Influence of Mycorrhizal Fungi and Microalgae Dual

Influence of Mycorrhizal Fungi and Microalgae Dual Inoculation on Basil Plants Performance Marieta Hristozkova, Lilyana Gigova, Maria Geneva, Ira Stancheva, Violeta Velikova & Gergana Marinova Gesunde Pflanzen Pflanzenschutz - Verbraucherschutz Umweltschutz ISSN 0367-4223 Gesunde Pflanzen DOI 10.1007/s10343-018-0420-5

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Author's personal copy Gesunde Pflanzen https://doi.org/10.1007/s10343-018-0420-5

ORIGINAL ARTICLE

Influence of Mycorrhizal Fungi and Microalgae Dual Inoculation on Basil Plants Performance Marieta Hristozkova1 · Lilyana Gigova2 · Maria Geneva1 · Ira Stancheva1 · Violeta Velikova3 · Gergana Marinova2 Received: 20 February 2018 / Accepted: 19 March 2018 © Springer-Verlag GmbH Deutschland, ein Teil von Springer Nature 2018

Abstract The rhizosphere microbial community is a complex of organisms interconnecting in multifold ways, acting upon each other and reacting to the surrounding environment. In the present research, we evaluated the influence of dual inoculation with arbuscular mycorrhizal fungi (AMF) and microalgae (Scenedesmus incrassatulus R83 and Synechocystis sp. R10) on basil plants performance. Different modes of basil inoculation (AMF, microalgae and a combination of both) were analyzed. We characterized AMF function (colonization and glomalin-related soil proteins), acid phosphatase activity (in root and soil), plant growth, photosynthetic parameters, secondary metabolites (fluorescence indices of leaf chlorophyll content; flavonols contents; nitrogen balance index), and the activity of plant enzymes linking nitrogen and carbon metabolism (glutamate synthase, aspartate aminotransferase and NADP-malic enzyme). The highest values of biometrical data were as a result of mycorrhiza application alone and in the mixed treatments with both microalgae strains. Dual inoculation with both microalgae and AMF, stimulated mycorrhizal function (concentration of glomalin-related proteins). Indexes of secondary metabolites (flavonols and anthocyanins) increased after treatment with Scenedesmus (Al1 and AM + Al1) compared to control plants. The addition of Synechocystis alone and in combination with fungi positively influenced nitrogen balance index. Different modes of inoculation increased gas-exchange parameters in all variations of inoculations compare to control plants. The results for activities of nitrogen-carbon metabolizing enzymes demonstrated close relationships with the plant growth. The mycorrhizal root colonization of basil may bear considerable economic importance. Thus, the addition of suitable AMF to the rhizosphere would significantly improve the growth and productivity of commercial Ocimum spp. cultivation.

Keywords Aboveground-belowground · Arbuscular mycorrhizal fungi · Soil microalgae · Ocimum basilicum L.

Marieta Hristozkova

[email protected] 1

Laboratory “Plant-Soil Interactions”, Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria

2

Laboratory “Experimental Algology”, Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria

3

Laboratory “Photosynthesis-Activity and Regulation”, Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria

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Author's personal copy M. Hristozkova et al.

