plants even stronger resistance to drought. These hypotheses were tested on three ornamental plant species, all originating from dry habitats of southern Africa and yet contrasting in their drought tolerance. While the two Pelargonium species chosen are considered tolerant (Chyliński et al., 2007), the Gazania plants are known to synchronize their life cycle with the precipitation period and are considered less drought resistant (Howis et al., 2009).

2.2. Substrate, plants and cultivation Round, plastic 700 ml pots were filled with a commercial peat-based substrate (garden substrate type “B”; manufacturer: Rašelina a.s., Soběslav, Czech Republic). This substrate contained highly decomposed and disintegrated dark peat mixed with a smaller quantity of fibrous white peat. The substrate had the following properties: pH 5.3 (according to European Union norm EN 13037); electric conductivity 0.63 mS cm−1 (EN 13038); plant available nutrients: NNH4 144 mg kg−1, NNO3 856 mg kg−1, P 45 mg kg−1, K 621 mg kg−1, Mg 498 mg kg−1 (all CAT-extractable, EN 13651), and Ca 975 mg kg−1 (waterextractable, EN 13652). One liter of the substrate dried to constant weight (moderately compressed, relevant to growing the plants) weights ca 346 g. If fully saturated, it can hold 611 ml of water. A biotest conducted prior to the experiment using Zea mays revealed the absence of AMF propagules in the substrate, as the roots of the biotest plants remained non-colonized by AMF after six weeks of cultivation in this substrate). Plants of G. rigens and P. peltatum were obtained from seeds purchased from a local retailer (manufacturer: SEMO a.s., Smržice, Czech Republic). The seeds were germinated in plastic multipots having 15 ml cells filled with a heat-sterilized (twice autoclaved at 121 °C for 30 min, 24 h apart) 1:1 (v/v) sand–zeolite mixture. Seeds of P. zonale (obtained from the Silva Tarouca Research Institute for Landscape and Ornamental Gardening, Průhonice, Czech Republic) were germinated, and plants were pre-planted in sterile conditions (Plavcová, 2009). The seedlings of all species were transplanted into experimental pots (one plant per pot) at the age of two cotyledon leaves and one true leaf. 2.3. Mycorrhizal inoculation and the application of the gel The inoculum Symbivit® (Symbiom, s.r.o., Lanškroun, Czech Republic) was used to inoculate the plants. This universal commercial AMF inoculum was preferred to a laboratory inoculum because it better represents possible practical application of AMF in the cultivation of ornamental plants, at least insofar as inoculum quantity and quality are concerned. This commercial AMF inoculum contains propagules (spores, mycelium and colonized root fragments) of six different AMF species on an inert carrier composed of zeolite and expanded clay. Importantly, by default this product contains also polyacrylamide water-binding gel (dosage ca 65 g of the gel per 1 kg of the final product) that is supposed to increase drought stress resistance of the plants by improving the water-holding capacity of the substrate. To test the potential effects of AMF and of the gel independently, the manufacturer kindly provided us with four product versions: 1) standard product including both AMF and the gel (this treatment is further referred to as “M + G”), 2) carrier including AMF but no gel (“M”), 3) carrier without AMF but including the gel (“NM + G”), and 4) carrier without AMF and without the gel (“NM”). The pots received an 8 ml dose of the respective product into cavities just below the transplanted seedlings, as in Püschel et al. (2014).

2. Material and methods 2.1. Experiment overview

2.4. Plant cultivation and watering Three species (varieties) of ornamental plants responding well to AMF inoculation (Püschel et al., 2014) were used in a greenhouse experiment using pots filled with a commercial peat-based substrate:

The experiment was established in May and conducted for 29 weeks in a heated greenhouse where temperature was maintained between 2

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27 °C (day) and 15 °C (night) by automatic ventilation and heating. Since September, natural light was supplemented with 400 W metal halide lamps set to a 14 h photoperiod. No fertilization was applied throughout the experiment, because the substrate was well fertilized from the manufacturer. During the first 14 weeks, all pots were watered regularly to establish uniform and healthy plants. For the next 15 weeks until harvest, a specific watering regime was established to expose certain treatments to moderate or strong drought. The watering encompassed three regimes differing in symptoms of plants´ wilting caused by drought. Under the “no-wilting” (NW) regime, the plants were watered regularly as before and were not affected by drought. Under the “initial wilting” (IW) regime, plants were not watered until clear symptoms of initial wilting were manifested (bending of leaves and/or ends of branches). Finally, under the “strong wilting” (SW) regime, the plants suffered from serious, yet recoverable wilting before they were watered. Because deciding whether to initiate watering or not just by observation of plant wilting symptoms would be highly subjective and insufficiently rigorous, watering decisions were actually based on measurement of substrate moisture (Moisture Meter HH2 with ThetaProbe ML2x type soil moisture sensor; manufacturer of both devices: Delta-T Devices Ltd, UK) and comparison of the results with specific values of substrate moisture termed “watering thresholds” (Table 1). These watering thresholds had been defined earlier in preliminary tests on another set of plants of the same species cultivated in the same conditions. In these earlier tests, symptoms of plant wilting were repeatedly observed and corresponding substrate moisture measured. All plant species manifested initial or strong wilting symptoms at different substrate moisture (Table 1). For simplicity’s sake, we use the terms NW, IW and SW in this article to refer to the different moisture levels (if necessary, please use Table 1 to translate each regime to exact substrate moisture conditions). Substrate moisture was measured every morning, including on weekends. If moisture lower than a watering threshold was recorded, watering was provided by pouring water into saucers placed below the pots and maintained there for 5 h during which water could elevate to the pots. After this time, the remaining water was removed from the saucers. This watering method increased substrate moisture to ca 50 vol %.

