Can mycorrhizal inoculation stimulate the growth and flowering of peat-grown ornamental plants under standard or reduced watering?

Applied Soil Ecology 80 (2014) 93–99

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Can mycorrhizal inoculation stimulate the growth and flowering of peat-grown ornamental plants under standard or reduced watering? David Püschel a,b,∗ , Jana Rydlová a , Miroslav Vosátka a a b

Department of Mycorrhizal Symbioses, Institute of Botany, AS CR, Pr˚ uhonice, Czech Republic Laboratory of Fungal Biology, Institute of Microbiology, AS CR, Prague, Czech Republic

a r t i c l e

i n f o

Article history: Received 18 December 2013 Received in revised form 26 March 2014 Accepted 2 April 2014 Keywords: Ornamental plants Arbuscular mycorrhizal fungi Peat-based substrate Mycorrhizal growth response Watering regime

a b s t r a c t Although the growth of plants is often successfully stimulated by inoculation with arbuscular mycorrhizal fungi (AMF), the question remains whether AMF are beneficial under the specific conditions of peat-based pot cultivation of ornamental plants. A series of two greenhouse experiments aimed on this question. In the first experiment, we tested the effect of inoculation with AMF, applied as a commercial inoculum, on various biometric parameters including the flowering of eight ornamental plant species. Capsicum annuum, Dimorphoteca sinuata, Gazania splendens, Impatiens hawkerii, Pelargonium peltatum, Pelargonium zonale, Sanvitalia procumbens and Verbena × hybrida were planted in pots with a peat-based substrate. AMF were naturally absent in this substrate. The plant species differed in their mycorrhizal growth response (MGR) evaluated as the effect of inoculation on shoot biomass. The MGR was positively correlated with the level of root colonization, which ranged from 17% to 68% depending on the plant species. Inoculation with AMF also significantly increased other growth parameters important for ornamental plants, namely the number of flowers (S. procumbens, Verbena × hybrida), flower size (I. hawkerii), shoot dry weight (P. peltatum, P. zonale and S. procumbens), root dry weight (G. splendens, P. peltatum and S. procumbens), the number of leaves (C. annuum, G. splendens, P. peltatum and P. zonale), plant length (C. annuum, P. zonale and S. procumbens), the number of branches (P. zonale and S. procumbens) and the total length of branches (S. procumbens). In the second experiment, P. zonale was used as a model plant grown under two watering regimes: standard and low. A strong positive effect of AMF on plants was observed under both watering regimes for all measured parameters (shoot fresh and dry weight, plant length, leaf area, number of branches and flowers). In all the parameters, inoculated low-watered plants performed significantly better than well-watered plants without AMF. We conclude that (1) inoculation with AMF improved the general vitality and visual quality of ornamental plants, and that (2) for P. zonale this stimulation occurred even under the low watering regime. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Arbuscular mycorrhizal fungi (AMF) establish symbiosis with most terrestrial plant species. Mycorrhiza is mutualistic symbiosis with a wide range of positive effects on host plants. Mycorrhizal plants are more efficient in the uptake of nutrients (Smith and Read, 2008), reproduce more successfully (Koide, 2010), exhibit improved post-transplant survival and growth (Vosátka, 1995), and are considered more resistant to certain pathogens (Dugassa et al., 1996). Inoculation with AMF is often regarded as a reasonable

∗ Corresponding author at: Institute of Botany, AS CR, Department of Mycorrhizal ˚ Symbioses, Zámek 1, 252 43 Pruhonice, Czech Republic. Tel.: +420 271 015 333; fax: +420 271 015 105. E-mail address: [email protected] (D. Püschel). http://dx.doi.org/10.1016/j.apsoil.2014.04.001 0929-1393/© 2014 Elsevier B.V. All rights reserved.

approach to improving plant growth and is being promoted for a wide range of applications (Feldmann et al., 2008; Gianinazzi et al., 2010), including cultivation of ornamental plants (Koltai, 2010; Vosátka and Albrechtová, 2008). AMF have been observed to increase shoot dry weight (Vosátka ˇ et al., 2000), the length or number of branches et al., 1999; Srámek (Meir et al., 2010) or the number and size of flowers in ornamental plants (AboulNasr, 1996; Perner et al., 2007; Long et al., 2010). They have also been found to accelerate flowering (Gaur et al., 2000; Garmendia and Mangas, 2012). Since ornamental potgrown plants are occasionally exposed to water deficiency, the reported positive effect of AMF on the water regime of plants (von Reichenbach and Schonbeck, 1995; Asrar et al., 2012) might be another benefit of mycorrhizal symbiosis. However, no definite conclusion regarding this matter has been reached because the exact mechanisms of how AMF affect the efficiency of water use by