Der Einfluss von Doppelinokulation mit Mykorrhizapilzen und Mikroalgen auf die Leistung von Basilikumpflanzen Zusammenfassung Die Mikrobengemeinschaft im Wurzelraum ist ein Komplex aus Organismen, die auf verschiedenste Weise miteinander verbunden sind, miteinander in Wechselwirkung stehen und auf ihre Umgebung reagieren. In der vorliegenden Studie wurde der Einfluss der Doppelinokulation mit Arbuskulären Mykorrhizapilzen (AMF) und Mikroalgen (Scenedesmus incrassatulus R83 und Synechocystis sp. R10) auf die Leistung von Basilikumpflanzen untersucht. Unterschiedliche Arten der Inokulation von Basilikum (AMF, Mikroalgen sowie eine Kombination aus beiden) wurden analysiert. Charakterisiert wurden die Funktion von AMF (Kolonisierung und Glomalin-verwandte Bodenproteine), die Aktivität saurer Phosphatase (in Wurzeln und im Boden), das Pflanzenwachstum, Photosyntheseparameter, sekundäre Metaboliten (Fluoreszenznachweis des Chlorophyllgehalts der Blätter, Flavonolgehalt, Stickstoffbilanz-Index) sowie die Aktivität von Pflanzenenzymen in der Verbindung des Stickstoff- und des Kohlenstoffmetabolismus (Glutamatsynthase, Aspartat-Aminotransferase und NADP-abhängiges Malatenzym). Die höchsten Werte dieser biometrischen Daten resultierten aus der Anwendung von Mykorrhiza allein sowie aus der gemischten Behandlung mit beiden Mikroalgenstämmen. Die doppelte Inokulation mit beiden Mikroalgen und AMF stimulierte die Mykorrhizafunktion (Konzentration Glomalin-verwandter Proteine). Die Indizes sekundärer Metaboliten (Flavonole und Anthocyane) stiegen nach der Behandlung mit Scenedesmus (Al1 und AM + Al1) im Vergleich mit Kontrollpflanzen. Die Zugabe von Synechocystis allein und in Kombination mit Pilzen hatte einen positiven Einfluss auf den Stickstoffbilanz-Index. Unterschiedliche Arten der Inokulation erhöhten Gasaustauschparameter in allen Variationen im Vergleich zu den Kontrollpflanzen. Die Ergebnisse zur Aktivität von Stickstoff-Kohlenstoff metabolisierenden Enzymen zeigten einen engen Zusammenhang mit dem Pflanzenwachstum. Die Wurzelbesiedelung von Basilikum mit Mykorrhiza könnte von beträchtlicher wirtschaftlicher Bedeutung sein. So würde die Zugabe geeigneter AMF in den Wurzelraum signifikant das Wachstum und die Produktivität im kommerziellen Anbau von Ocimum spp. verbessern.

Schlüsselwörter Oberirdisch-unterirdisch · Arbuskuläre Mykorrhizapilze · Bodenmikroalgen · Ocimum basilicum L.

Introduction Interactions in the rhizosphere are essential for many interactions and their corresponding evolution (Lambers et al. 2009). Plants associated with a rich diversity of microorganisms during their entire development. Arbuscular mycorrhizal fungi (AMF) of the phylum Glomeromycota live in symbiosis with a majority (over 80%) of land plants (Smith and Read 1997). Mycorrhizal fungi interact with a broad range of soil organisms, in the root, in the rhizosphere and the bulk soil. These associations may be inhibitory or stimulatory; some are apparently competitive, others may be mutualistic. Mycorrhizal fungi also adjust the communications of plants with other soil organisms (Fitter and Garbaye 1994). AMF may consume 4–16% of their host plants recently photosynthetically-fixed carbon to support their growth, activity and reserves. These symbiotic fungi stimulate the growth and the photosynthetic ability of the host plants by improving nutrient uptake and CO2 assimilation (Kaschuk et al. 2009). Other soil organisms that have a potential to enhance productivity in a variety of agricultural and ecological conditions are microalgae. Microalgae are photoautotrophic, aerobic organisms that use CO2 from the atmosphere and energy from sunlight to synthesized organic compounds. Microalgae commonly found in the soil belong to the phyla Cyanophyta (cyanobacteria, blue-

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green algae) and Chlorophyta (green algae). Both cyanobacteria and green algae play a significant role in intensifying soil fertility and crop yield by adding organic matter, nitrogen, carbon, phosphorus, potassium, zinc and micronutrients (Abdel-Raouf et al. 2012). Most soil algae, especially blue-green algae act as cementing agents in binding soil particles and thereby prevent soil erosion. They generate a large quantity of oxygen through photosynthesis and thus facilitate the aeration in submerged soils (Pelczar et al. 2003). Literature concerning the ability of cyanobacteria and green algae to improve physiological performance, metabolic activity and thus plant development is limited. Grzesik and Romanowska-Duda (2015) reported that the application of monocultures of Microcystis aeruginosa MKR 0105, Anabaena sp. PCC 7120 and Chlorella sp. increased net photosynthesis, transpiration, stomatal conductance and activated dehydrogenases, acid, and alkaline phosphatase in the leaves of rooted cuttings and willow (Salix viminalis L.) plants. The present study is focused on an important member of Lamiaceae family-sweet basil (Ocimum basilicum L.) which has been used since ancient times as a spice and to treat many ailments. Basil plants are known as sources of active compounds that are widely sought after worldwide for their natural properties. While the independent stimulating effects of AMF and soil algae on plant growth and development are al-