Table 1 Overview of soil moisture thresholds (in vol%) at which watering of model plants was initiated in relevant wilting regime treatment. Thresholds were set individually for the tested species based on physiological response of the plants to the soil water content. NW – no wilting, IW – initial wilting manifested by bending of leaves and/or ends of branches, SW – strong, yet recoverable wilting.

NW threshold IW threshold SW threshold

Gazania rigens

Pelargonium peltatum

Pelargonium zonale

29 % 13 % 7%

16 % 11 % 4%

11 % 8% 4%

system. 2.6. Statistical analyses The results were analyzed using STATISTICA 12.0 software (StatSoft Inc., USA). Data were tested for normality (Kolmogorov–Smirnov test) and homogeneity of variance (Levene’s test). Data not meeting these criteria were arcsine (mycorrhizal colonization in all species) or log transformed (leaf area, RDW and P concentration in P. peltatum). Threeway ANOVA was used to determine the effects of individual factors. Mean separation was done using Tukey’s HSD test at significance level p < 0.05. Data on number of branches in P. peltatum and P. zonale were analyzed using generalized linear models with Poisson distribution. 3. Results 3.1. Plant growth and flowering Plant growth was strongly affected by all experimental factors, yet the plants did not respond in a uniform manner (Table 2). Drought negatively affected growth of all plants except P. peltatum (Tables 3–5), in which case no effect on SDW, RDW or number of branches was found and its leaf area was even increased by drought (Table 4). Mycorrhizal symbiosis caused positive growth response in all plants, as apparent in most measured biometric parameters for G. rigens and P. peltatum (Fig. 1, Tables 3 and 4) and in all biometric parameters of P. zonale (Fig. 1, Table 5). In contrast, the addition of hydrogel had no significant effect on any biometric parameter of any plant species (Table 2). Flowering of the tested ornamental plants also was strongly, albeit inconsistently, affected by all experimental factors (Table 2). The inoculation with AMF significantly stimulated flowering of G. rigens. This species produced the largest flowers if colonized by AMF, regardless of drought (Fig. 2). The quantity of flowers also tended to increase in mycorrhizal plants, except that under the NW regime no differences were found (Table 3). The addition of hydrogel brought no additional benefit to the number of flowers of G. rigens (Fig. 2) and it decreased the largest flower’s diameter (Tables 2 and 3). In the case of P. peltatum, AMF had no effect on flowering and the addition of hydrogel affected the flowering negatively (Table 2). The number of P. peltatum flowers tended to be increased by AMF under the SW regime and at the same time it tended to be decreased by the presence of hydrogel (Fig. 2, Table 2). Flowering of P. zonale was strongly stimulated by inoculation with AMF, and this pattern was observed across all watering regimes (NW, IW, as well as SW; Fig. 2, Table 2). Whereas no effect of water deficiency on the number of flowers was observed for P. zonale plants, the addition of hydrogel acted negatively in this regard (Table 2).

2.5. Harvest Several biometric parameters were measured to characterize growth of the tested ornamental plants: plant height (or total branch length for P. peltatum), number of branches (P. zonale and P. peltatum) and total leaf area. The visual qualities were characterized by counting the number of flowers1 and measuring the diameter of the largest flower (only for G. rigens). For simplicity, these two parameters are termed “flowering” in the text. Shoot dry weight (SDW) was determined after drying the shoot biomass at 70 °C to constant weight. The dry biomass was then ground, digested in nitric and perchloric acids (Kopáček and Hejzlar, 1995) and the concentration of P was assessed spectrophotometrically (ISO 15681-1; FIA QuickChem 8500 FIA System, Lachat Instruments, Loveland, CO, USA). The root system was carefully washed of the substrate, after which root subsamples (ca 0.5 g) were weighed and stained with 0.05% Trypan blue in lacto-glycerol (Koske and Gemma, 1989) to quantify mycorrhizal colonization under a dissecting microscope at 100× magnification and using the gridline intersect method (Giovannetti and Mosse, 1980). The remaining roots were first weighed fresh and then after drying at 70 °C to constant weight. Total root dry weight (RDW) was calculated for the whole root