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plants are still unexplained (Auge, 2001). Mycorrhizal symbiosis is indeed a very complex relationship, and plants can even respond negatively to AMF inoculation (Johnson et al., 1997; Klironomos, 2003). Neutral or even negative effects of AMF have been reported for some ornamental plants (Koide et al., 1999; Linderman and Davis, 2004; Gaur and Adholeya, 2005). The inconsistent effects of AMF inoculation may be caused by the diversity of host plants and fungal symbionts or by variable cultivation conditions (Fester and Sawers, 2011). Plants can differ in their response to AMF even on the level of cultivars (Linderman and Davis, 2004) or genotypes (Sensoy et al., 2007). Similarly, different AMF species (Klironomos, 2003) or even isolates of the same AMF species (Munkvold et al., 2004) differ in their effects on plants. Obviously, the complex characteristics of the soil environment and of cultivation substrates also contribute to the varied effects of mycorrhizal symbiosis. Ornamental plants are usually planted in peat-based substrates. Although some ecological concerns about the use of peat have been brought up in recent years, alternative substrates are not becoming prevalent, so peat will arguably remain the key component of substrates for years to come. Peat-based substrates are, however, well-known for the absence of beneficial soil microorganisms including AMF (Linderman and Davis, 2003a; Perner et al., 2007; Koltai, 2010). Even subsequent introduction of AMF into peatbased substrates can be difficult due to possible negative effects of some types of peat on various aspects of AM symbiosis such as germination and early mycelial growth (Ma et al., 2006), colonization establishment (Calvet et al., 1992), the development of intraradical colonization (Linderman and Davis, 2003b; Ma et al., 2007) or the effectiveness of the symbiosis itself (Vestberg et al., 2005). Inoculation with AMF is nevertheless being seriously considered as a practice for amending substrates. Moreover, a specialized peat substrate for ornamental plants containing AMF has recently appeared on the market. Similar products from other manufacturers can be expected before long. We aimed to find out whether AMF inoculation is an effective means of stimulating the growth of ornamental plants grown in peat-based substrates. In the first greenhouse experiment, we cultivated eight ornamental plant species in a standard commercial peat-based substrate and inoculated them with a commercially available inoculum of AMF, keeping non-inoculated plants as controls. We aimed to answer the question whether mycorrhizal inoculation stimulates various aspects of plant growth perceived together as the visual quality of ornamental plants. In the second experiment, we grew Pelargonium zonale (arguably one of the most popular ornamental plant species in the Czech Republic) under two watering regimes. Our goal was to find out whether the effect of mycorrhizal symbiosis changes under limited moisture conditions.

2. Material and methods 2.1. Screening experiment Round plastic 700 ml pots were filled with a commercial peat substrate (horticultural substrate–type B; manufacturer: Raˇselina a.s., Sobˇeslav, Czech Republic). This substrate contained highly decomposed and disintegrated dark peat mixed with a lower 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), 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), Ca 975 mg kg−1 (water-extractable, EN 13652). We selected