Author's personal copy Influence of Mycorrhizal Fungi and Microalgae Dual Inoculation on Basil Plants Performance

ready established, little research has addressed how belowground functional interactions between mycorrhizal fungi and microalgae may influence plant development. Therefore, the present investigation aimed to reveal the effect of dual inoculation on basil plant growth and to characterize the physiological basis by determination of the following indicators: mycorrhizal colonization; glomalinrelated soil protein; acid phosphatase activity (in root and soil); gas-exchange parameters; fluorescence indices of leaf chlorophyll content; secondary metabolites (flavonols and anthocyanins); nitrogen balance index; carbon-nitrogen metabolic status.

twice after seed germination instead of watering with sterilized water (Petkov 1995). Six treatments were compared: 1—control, non-inoculated plants (C), 2—plants, inoculated with Claroideoglomus claroideum EEZ 54 (AM), 3—plants inoculated with Scenedesmus incrassatulus R83 (Al1), 4—plants inoculated with Synechocystis sp. R 10 (Al2), 5—plants coinoculated with Claroideoglomus claroideum EEZ 54 and Scenedesmus incrassatulus R83 (AM + Al1) and 6—plants co-inoculated with Claroideoglomus claroideum EEZ 54 and Synechocystis sp. R10 (AM + Al2).

Determination of Root Colonization and Plant Biomass Production

Materials and Methods Biological Materials and Growth Conditions Basil plants (Ocimum basilicum L. var. purpurascens Benth.) were grown from seeds in 2 kg transparent plastic boxes (5 plants per box for each treatment) of polyethylene glycol terephthalate (PET) with 92% transmittance in the range of 380–710 nm in a growth chamber from May to September under a light intensity of 320 µmol m–2 s–1 at 21–25 °C (night-day) and a 15 h photoperiod. The relative humidity ranged from 40 to 65%. The plants developed on a soil/ perlite substrate (3:1, v/v). All pots were adjusted daily to 60% water holding capacity. The soil (leached cinnamonic forest soil (Chromic Luvisols, FAO), 30–40 cm depth) had the following agrochemical characteristics: pH (H2O) = 6.2; 8 mg kg–1 soil total mobile nitrogen (N-NO3– + N-NH4+); 30 mg kg–1 soil P2O5; 120 mg kg–1 soil K2O. The mycorrhizal strain (Claroideoglomus claroideum, ref. EEZ 54) was kindly provided by the AMF collection of Estación Experimental del Zaidín (CSIC Granada, Spain). Mycorrhizal inoculation was done by placing the seeds over a thin layer of the AMF inoculum (2 g kg–1 soil substrate) following the layering method (Jackson et al. 1972). The inoculum consisted of colonized roots and soil from 4-month-old oat pot cultures. Algae strains derived from the culture collection of the Experimental Algology Department (IPPG-BAS, Bulgaria). Monoalgal, non-axenic cultures of Scenedesmus incrassatulus R83 and Synechocystis sp. R10 were grown autotrophically in a medium after Šetlik (1967), modified by Georgiev et al. (1978) with a ½ concentration of nutrients and mineral medium after Aiba and Ogawa (1977), respectively. The final concentration of algal suspensions used for watering was 0.5 g L–1. The algae were maintained and prepared for watering of basil plants with uninterrupted illumination from luminescent lamps (75 µmol m–2 s–1 light intensity), and bubbling with 3 cm3 s–1 air, enriched with 0.5% CO2 (Petkov 1995). Algal suspension for inoculation was added

At harvest (5th months old plants), the root system was separated from the shoot and the biometrical data (plant height, a fresh and dry weight of shoots and roots) was determined. The extent of mycorrhizal root colonization was estimated using the gridline intersect method (Giovanetti and Mosse 1980). To visualize the AMF colonization, roots (approximately 1 g DW) were cleared in 10% KOH and stained with 0.05% Trypan blue in lactic acid (v/v), according to Phillips and Hayman (1970).

Acid Phosphatase Activity Acid phosphatase activity (APA, EC 3.1.3.2) was measured according to the method of Schneider et al. (2000), based on the original one of Tabatabai and Bremner (1969). Fresh root tissue (0.5 g) was homogenized with 0.1 M sodium acetate buffer (pH 5.0). After centrifugation, the supernatant was assayed for the enzyme activity by incubation in 5 mM p-nitrophenyl phosphate and 0.1 M sodium acetate buffer (pH 5.0). The reaction was stopped by the addition of 0.2 M NaOH, and absorbance was measured at 405 nm. Soil phosphatase activity was assayed by colorimetric estimation of the p-nitrophenol released by phosphatase activity when the mixed sample of fresh soil (1 g) was incubated with buffered (pH 6.5) sodium p-nitrophenyl phosphate solution and toluene at 37 °C for 1 hr.