3.2. Plant P concentration and mycorrhizal colonization All plant species in this study create inflorescences instead of flowers. However, for simplicity and convenience of the readers, we use the term flowers throughout the manuscript. 1

Mycorrhizal symbiosis increased P concentration in shoots of all plant species (Table 2). The addition of hydrogel had no effect on P 3

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Table 2 Effects of factors (1 – watering, 2 – arbuscular mycorrhizal fungi (AMF) inoculation, 3 – hydrogel) and their interactions on various growth parameters and mycorrhizal colonization of roots of model plants according to three-way ANOVA (presented as F value and significance) or according to generalized linear model for number of branches (presented as Wald statistics and significance): * p < 0.05, ** p < 0.01, *** p < 0.001; ns – no significant effect. Arrows indicate positive (↗) or negative (↘) effect, if clearly identifiable. Plant

Factor

Number of flowers

Diameter of largest flower

Shoot dry weight

Root dry weight

Plant height / Total length of branches (P.peltatum)

Number of branches

Total leaf area

Shoot P concentration

Mycorrhizal colonization

Gazania

1) watering

20.2 *** ↘

ns

5.5 ** ↘

ns

4.5 * ↘

—

5.6 ** ↘

18.9 *** ↗

rigens

2) AMF

31.1 *** ↗

86.5 *** ↗

ns

76.5 *** ↗

—

13.8 *** ↗

—

Pelargonium peltatum

3) gel 1×2 1×3 2×3 1×2×3 1) watering 2) AMF

ns ns ns ns ns 6.6 ** ↗ ns

4.9 * ↘ 3.4 * ns ns ns — —

20.0 *** ↗ ns 6.7 ** ns ns ns ns 6.2 * ↗

ns ns ns ns ns ns ns

ns 14.2 *** ns ns ns 4.6 * 11.4 ** ↗

— — — — — ns ns

ns ns ns 5.7 * ns 3.8 * ↘ 14.5 *** ↗

ns — ns — — 42.5 *** ↗ —

3) gel 1×2 1×3 2×3 1×2×3

6.7 * ↘ 3.3 * ns ns ns

— — — — —

ns ns ns ns ns

ns ns ns ns ns

ns ns ns 6.8 * ns

ns ns ns ns ns

8.2 *** ↘ 40.5 *** ↗ ns 5.5 ** ns ns ns 7.0 ** ↗ 25.0 *** ↗ ns 3.9 * ns 14.2 *** ns

ns ns ns ns ns

10.4 ** ↘ — ns — —

Pelargonium

1) watering

ns

—

ns

26.8 ***

4.4 * ↗

12.2 *** ↗

2) AMF

148.5 *** ↗

—

378.0 *** ↗

62.4 *** ↗

—

7.3 ** ↘ ns ns ns ns

— — — — —

ns 9.3 *** ns 6.5 * ns

ns ns ns ns ns

442.5 *** ↗ ns 12.1 *** 5.6 ** ns ns

52.5 *** ↗

3) gel 1×2 1×3 2×3 1×2×3

24.4 *** ↘ 190.4 *** ↗ ns 7.5 *** ns ns 4.4 *

13.4 ***

zonale

11.5 *** ↘ 169.9 *** ↗ ns ns ns ns ns

ns ns 3.6 * 7.9 ** ns

ns — 8.7 *** — —

Table 3 Effect of presence (M) or absence (NM) of arbuscular mycorrhizal fungi together with presence (+G) or absence of water-binding gel on various biometric parameters of Gazania rigens and on phosphorus (P) concentration in its shoot biomass under different watering regimes. NW – no wilting, IW – initial wilting manifested by bending of leaves and/or ends of branches, SW – strong, yet recoverable wilting. The data are means of ten replicates ± SE. Different letters within each watering treatment indicate significant differences based on Tukey’s HSD test (p < 0.05); ns – no significant effect. Drought

Treatment

Diameter of largest flower [cm]

Root dry weight [g]