this substrate for the study because it is a universal substrate for horticulture with a good price/quality ratio that is usable in a wide range of applications. Prior to the experiment, the peat-based substrate was tested for the presence of AMF propagules with negative results (the roots of Zea mays – a universal host plant used in this bioassay – remained uncolonized after six weeks of cultivation in the tested substrate). In our experiment, we included eight of common species of ornamental plants: Capsicum annuum, Dimorphoteca sinuata, Gazania splendens (var. Daybreak Red Stripe), Impatiens hawkerii (var. Divine), Pelargonium peltatum (var. Tornado), P. zonale (var. Gizela), Sanvitalia procumbens (var. Sprite) and Verbena × hybrida. The plants germinated from seeds purchased from a local retailer (manufacturer: SEMO a.s., Smrˇzice, Czech Republic) in plastic multipots with 15 ml cells filled with a heat-sterilized (autoclaved twice at 121 ◦ C for 30 min, 24 h apart) sand–zeolite mixture 1:1 (v/v). Young plants were transplanted into experimental pots (one plant per pot) when they had two cotyledons and one true leaf. The mycorrhizal inoculation comprised two treatments: (1) AM—treatment inoculated with a commercial AMF inoculum and (2) NM—non-mycorrhizal control treatment. The inoculum Symbivit® (manufacturer: Symbiom s.r.o., Lanˇskroun, Czech Republic) consists of a mixture of zeolite and expanded clay that acts as a carrier of propagules (spores, mycelium and colonized root fragments) of six different Glomus species. We used a universal commercial AMF inoculum because it better corresponds to its potential commercial application as far as its quantity and quality is concerned. For the control treatment, a custom-made carrier of the same properties but without AMF propagules was provided by the manufacturer of the inoculum. Eight millilitre of the inoculum or the AMF-free carrier, respectively, per pot were poured into holes below the transplanted seedlings. The experiment comprised 10 replicates per treatment per plant species, AM and NM pots were randomized in blocks of the same species. The experiment was established in a greenhouse at the beginning of September, and lasted for three months. The ambient light was supplemented with 400 W metal halide bulbs switched on for photoperiods of 14 h. The light intensity measured at the top of the pots reached 6000 lx (measured in the middle of the experiment, i.e. at the end of October; Testo 435-2 with probe 0635 0545, Testo AG, Germany) when no ambient light was present (i.e. at the beginning and end of the set photoperiod). Combined with daylight, the light intensity ranged from 12,000 to 20,000 lx. The temperature was maintained between 27 ◦ C (day) and 15 ◦ C (night) by an automatic ventilation and heating (in later autumn) system installed in the greenhouse. The experiment was watered as necessary to ensure that the plants would not be exposed to drought stress. Manufacturers commonly amend commercial peat-based substrates with significant amounts of fertilizer to cover the nutrient demands of plants for several weeks. Because the study was intended to simulate real-life conditions in which plants receive little maintenance, no additional fertilizer was applied throughout the experiment. The experiment was harvested at the flowering stage of each species (with the exception of P. peltatum, whose flowering was delayed and expected in several weeks). Rigorous quantification of the ornamental plants’ visual quality, a highly subjective matter, is quite a challenging task because it is perceived as a combination of multiple plant traits. To accomplish this task as best as possible, we opted to measure the following biometric or flowering-related parameters (optimized on a per-species basis to reflect the morphology of each given plant species) at harvest: number of leaves (C. annuum, D. sinuata, G. splendens, I. hawkerii, P. peltatum and P. zonale), number of branches (C. annuum, D. sinuata, I. hawkerii, P. peltatum, P. zonale, S. procumbens and Verbena × hybrida),

D. Püschel et al. / Applied Soil Ecology 80 (2014) 93–99

plant length (all species), total length of branches (D. sinuata, P. peltatum, S. procumbens and Verbena × hybrida), number of flowers1 (C. annuum, D. sinuata, G. splendens, P. zonale, S. procumbens and Verbena × hybrida) and diameter of the largest flower (I. hawkerii). At harvest, shoot dry weight (SDW) and root dry weight (RDW) were determined for all plant species after drying the biomass at 70 ◦ C to constant weight. Roots were washed to remove the substrate and then weighed. Random root samples were taken from multiple parts of the root system (ca 0.7 g of fresh weight in each sample) 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 using the gridline intersect method (Giovannetti and Mosse, 1980). Colonization was expressed as the percentage of root length colonized by AMF. The remaining roots were weighed after drying at 70 ◦ C to constant weight. The weight of the root sub-sample used for the determination of colonization was added to RDW after recalculation of its fresh weight to dry weight. To compare the growth response of different plant species to inoculation, mycorrhizal growth response (MGR) was calculated for shoot dry weight according to the equation MGR = (M − NMmean )/NMmean × 100%, where M is the value of SDW recorded for a given inoculated plant and NMmean is the mean value of SDW of plants in the corresponding non-inoculated treatment (Gange and Ayres, 1999). The results were analyzed using STATISTICA 12 (StatSoft Inc., USA). The data were checked for normality of distribution and homogeneity of variance. The continuous biometric data were either square-root (SDW, plant length, total length of branches) or logarithmically (RDW) transformed to improve the homogeneity of variances and then subjected to two-way analysis of variance (ANOVA). T-test was used to find significant differences between inoculated and control plants. Discrete data (number of leaves, branches or flowers) were analyzed using Generalized Linear Models (GLM) with Poisson distribution. The data on mycorrhizal colonization did not meet the criteria of homogeneity of variances even after several attempted transformations, and were thus analyzed using a non-parametric Kruskal–Wallis test followed by a multiple-comparisons z-value test. The data on MGR were logarithmically transformed and then analyzed using a one-way ANOVA and Fisher’s LSD test. The relationship between MGR and root colonization was assessed using linear regression performed on the means of the two parameters. 2.2. Watering experiment In the follow-up experiment, the same material and methods were used as in the Screening experiment with regard to the substrate, pots, inoculation and no added fertilizer. Seeds of P. zonale (var. Gizela) were germinated, and plants were pre-planted in sterile conditions (Plavcová, 2009). A single plant was transplanted into each experimental pot at the stage when it had two cotyledons and one true leaf. The experiment was started at the beginning of May 2009 and conducted in the greenhouse for 28 weeks until the half of November. No additional lighting was provided from May to September. In the autumn period, additional lighting and heating maintained conditions described in Experiment I. In the first stage, the plants were watered as necessary for the first nine weeks. Then the experiment was split into two watering treatments: In the standard watering treatment (SW), the plants were