Easily Extracted and Total Extracted GlomalinRelated Soil Proteins (EE-GRSP and TE-GRSP) The extraction procedure followed the method reported by Wright and Upadhyaya (1996). The soil (2 g) was mixed with 8 ml of 20 mM sodium citrate at pH 7.0. The samples were autoclaved for 30 min (121 °C) and immediately centrifuged at 5000 × g for 15 min. The supernatant represented the easily extractable glomalin-related soil proteins (EE-GRSP). The procedure for extracting total glomalin-related soil proteins (TE-GRSP) consisted of autoclaving 2 g

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soil in 8 ml of 50 mM sodium citrate at pH 8.0 for 60 min. Immediately, after autoclaving, centrifugation at 5000 × g for 15 min was done; the supernatant was stored at 4 oC until needed for analysis. Glomalin quantification (EE-GRSP and TE-GRSP) was done by the method of Bradford (1976) using protein dye reagent and bovine serum albumin (BSA, Sigma) as a standard.

Gas Exchange Measurements The measurements were done before the harvest time of 9th fully developed leaves for each box (Brilli et al. 2013). Intercellular CO2 concentration (ci CO2, ppm), net CO2 assimilation (A, µmol CO2 m–2 s–1), transpiration rate (Tr, mmol H2O m–2 s–1) and stomatal conductance (Gs, mol m–2 s–1) were measured simultaneously with a LI-6400 IRGA (LICOR, Lincoln, NE, USA), by enclosing a portion of the leaf in a 6 cm2 cuvette with a transparent upper Teflon window. All gas exchange measurements were made in the morning at saturating photosynthetic photon flux density (PPFD) (1000 μmol m–2 s–1), with a relative humidity of the air within the leaf cuvette ranging between 45–55%, air temperature of 27 °C and 380 μmol ambient concentration of CO2. Water use efficiency (WUE) was calculated according to Nijs et al. (1997): μmol CO2/mmol H2O = Pn/Tr.

DUALEX Measurements Concentrations of leaf chlorophyll (Simple Fluorescence Ratio, SFR), flavonols (FLAV), anthocyanins (ANTH) and their ratio (Nitrogen Balance Index, NBI) were estimated by field-portable leaf-clip instrument Dualex® Scientific (Force-A, Orsay, France). The measurements were done before the harvest time of 9th fully developed leaf for each box. Based on the characteristics of UV transmittance of the epidermis, the device can estimate leaf phenolic compounds from the measurement of UV (375 nm) absorption of the leaf epidermis by the double excitation of Chl fluorescence as described by Goulas et al. (2004).

Native PAGE and Enzyme Activity Staining For the determination of glutamate synthase (GOGAT, EC 1.4.1.14), aspartate aminotransferase (AAT, EC 2.6.1.1) and malic enzyme (NADP-ME, EC 1.1.1.40) activities, fully developed 5th months fresh leaves (0.50 g FW) were homogenized in 0.1 mM K-phosphate buffer, pH 7.8, containing 2.0 mM Na2-EDTA, 1 mM PMSF (phenyl methyl sulfonyl fluoride), 2% polyvinylpyrrolidone K-40 (w/v) and 10% glycerol. The homogenate was centrifuged at 12,000 × g for 30 min and the supernatant was used as a crude enzyme extract. All steps in the preparation of the enzyme extract were carried out at 0–4oC. Protein content was de-