NW

M M+G NM NM + G M M+G NM NM + G M M+G NM NM + G

4.34 4.01 3.56 3.20 4.28 4.00 2.75 3.03 4.20 3.83 3.47 3.23

0.77 0.71 0.87 0.96 1.19 0.88 0.80 0.70 0.53 0.88 0.84 0.62

IW

SW

± 0.10 ± 0.19 ± 0.18 ± 0.15 ± 0.22 ± 0.18 ± 0.16 ± 0.17 ± 0.14 ± 0.14 ± 0.16 ± 0.19

a ab bc c a a b b a ab b b

± 0.16 ± 0.12 ± 0.15 ± 0.13 ± 0.21 ± 0.12 ± 0.13 ± 0.14 ± 0.10 ± 0.08 ± 0.15 ± 0.08

ns

ns

ns

13.2 14.6 11.8 12.1 14.4 15.9 8.4 9.3 12.5 12.4 10.7 10.4

± 0.41 ± 0.40 ± 0.94 ± 0.33 ± 1.07 ± 0.77 ± 0.54 ± 0.53 ± 0.71 ± 0.67 ± 0.66 ± 0.55

Shoot P concentration [mg. kg−1]

Total leaf area [cm2]

Plant height [cm] ab a b b a a b b ns

106 102 70 67 84 113 23 33 51 77 46 45

± 16.3 ± 13.2 ± 13.9 ± 9.2 ± 14.3 ± 12.3 ± 4.4 ± 4.6 ± 7.5 ± 9.9 ± 7.6 ± 14.3

ns

a a b b ns

2788 2461 2053 2375 2303 2039 1982 1813 2883 2471 1958 2349

± 261 ± 271 ± 237 ± 185 ± 222 ± 71 ± 100 ± 102 ± 193 ± 131 ± 103 ± 223

ns

ns

a ab b ab

showed generally strong resistance to decreased water supply. Nevertheless, there were considerable differences among the individual species: G. rigens proved to be the least and P. peltatum the most drought-resistant of the three species studied. In G. rigens, the SW regime negatively affected shoot biomass, plant height, as well as leaf area and, most importantly for ornamental plants, also the number of flowers. By contrast, P. peltatum did not seem to be affected by reduced watering at all, and its total leaf area and the number of flowers were even stimulated by the SW regime. The biomass of P. zonale plants was somewhat reduced by drought, but the flowering was unaffected. These observed differences in drought tolerance probably result from individual life strategies of the species we tested. In its natural habitats, G. rigens is a perennial herb that invests most of its resources into growth and flowers mainly after rainfall events (Howis et al., 2009). In contrast, P. zonale is a shrub with almost succulent stems that become

concentration in plant shoots, whereas drought decreased P concentration in shoots of G. rigens and P. peltatum and increased P concentration in shoots of P. zonale (Tables 2–5). The roots of all inoculated plants were intensively colonized by AMF (Table 6), whereas non-inoculated control plants remained non-colonized. Intensified drought stimulated mycorrhizal colonization of roots of all tested ornamental plants species. On the contrary, hydrogel reduced mycorrhizal colonization of P. peltatum plants (Tables 2 and 6).

4. Discussion 4.1. Plants response to drought The three tested species of ornamental plants originate from adverse dry habitats of southern Africa. Perhaps for this reason, all these species 4

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Table 4 Effect of presence (M) or absence (NM) of arbuscular mycorrhizal fungi together with presence (+G) or absence of water-binding gel on various biometric parameters of Pelargonium peltatum and on phosphorus (P) concentration in its shoot biomass under different watering regimes. NW – no wilting, IW – initial wilting manifested by bending of leaves and/or ends of branches, SW – strong, yet recoverable wilting. The data are means of ten replicates ± SE. Different letters within each watering treatment indicate significant differences based on Tukey’s HSD test or generalized linear model (number of branches; p < 0.05); ns – no significant effect. Drought

Treatment

Root dry weight [g]

NW

M M+G NM NM + G M M+G NM NM + G M M+G NM NM + G

0.56 0.64 0.49 0.54 0.58 0.59 0.49 0.52 0.65 0.91 0.50 0.57

IW

SW

± 0.08 ± 0.10 ± 0.05 ± 0.04 ± 0.08 ± 0.07 ± 0.06 ± 0.05 ± 0.07 ± 0.44 ± 0.05 ± 0.05

ns

ns

ns

Number of branches

Total length of branches [cm]

Total leaf area [cm2]

Shoot P concentration [mg. kg−1]