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Fig. 1. Mycorrhizal colonization (MC) of roots of different ornamental plant species. CA—Capsicum annuum, DS—Dimorphoteca sinuata, GS—Gazania splendens, IH—Impatiens hawkerii, PP—P. peltatum, PZ—Pelargonium zonale, SP—Sanvitalia procumbens, VH—Verbena × hybrida. The presented data are means of 10 replicates ± standard error of mean. Different letters above the columns indicate significant differences (p < 0.05) according to multiple comparisons z-value test (screening experiment).

watered approximately every five days when the soil moisture dropped to 13–15% (vol.) of water content (Moisture Meter HH2 with ThetaProbe ML2x type soil moisture sensor, Delta-T Devices Ltd, UK); in the low watering treatment (LW), the plants were watered approximately every nine days when water content reached 3–5%. This threshold was determined by observing physiological symptoms of strong yet reversible wilting and by measuring the corresponding water content in a previous preliminary test with P. zonale. The watering was carried out based on regular measurements of soil moisture to avoid subjective error. The experiment comprised 10 replicates per treatment. AM and NM pots were randomized in blocks of the same watering treatment. At harvest, shoot fresh weight (SFW), SDW, plant length, leaf area (using an area meter LI-3100, LI-COR, USA), the number of branches and the number of flowers were measured. The root samples were stained, and mycorrhizal colonization was determined as in the Screening experiment. The data for all measured parameters passed the tests of normality and homogeneity of variances. A two-way ANOVA was used to determine the effects of individual factors. The means of continuous parameters were separated using Fisher’s LSD test; discrete data (number of branches and flowers) were analyzed using Generalized Linear Models (GLM) with Poisson distribution. 2.3. Economic feasibility of AMF inoculation The cost of mycorrhizal inoculation was expressed as the relative cost increase per one plant cultivated in 1 l of substrate. The price of the mycorrhizal inoculum and the recommended dosage as well as the prices of various peat substrates usable for ornamental plants, including the new product containing AMF, were obtained from the on-line shops of the two manufacturers, Symbiom, s.r.o. and Raˇselina, a.s., respectively. Calculations of all prices are based on the largest – most cost-efficient – packaging available to consumers (shipping costs were not included). 3. Results 3.1. Screening experiment

1

C. annuum and I. hawkerii produce flowers whereas all other species produce ¨ all species in ¨ of flowersfor inflorescences. For simplicity, we use the term number the rest of the manuscript.

Mycorrhizal inoculation resulted in root colonization of all the ornamental plant species (Fig. 1), while the roots in the control

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Fig. 2. Mycorrhizal growth response (MGR) of ornamental plants expressed as percentage increases of shoot dry weight. CA—Capsicum annuum, DS—Dimorphoteca sinuata, GS—Gazania splendens, IH—Impatiens hawkerii, PP—Pelargonium peltatum, PZ—P. zonale, SP—Sanvitalia procumbens and VH—Verbena × hybrida. The presented data are means of 10 replicates ± standard error of mean. Different letters above the columns indicate significant differences (p < 0.05) according to Fisher’s LSD test (screening experiment).