4.9 4.8 3.7 4.2 5.1 4.5 4.0 4.7 5.9 4.3 4.8 4.2

190 182 159 200 199 175 126 129 366 185 160 173

55 41 36 39 66 45 10 36 204 77 35 57

2317 2571 1822 1319 2187 2020 1300 1543 1374 1708 997 1453

± 0.59 ± 0.49 ± 0.30 ± 0.29 ± 0.41 ± 0.50 ± 0.33 ± 0.15 ± 0.54 ± 0.21 ± 0.29 ± 0.32

ns

ns

a ab ab b

± 37.6 ± 24.8 ± 21.8 ± 27.7 ± 49.0 ± 27.9 ± 12.1 ± 7.8 ± 52.7 ± 19.1 ± 18.8 ± 27.6

ns

ns

a b b b

± 12.4 ± 10.1 ± 8.5 ± 11.0 ± 17.0 ± 10.2 ± 1.6 ± 6.5 ± 30.1 ± 16.8 ± 11.0 ± 18.5

ns

a a b a a ab b b

± 608 ± 475 ± 200 ± 225 ± 422 ± 279 ± 125 ± 300 ± 93 ± 65 ± 94 ± 230

ns

ns

ab a b ab

Table 5 Effect of presence (M) or absence (NM) of arbuscular mycorrhizal fungi together with presence (+G) or absence of water-binding gel on various biometric parameters of Pelargonium zonale and on phosphorus (P) concentration in its shoot biomass under different watering regimes. NW – no wilting, IW – initial wilting manifested by bending of leaves and/or ends of branches, SW – strong, yet recoverable wilting. The data are means of ten replicates ± SE. Different letters within each watering treatment indicate significant differences based on Tukey’s HSD test or generalized linear model (number of branches; p < 0.05); ns – no significant effect. Drought

Treatment

Root dry weight [g]

NW

M M+G NM NM + G M M+G NM NM + G M M+G NM NM + G

5.45 5.02 3.76 4.46 5.81 6.04 4.08 3.51 4.75 5.11 2.46 2.83

IW

SW

± 0.21 ± 0.22 ± 0.24 ± 0.29 ± 0.22 ± 0.23 ± 0.23 ± 0.17 ± 0.27 ± 0.32 ± 0.17 ± 0.13

Plant height [cm] a ab c bc a a b b a a b b

28.2 24.8 17.4 16.8 29.1 29.4 18.9 21.0 30.0 27.1 13.4 14.6

± 0.67 ± 1.05 ± 0.83 ± 1.21 ± 0.63 ± 0.68 ± 1.49 ± 1.29 ± 1.05 ± 1.30 ± 0.57 ± 0.40

a a b b a a b b a a b b

4.2 3.6 1.2 1.1 4.5 3.4 1.1 1.1 3.8 2.6 1.6 1.7

± 0.13 ± 0.22 ± 0.13 ± 0.10 ± 0.17 ± 0.16 ± 0.10 ± 0.10 ± 0.22 ± 0.34 ± 0.22 ± 0.15

Shoot P concentration [mg. kg−1]

Total leaf area [cm2]

Number of branches a b c c a b c c a b c bc

191 154 53 71 295 262 93 73 207 233 58 92

± 15.1 ± 13.8 ± 6.5 ± 10.6 ± 15.6 ± 10.6 ± 15.3 ± 7.8 ± 11.5 ± 17.5 ± 5.7 ± 11.7

a a b b a a b b a a b b

1335 880 796 734 1384 1350 731 900 1445 1333 748 1097

± 173 ± 104 ± 141 ± 105 ± 105 ± 108 ± 58 ± 142 ± 25 ± 84 ± 71 ± 101

a ab ab b a a b b a ab c b

Fig. 1. Shoot dry weight (SDW) of Gazania rigens (A), Pelargonium peltatum (B) and P. zonale (C) produced under three watering regimes during the second stage of the experiment. NW – no wilting, IW – initial wilting manifested by bending of leaves and/or ends of branches, SW – strong, yet recoverable wilting. Dark columns represent mycorrhizal treatments, light columns control non-mycorrhizal treatments. Diagonal pattern indicates presence of water-absorbing gel. The data are means of ten replicates ( ± SE). Different letters indicate significant differences (within respective watering regime) according to Tukey’s HSD test (p < 0.05); ns – no significant difference.

soil water deficiency (Álvarez et al., 2013; Boyle et al., 2016; Eiasu et al., 2012; Sánchez-Blanco et al., 2009). In our experiment, these traits most likely contributed to stronger resistance to drought of both Pelargonium species compared to G. rigens plants.

woody with age and P. peltatum is a trailing evergreen perennial with fleshy stem branches and succulent leaves (van der Walt, 1979). Both Pelargonium species keep their biomass throughout the whole year and can flower at all times of the year, though they flower particularly in spring after winter rainfalls. Pelargonium species, reported to tolerate moderate (Amiri et al., 2017; Henson et al., 2006; Sánchez-Blanco et al., 2009) to severe (Chyliński et al., 2007) levels of drought, are better adapted to drought due to their partial succulence, but possibly also due to mechanisms related to effective stomatal closure under conditions of

4.2. Plant response to arbuscular mycorrhizal fungi Positive effects of AMF on ornamental plants have been widely reported, be that through increase of growth (Amiri et al., 2017; Csima 5

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Fig. 2. Number of flowers of Gazania rigens (A), Pelargonium peltatum (B) and P. zonale (C) produced under three watering regimes during the second stage of the experiment. NW – no wilting, IW – initial wilting manifested by bending of leaves and/or ends of branches, SW – strong, yet recoverable wilting. Dark columns represent mycorrhizal treatments, light columns control non-mycorrhizal treatments. Diagonal pattern indicates presence of water-absorbing gel. The data are means of ten replicates ( ± SE). Different letters indicate significant differences (within respective watering regime) according to Tukey’s HSD test (p < 0.05); ns – no significant difference.

there water containing nutrients (Allen, 2007). In contrast, the mycelia growing out from colonized roots of mycorrhizal plants reach well beyond the nutrient depletion zone of plant roots. As the hyphae often are only about 2 μm in diameter (Friese and Allen, 1991) and can thus penetrate even into some of the micropores and exploit the nutrients available there, AMF increase surface area per unit biomass by as much as two orders of magnitude compared to plant roots alone (Raven and Edwards, 2001). Furthermore, the AMF mycelium is thought to contribute to water movement in the soil by increasing soil hydraulic conductivity (Bitterlich et al., 2018a). While water movement pathways to roots are impeded by air-filled pore spaces, the hyphal network bridges these empty pores and connects moistened pores with root surfaces (Miller and Jastrow, 2000). We believe that these mechanisms facilitated acquisition of water and nutrients by mycorrhizal plants and contributed not only to their increased growth but also to flowering compared to their non-mycorrhizal counterparts. As quantity of flowers has been reported to be proportional to plant size and nutrient content (Gaur et al., 2000; Lu and Koide, 1994), the increased flowering could thus be caused simply by larger biomass and better nutrient uptake of mycorrhizal plants. Moreover, AMF can also influence the balance of plant hormones (Perner et al., 2007), including the production of gibberellin-like substances, which can influence flowering (Allen et al., 1982). Thus, mycorrhizal symbiosis probably engaged several mechanisms that contributed in concert to the strong effects that we observed. Mycorrhizal colonization of all three plant species intensified with water deficiency. This could indicate that plants invested more resources into their AMF partners in conditions where mycorrhiza became more important for plant uptake of nutrients, water or both. Although such stimulation of colonization seems logical and similar results have been reported elsewhere, this is not a general, uniform trend. Mycorrhizal colonization often remains unchanged or is even significantly decreased under conditions of water deficiency. Augé (2001) has provided a comprehensive overview (with additional references) of the observed positive, neutral and negative responses regarding water relations and mycorrhizal colonization and their effects on host plants growth. Although AMF are probably more tolerant to drought than are the host plants themselves (Lenoir et al., 2016), some amount of water is necessary for mycorrhiza to develop successfully (Augé, 2001). The reduced colonization by AMF found in some studies was probably observed under conditions wherein the drought tolerance of AMF was exceeded. In our study, mycorrhizal colonization increased even under conditions where water deficiency had a negative impact on biomass of some plants (G. rigens and P. zonale). These results thus indicate that our HW regime was still within the tolerance of AMF (and of P. peltatum) but was outside the tolerance of G. rigens and P. zonale

Table 6 Effect of watering regime and absence (−G) or presence (+G) of water-binding gel on mycorrhizal colonization of roots of Gazania rigens¸ Pelargonium peltatum and P. zonale. NW – no wilting, IW – initial wilting manifested by bending of leaves and/or ends of branches, SW – strong, yet recoverable wilting. The data are means of ten replicates ± SE. Different letters indicate significant differences based on Tukey’s HSD test (p < 0.05). Plant

Drought

Gel treatment

Mycorrhizal colonization [%]