SDW [g] Capsicum annuum Dimorphoteca sinuata Gazania splendens Impatiens hawkerii Pelargonium peltatum Pelargonium zonale Sanvitalia procumbens Verbena × hybrida Effect of factors (1) Plant species (2) Inoculation Interaction

AM NM AM NM AM NM AM NM AM NM AM NM AM NM AM NM

2.9 (±0.36) 2.9 (±0.32) 4.4 (±0.38) 4.7 (±0.32) 3.0 (±0.30) 2.4 (±0.22) 2.1 (±0.37) 2.3 (±0.38) 6.3 (±0.81) 4.1 (±0.56) 9.5 (±0.64) 7.6 (±0.58) 1.5 (±0.21) 0.8 (±0.13) 5.2 (±0.24) 4.3 (±0.49) F 64.21 10.32 1.74

RDW [g] ns ns ns ns *

*

*

ns

*** ***

ns

1.09 (±2.35) 0.78 (±0.11) 0.55 (±0.06) 0.43 (±0.04) 0.32 (±0.05) 0.15 (±0.02) 0.38 (±0.08) 0.42 (±0.07) 0.27 (±0.04) 0.13 (±0.02) 1.36 (±0.17) 1.30 (±0.11) 0.55 (±0.06) 0.35 (±0.08) 0.57 (±0.09) 0.38 (±0.06) F 29.70 13.89 1.62

Plant length [cm] ns ns *

ns **

ns *

ns

*** ***

ns

23.4 (±1.10) 18.8 (±0.63) 71.1 (±6.01) 74.0 (±1.00) 11.2 (±1.49) 7.2 (±1.65) 13.9 (±1.46) 12.9 (±1.63) 51.9 (±4.37) 46.3 (±4.32) 38.9 (±2.51) 32.3 (±1.49) 19.2 (±2.05) 13.8 (±1.70) 45.7 (±3.02) 39.3 (±2.82) F 119.84 10.90 1.07

**

ns ns ns ns *

ns ns

*** **

ns

No. of branches 8.9 (±1.31) 8.3 (±0.76) 14.5 (±1.73) 14.8 (±1.01) –

ns

6.6 (±0.72) 6.6 (±0.37) 5.5 (±0.57) 4.9 (±0.64) 2.4 (±0.31) 0.9 (±0.18) 11.1 (±1.04) 6.4 (±0.54) 19.5 (±1.36) 16.3 (±2.53) WS 369.36 12.89 14.88

ns

ns

ns *

***

ns

*** *** *

No. of leaves 113 (±9.3) 101 (±6.40) 307 (±42.3) 294 (±28.8) 45.6 (±4.37) 31.9 (±2.93) 78.1 (±11.09) 65.4 (±2.68) 52.1 (±3.70) 36.8 (±4.85) 26.3 (±2.09) 19.3 (±1.61) nd

No. of flowers *

ns **

ns ***

nd

**

2.2 (±0.25) 1.5 (±0.17) 41.2 (±5.87) 24.8 (±3.76) 15.7 (±1.98) 4.2 (±1.76) WS 825.97 23.62 39.85

nd WS 7722.21 67.73 33.14

36.3 (±1.15) 32.8 (±4.78) 8.4 (±2.03) 8.7 (±2.56) 1.5 (±0.58) 0.6 (±0.27) nd

*** *** ***

ns ns ns

ns ***

***

*** *** ***

D. Püschel et al. / Applied Soil Ecology 80 (2014) 93–99

treatment remained uncolonized. There were significant (p < 0.05) differences among the plant species in the percentage of root length colonized. The highest mycorrhizal colonization was found in the roots of S. procumbens, G. splendens and P. peltatum (60–70% of root length). Low levels of colonization (around 20% of root length) were observed in the roots of C. annuum, D. sinuata and I. hawkerii (Fig. 1). MGR calculated for SDW differed significantly (p < 0.05) among the plant species tested (Fig. 2). Our findings indicate a positive relationship (p = 0.0055, r = 0.8654) between the mean level of root colonization and the observed mean MGR (Fig. 3). The eight species of ornamental plants responded differently to inoculation in a spectrum of measured parameters, ranging from significantly positive to neutral (Table 1). In S. procumbens, P. zonale and P. peltatum, the stimulatory effect of AMF was observed in most traits. The inoculation of S. procumbens increased SDW and stimulated various parameters related to branching: it almost doubled the number of branches, significantly increased plant length (Table 1) and also more than doubled the total length of branches (139 cm vs 63 cm; p = 0.004). The inoculation also increased the number of flowers by ca 66%

Fig. 3. Relationship between mean values of mycorrhizal colonization (MC) of roots and mycorrhizal growth response (MGR) of tested ornamental plants (screening experiment).