Gazania rigens

NW

–G +G –G +G –G +G –G

51 58 69 71 65 66 42

± 1.81 ± 3.81 ± 2.28 ± 2.95 ± 2.42 ± 1.49 ± 2.53

c bc ab a ab ab b

+G –G +G –G +G –G +G –G +G –G +G

39 43 32 59 55 74 66 72 77 76 81

± 2.82 ± 2.78 ± 1.79 ± 1.80 ± 1.06 ± 1.20 ± 3.43 ± 1.37 ± 1.21 ± 1.17 ± 0.85

bc b c a a b c bc ab ab a

IW SW Pelargonium peltatum

NW

IW SW Pelargonium zonale

NW IW SW

et al., 2012; Nowak, 2004; Püschel et al., 2014) or stimulation of flowering (Asrar and Elhindi, 2011; Perner et al., 2007; Püschel et al., 2014; Sohn et al., 2003). It is also well known that mycorrhizal symbiosis can be particularly beneficial for plants growing in less than favorable conditions (Latef et al., 2016; Smith and Read, 2008) and our team has already observed positive effects of AMF on plants exposed to drought stress (Doubková et al., 2013; Voříšková et al., 2019). For these reasons, we were curious to test the hypothesis that AMF can mitigate drought stress in ornamental plants. Our current findings clearly confirmed this hypothesis. During the second half of the experiment, when not only the original nutrient resources became depleted but also drought impacted the plants, AMF increased growth and P acquisition in all three plant species and also enhanced flowering of G. rigens and P. zonale plants. The question arises as to what mechanisms were acting here. If the soil water is depleted, the accessibility of water and solutes (particularly of low-mobility P) by plant roots is consequently reduced, as well, because with progressing drought water increasingly resides in pores of smaller sizes. The plant roots cannot reach to water reservoirs in soil micropores (i.e., pores with diameter < 30 μm) and acquire from 6

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plants.

comprehensively explored and understood.

4.3. Plant response to hydrogel

Declaration of Competing Interest

Our hypothesis concerning positive effect of hydrogel on plant growth under drought was not confirmed. The addition of hydrogel had no positive effect on plant growth or P uptake and it even negatively affected flowering of all three plant species. Although hydrogels can improve some physical soil properties, mainly water retention, these changes are not always reflected in plant growth (Bres and Weston, 1993; Clemente et al., 2004; Martinez et al., 2001). There are several possible explanations for this inconsistency. First, the effect of the hydrogels can be only transitory. Some studies have found that positive effects of the gel on soil properties last from several hours to several months (Holliman et al., 2005; Jobin et al., 2004; Kim et al., 2010; Martinez et al., 2001). This could have been the case in our study. Second, frequent dehydration and hydration cycles decrease or eliminate positive effect of hydrogel on water retention in the soil (Montesano et al., 2015). Finally, sufficient concentration of the gel is also crucial for its positive action in the soil (Demitri et al., 2013; Hüttermann et al., 1999; Martinez et al., 2001). It is possible that the manufacturer had added too little of the gel to have any positive effects on soil properties and consequently on plant growth. As mentioned before, we observed that the hydrogel negatively affected flowering of all studied plant species. Although it earlier had been found that hydrogel can severely damage experimental plants (Chen et al., 2017), this occurred if the plants were cultivated in pure hydrogel. It should not be the case if the hydrogels are applied as soil amendments, because these have been determined relatively stable with respect to the production of potentially toxic decomposition substances (Holliman et al., 2005) and no toxic compounds originating from hydrogel have been detected in cultivated plants (Suresh et al., 2018). We thus cannot propose any reasonable explanation for the negative effect of hydrogels on flowering observed in our study. It is noteworthy that the addition of hydrogel decreased some growth parameters and P uptake of inoculated plants even as no such effect was observed for non-inoculated control plants (see the interaction of AMF and gel in Table 2). This indicates that the gel negatively affected AMF, that this was projected into decreased colonization of roots (significant for P. peltatum), and that it decreased growth of mycorrhizal plants. To our knowledge, potential toxicity of polyacrylamide gels vis-à-vis beneficial soil microorganisms, including AMF, has not been much explored. Holliman et al. (2005) observed some low microbial colonization (fungal hyphae, fungal spores and bacterial rods) present on the surface and within the matrix of field-conditioned polyacrylamide gels, so the gel could have acted as a matrix for microorganisms potentially antagonistic to AMF and thereby suppressed their positive effect on the plants.

The authors declare they have no conflict of interests. The authors declare that the presented data have neither been already published nor are they being considered for publication elsewhere. Acknowledgements The authors are grateful to Dr. Martin Dubský and Ing. Otka Plavcová (Silva Tarouca Research Institute for Landscape and Ornamental Gardening, Průhonice, Czech Republic) for chemical analyzes of the substrate and pre-planting of P. zonale, respectively, and to our technician Soňa Zvolenská for the enormous work associated with daily measurement of substrate moisture and selective watering. The study was supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant no. 1M0571), Czech Science Foundation (project 17-12166S) and by the Czech Academy of Sciences within the long-term research development project no. RVO67985939. References Allen, M.F., 2007. Mycorrhizal fungi: highways for water and nutrients in arid soils. Vadose Zone J. 6, 291–297. Allen, M.F., Moore, T.S., Christensen, M., 1982. Phytohormone changes in Bouteloua gracilis infected by vesicular arbuscular mycorrhizae .2. Altered levels of gibberellinlike substances and abscisic-acid in the host plant. Can. J. Bot. 60, 468–471. Álvarez, S., Bañón, S., Sánchez-Blanco, M.J., 2013. Regulated deficit irrigation in different phenological stages of potted geranium plants: water consumption, water relations and ornamental quality. Acta Physiol. Plant. 35, 1257–1267. Amiri, R., Nikbakht, A., Rahimmalek, M., Hosseini, H., 2017. Variation in the essential oil composition, antioxidant capacity, and physiological characteristics of Pelargonium graveolens L. inoculated with two species of mycorrhizal fungi under water deficit conditions. J. Plant Growth Regul. 36, 502–515. Asrar, A.W.A., Elhindi, K.M., 2011. Alleviation of drought stress of marigold (Tagetes erecta) plants by using arbuscular mycorrhizal fungi. Saudi J. Biol. Sci. 18, 93–98. Augé, R.M., 2001. Water relations, drought and vesicular-arbuscular mycorrhizal symbiosis. Mycorrhiza 11, 3–42. Augé, R.M., 2004. Arbuscular mycorrhizae and soil/plant water relations. Can. J. Soil Sci. 84, 373–381. Augé, R.M., Moore, J.L., Cho, K.H., Stutz, J.C., Sylvia, D.M., Al-Agely, A.K., Saxon, A.M., 2003. Relating foliar dehydration tolerance of mycorrhizal Phaseolus vulgaris to soil and root colonization by hyphae. J. Plant Physiol. 160, 1147–1156. Bitterlich, M., Franken, P., Graefe, J., 2018a. Arbuscular mycorrhiza improves substrate hydraulic conductivity in the plant available moisture range under root growth exclusion. Front. Plant Sci. 9, 11. Bitterlich, M., Sandmann, M., Graefe, J., 2018b. Arbuscular mycorrhiza alleviates restrictions to substrate water flow and delays transpiration limitation to stronger drought in tomato. Front. Plant Sci. 9, 15. Boyle, R.K.A., McAinsh, M., Dodd, I.C., 2016. Stomatal closure of Pelargonium x hortorum in response to soil water deficit is associated with decreased leaf water potential only under rapid soil drying. Physiol Plantarum 156, 84–96. Bres, W., Weston, L.A., 1993. Influence of gel additives on nitrate, ammonium, and waterretention and tomato growth in a soilless medium. Hortscience 28, 1005–1007. Calvet, C., Estaun, V., Camprubi, A., 1992. Germination, early mycelial growth and infectivity of a vesicular-arbuscular mycorrhizal fungus in organic substrates. Symbiosis 14, 405–411. Clemente, A.S., Werner, C., Maguas, C., Cabral, M.S., Martins-Loucao, M.A., Correia, O., 2004. Restoration of a limestone quarry: Effect of soil amendments on the establishment of native Mediterranean sclerophyllous shrubs. Restor. Ecol. 12, 20–28. Csima, G., Hernadi, I., Posta, K., 2012. Effects of pre- and post-transplant inoculation with commercial arbuscular mycorrhizal (AM) fungi on pelargonium (Pelargonium hortorum) and its microorganism community. Agric. Food Sci. 21, 52–61. Demitri, C., Scalera, F., Madaghiele, M., Sannino, A., Maffezzoli, A., 2013. Potential of cellulose-based superabsorbent hydrogels as water reservoir in agriculture. Int. J. Polym. Sci. 6. Doubková, P., Vlasáková, E., Sudová, R., 2013. Arbuscular mycorrhizal symbiosis alleviates drought stress imposed on Knautia arvensis plants in serpentine soil. Plant Soil 370, 149–161. Eiasu, B.K., Steyn, J.M., Soundy, P., 2012. Physiomorphological response of rose-scented geranium (Pelargonium spp.) to irrigation frequency. S. Afr. J. Bot. 78, 96–103. Friese, C.F., Allen, M.F., 1991. The spread of VA mycorrhizal fungal hyphea in the soil inoculum types and external hyphal architecture. Mycologia 83, 409–418. Gaur, A., Gaur, A., Adholeya, A., 2000. Growth and flowering in Petunia hybrida, Callistephus chinensis and Impatiens balsamina inoculated with mixed AM inocula or chemical fertilizers in a soil of low P fertility. Sci. Hort. Amst. 84, 151–162.

5. Conclusions Our study illustrates that arbuscular mycorrhiza increased P uptake and can stimulate growth and flowering of ornamental plants exposed to water deficiency. While some of the studied species were particularly responsive to mycorrhiza both in terms of growth and flowering (P. zonale and G. rigens), another responded positively only with respect to growth (P. peltatum). On the other hand, the addition of hydrogel had no positive effect on any parameter of any tested plant species. Conversely, it negatively affected flowering of the three plant species and reduced root colonization of P. peltatum. For this reason, the expected synergy of AMF and hydrogel was not manifested. Whereas inoculation with AMF can be advantageous for the growth and visual qualities in pot-grown ornamental plants experiencing periods of drought and can be recommended for horticultural application, addition of hydrogel should be rather avoided, at least until the mechanisms of its effects in the soil as well as possible pitfalls of its application are 7

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