Table 1 Effect of mycorrhizal inoculation on different parameters of eight species of ornamental plants. Effects of factors according to two-way ANOVA for SDW, RDW and plant length (presented as F values and significance) or according to GLM for the number of branches, leaves and flowers (presented as Wald statistic and significance): * p < 0.05, ** p < 0.01, *** p < 0.001, ns—non-significant effect. Comparison of inoculated (AM) and control (NM) treatment using t-test or GLM at p < 0.05. nd—not determined. Data are means of 10 replicates (±standard errors; screening experiment).

ns

***

ns

***

ns

ns

***

ns

7.2 6.4 8.6 5.0 WS 0.41 18.24 1.26 ***

ns

ns

***

***

5.2 4.4 7.5 2.1 WS 0.43 51.84 0.43 ***

***

ns

***

ns

***

530 440 725 245 F 23.66 674.04 2.07 ns

***

***

***

***

37.3 36.1 44.1 29.3 F 1.33 194.27 2.92 ***

***

***

***

***

11.2 9.7 13.6 7.3 F 16.79 308.89 35.85 ***

48.2 42.3 64.1 26.4 F 24.34 967.94 24.17 Effect of factors (1) Watering (2) Inoculation Interaction

SW LW AM NM Inoculation

LW

Mean values per treatment Watering

a b a b

No. of flowers

9.5 (±0.40) 4.8 (±0.33) 7.6 (±0.34) 5.1 (±0.23) a b a b

No. of branches

8.3 (±0.34) 2.1 (±0.23) 6.6 (±0.27) 2.1 (±0.23) a c b d 783 (±15.1) 276 (±22.6) 667 (±16.8) 213 (±18.6) a b a b AM NM AM NM

70.0 (±1.73) 26.4 (±1.35) 58.1 (±0.77) 26.4 (±0.66)

a c b c

15.3 (±0.31) 7.0 (±0.50) 11.8 (±0.23) 7.6 (±0.32)

a c b c

45.6 (±1.08) 29.0 (±1.02) 42.6 (±1.07) 29.6 (±1.08)

Leaf area [cm2 ] Plant length [cm]

SW

All species of ornamental plants tested in our study were successfully inoculated with AMF when planted in the commercial peat-based substrate, which is an important precondition for potential practical use. This indicates that the three components involved in the interaction – the plants, AMF and substrate – were compatible and that mycorrhizal symbiosis was established. Moreover, we revealed that species with higher levels of root colonization responded positively to AMF inoculation in more traits than plants with roots colonized to a lesser extent. The arguably most desired potential effect of mycorrhizal inoculation of ornamental plants is the stimulation of flowering. We found a clearly positive effect of AMF on the flowering of two of the species tested, specifically an increase in the number of flowers in S. procumbens and Verbena × hybrida. In addition, we observed a tendency towards a higher number of flowers in G. splendens. The effect of AMF on the number of flowers is well documented for a whole array of plant species, such as P. peltatum (Perner et al., 2007), Verbena sp. (Vosátka et al., 1999), Antirrhinum majus (Asrar et al., 2012), Chrysanthemum morifolium or Tagetes erecta (Vaingankar and Rodrigues, 2012). AMF can also increase the size of flowers (Sohn et al., 2003; Asrar and Elhindi, 2011), an effect we observed in I. hawkerii. In many plant species, the quantity of flowers is proportional to plant size and nutrient content (Lu and Koide, 1994; Gaur et al., 2000). The increased flowering of inoculated plants could be thus related to their larger biomass and probably also to better nutrient uptake. However, the flowering of Verbena × hybrida and I. hawkerii was stimulated despite the fact that their biomass was not increased by AMF at the same time. This points to rather different mechanisms of mycorrhizal stimulation of flowering in these species, for example, effects on the plant hormonal profile including the production of gibberellin-like substances, which can influence

SDW [g]

4. Discussion

SFW [g]

Root colonization of P. zonale did not significantly differ between the SW and LW treatment (25% and 20%, respectively). The roots of the control treatment remained uncolonized under both watering regimes. The mycorrhizal inoculation significantly increased all the biometric parameters measured. SFW, SDW and leaf area were also significantly affected by the watering factor with smaller plants found in the LW treatment. A significant interaction of factors was observed for SFW and SDW, indicating that the mycorrhizal growth stimulation was higher under the SW treatment than under the LW treatment (Table 2).

Inoculation

3.2. Watering experiment

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Watering

(Table 1). In P. zonale, the inoculation stimulated SDW, increased the number of leaves, more than doubled the number of branches and increased plant length (Table 1). Finally, in the case of P. peltatum, the inoculation significantly increased SDW, RDW as well as number of leaves (Table 1). At the time of the harvest, the plants were not yet flowering at a rate that would allow statistical analysis; we found only three flowering plants in the AM treatment and one plant with flower buds in the NM treatment. The inoculation increased RDW and the number of leaves of G. splendens as well as plant length of C. annuum (Table 1). A positive effect on flowering was observed for both Verbena × hybrida, where the inoculation increased the number of flowers almost fourfold (Table 1), and I. hawkerii, where it significantly increased the diameter of the largest flower (6.4 cm vs 4.8 cm; p = 0.003). AMF also tended to stimulate the flowering of G. splendens (p = 0.058). The inoculation did not significantly affect the growth of D. sinuata in any of the measured parameters (Table 1).

Table 2 Effect of AMF inoculation on different parameters of Pelargonium zonale when grown under two watering regimes. Effects of factors according to two-way ANOVA for SFW, SDW, plant length and leaf area (presented as F values and significance) or according to GLM for the number of branches or flowers (presented as Wald statistic and significance): * p < 0.05, ** p < 0.01, *** p < 0.001, ns—non significant effect. Comparison of inoculated (AM) and control (NM) treatment under standard watering (SW) or low watering (LW). Data are means of 10 replicates (±standard errors). Different letters within each parameter indicate significant differences according to Fisher’s LSD test or GLM, respectively, at p < 0.05 (watering experiment).

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flowering (Allen et al., 1982). Perner et al. (2007) hypothesized that mycorrhizal colonization may directly or indirectly (through altered plant nutrient status) influence the balance of plant hormones. On the other hand, AMF need not in all cases affect the flowering of ornamental plants positively, as observed, for example, by Dubsky´ et al. (2002), Linderman and Davis (2004) or Nowak (2004). Regrettably, we were unable to determine the effect of AMF on the flowering of P. peltatum because this species had not started flowering by the time of the harvest. We, however, observed that the inoculated plants were more advanced in this regard than the control plants (three inoculated plants were already flowering), in which the development of flower buds was visibly delayed. A similar effect of AMF on expedited flowering time has been reported for various ornamental plants (AboulNasr, 1996; Gaur et al., 2000; Sohn et al., 2003; Garmendia and Mangas, 2012). Nowak (2004), by contrast, found AMF to delay the flowering of P. zonale. In a study of Dubsky´ et al. (2002), AMF delayed the beginning of flowering of Cyclamen persicum but increased the number of flower buds. In addition to the onset of flowering, AMF can also influence its duration. Our knowledge of this effect is fragmentary, however. Scagel and Schreiner (2006), for instance, reported prolonged flowering of the calla lily (Zantedeschia) caused by Glomus intraradices. Besides flowering, the second most interesting parameter for growers is the general size of ornamental plants and how it is affected by AMF. There are numerous reports of increased SDW in ornamental plants (Vosátka et al., 1999; Gaur et al., 2000; ˇ Srámek et al., 2000; Asrar and Elhindi, 2011; Asrar et al., 2012). Nowak (2004) found increased plant height as well as the number and length of branches. However, from a visual standpoint, the increased plant height or branch length may not necessarily be positive if shoot biomass is not increased at the same time. Such plants may look too spindly, as was the case of C. annuum in our experiment. Importantly, AMF can also suppress the growth of ornamental plants, as reported, for example, by Koide et al. (1999), who found growth depressions in all plant species studied under certain inoculation treatments. These negative effects of inoculation probably suggest a certain level of incompatibility among plants, AMF and their substrate. The cost-benefit balance – determined by carbon drain and AMF-mediated nutrient uptake – can thus be negative, resulting in an AMF-induced depression in plant growth (Smith et al., 2009). The results of our second experiment indicate that P. zonale is a species tolerant to water deficiency. In non-inoculated plants, none of the measured traits, excluding leaf area, was negatively affected by the LW treatment. In mycorrhizal plants, by contrast, the reduced watering decreased shoot biomass and leaf area. Interestingly, inoculated plants under the LW treatment still performed significantly better for all measured traits than non-inoculated plants grown under the SW treatment. The observed growth reduction of inoculated plants can probably be ascribed to higher water demands of larger inoculated plants. Our results also indicate that lower water demands of generally smaller non-mycorrhizal plants were met in both the SW and LW treatment. Mycorrhizal plants, by contrast, were larger in size, and the LW treatment could not meet their water demands. This resulted in the differences between the watering treatments which we observed in some traits of mycorrhizal plants. Our results are similar to those of other studies dealing with ornamental plants (Asrar and Elhindi, 2011; Asrar et al., 2012). In these studies, however, water deficiency also affected non-inoculated plants (T. erecta and A. majus). This can probably be explained by a higher drought sensitivity of the plant species used by others (Asrar and Elhindi, 2011; Asrar et al., 2012) or a higher degree of water deficiency the plants were exposed to. The possible mechanisms behind the higher resistance of inoculated plants to water deficiency are both direct and indirect. Direct

mechanisms include a larger volume of soil that is accessible for water extraction thanks to the hyphal network (Ruiz-Lozano and Azcon, 1995; Cho et al., 2006) or stomatal regulation through hormonal signals (Goicoechea et al., 1996). Indirect mechanisms are, for example, generally increased nutritional status or enhanced photosynthetic efficacy of mycorrhizal plants (Asrar and Elhindi, 2011). The question arises whether the promising positive effects of AMF are worth the cost of mycorrhizal inoculation. If we, for simplicity, assume that 1 l of peat-based substrate is used per pot per plant and that the mycorrhizal inoculum is applied according to the manufacturer’s recommended dosage, the additional costs of mycorrhizal inoculation represent roughly 130% of the price of the substrate. These costs can, however, be reduced by several means. First, the manufacturer’s recommended dosage was probably set high for safety reasons and can be lowered in practice (assuming the inoculum is of sufficiently high and consistent quality). We used approximately half the recommended dose in our study and still observed good colonization rates with positive effects. Second, manufacturers are recently starting to introduce new ranges of specialized substrates containing AMF. With these products, we can estimate the price increase to be in the range of ca 60–90%, depending on the substrate used as a reference. Finally, the most cost-efficient approach is indisputably pre-inoculation of plant seedlings at the propagation stage, in which the costs of inoculation are a fraction of the costs outlined above. We can conclude that mycorrhizal inoculation can be conditionally recommended for cultivation of ornamental plants in peat-based substrates. While inoculation with AMF can have a noticeable positive effect on the visual quality of some ornamental plant species, the response of other plant species is not as convincing. Inoculation with AMF should be assessed on a per-species basis when deciding whether its results are worth the costs. The clear positive effects of mycorrhizal inoculation on P. zonale were maintained even under partial water deficiency, to which home-grown ornamentals are commonly subjected. Though the water deficiency affected most traits related to plant growth, mycorrhizal plants were still distinctly more vital than their nonmycorrhizal counterparts.

Acknowledgements We are grateful to Dr. Jan Jansa for his critical commentary on the manuscript, to the company Raˇselina a.s., Sobˇeslav, Czech Republic for material and financial support of our research, to Dr. Martin Dubsky´ and Ing. Otka Plavcová (Silva Tarouca Research Institute for ˚ Czech Republic) Landscape and Ornamental Gardening, Pruhonice, for chemical analyzes of the substrate and pre-planting of P. zonale, respectively. The help of Doc. Zuzana Münzbergová with statistical analyses is gratefully acknowledged. Financial support for this study was provided by the Ministry of Education, Youth and Sports of the Czech Republic (grant 1M0571). This work was also supported as part of the long-term research development projects No. RVO 67985939 and No. RVO 61388971 and by the Joint working group of the Institute of Microbiology AS CR and the Institute of Botany AS CR.

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