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termined by the method of Bradford (1976) using bovine serum albumin (BSA, Sigma) as a standard. Equal amounts (35 µg) of leaf proteins from plants exposed to different treatments were subjected to discontinuous PAGE under non-denaturing, non-reducing conditions essentially as described by Laemmli (1970), except that SDS was omitted. Electrophoretic separation was performed on 10% polyacrylamide gels, for 3–4 h at a constant current of 35 mA per gel. After completion of electrophoresis, separate gels were stained for the activities of GOGAT, AAT and NADP-ME. Glutamate synthase staining was carried out by submerging the gel in 0.1 M phosphate buffer (pH 7.5), containing 0.03 M NADH, 0.015 M α-ketoglutarate and 0.015 M glutamine for 60 min at room temperature. Then the gel was pre-equilibrated in 0.1 M Tris-HCl (pH 8.5) and incubated in a staining solution, containing 0.05 M Tris-HCl (pH 8.5), 5 mg ml–1 nitro-blue tetrazolium chloride (NBT) and 0.6 mg ml–1 phenazine methosulfate (PMS), following Matoh et al. (1980). The activity band showed a clear zone against a purple background. Aspartate aminotransferase activity was detected by the method of Griffith and Vance (1989). The resolved gel was incubated in 0.1 M Tris-HCl buffer (pH 7.5), containing 0.04 M aspartate, 0.005 M 2-oxoglutarate and 1 mg ml–1 Fast Violet B salt in darkness for 30 min at 40 °C. Activity staining for NADP-ME was proceeded by incubating the gel in staining solution containing 0.05 M Tris-HCl (pH 7.5), 0.01 M L-malate, 0.01 M MgCl2, 0.0005 M NADP, 35 µg ml–1 NBT, and 0.85 µg ml–1 PMS at 30 °C (Gerrard Wheeler et al. 2005), until the appearance of intense dark bands. All reagents used for enzyme activity staining were purchased from Sigma (Sigma Inc., St. Louis, MO, USA). Gel patterns were photographed immediately after staining using the UVItec gel documentation system (Cambridge, UK). Image analysis of the gels was performed on a PC using Gel-Pro32 Analyzer software (Media Cybernetics Inc., Bethesda, MD, USA). The activity of each isoenzyme (band) was measured as total integrated optical density (IOD), in arbitrary units. As the studied enzymes had multiple lines, the sum of their IOD values was considered as a total enzyme activity for a particular treatment.

Statistics The experiment had a completely randomized block design with four replications. The data were subjected to one-way ANOVA for comparison of means, and significant differences were calculated according to Fisher’s least significance difference (LSD) test at the 5% significance level using a statistical software package (Statgraphics Plus, version 5.1 for Windows). Data are presented as means ± standard error.

Author's personal copy Influence of Mycorrhizal Fungi and Microalgae Dual Inoculation on Basil Plants Performance

Table 1 Biometrical data (plant height, fresh and dry weight) and percentage of mycorrhization of non-inoculated (control) and inoculated (AMF and/or algae) Ocimum basilicum L. five months old plants

C AM Al1 Al2 AM + Al1 AM + Al2

Plant height (cm)

Shoot FW (g plant–1)

Shoot DW (g plant–1)

Root FW (g plant–1)

Root DW (g plant–1)

Mycorrhizal colonization (%)

13.17aa 22.19d 17.57c 15.25b 30.04e 29.58e

1.19a 2.13c 1.54b 1.68b 2.75e 2.50d

0.08а 0.20c 0.16b 0.17b 0.33е 0.23d

0.69b 1.00d 0.54a 0.64b 0.87c 1.32d

0.03a 0.07c 0.027a 0.03a 0.04b 0.07c

– 68 – – 56 59

Values are means and letters in common within a column indicate no significant differences assessed by Fisher LSD test (P Ä 0.05) after performing ANOVA (n = 9)

a

acid phosphatase - soil

AP activity (mM pNP gFW-1 h-1)

AP activity (mM pNP g soil-1 h-1)

7

6

5

c

c

b

1,5 1,0

a

a

a

0,5 0,0

C

AM

AL

1

AL 2

AM

AM

7

d

5

c

c b

4 3

a

a

2 1 0

2,5

EE-GRSP

acid phosphatase - root

6

+AL 1 + AL 2

2,5

concentration (mg g-1 soil)

Fig. 1 Acid phosphatase activity (soil and roots), easily extractable glomalin related soil proteins (EE-GRSP), total extractable glomalin related soil proteins (TE-GRSP) and total protein concentration (root and shoot) of non-inoculated (control) and inoculated (AMF and/or algae) Ocimum basilicum L. plants (5-months old). Values are means ± SE; letters in common within a graph indicate no significant differences assessed by Fisher LSD test (P Ä 0.05) after performing ANOVA (n = 9)

C

AM

AL 1

AL 2

AM

AM

+AL 1 + AL 2

T-GRSP

2,0

e d

2,0

c

1,5

e 0,75

c b

0,50 0,25 0,00

0,5

AM

AL 1

Results Following different modes of inoculation, we traced the development of basil plants until five months old flowering plants. Development of soil algae was visually detected. We found growing green spots on the walls of the transparent plastic boxes ten days after inoculation. Biometrical data analyses (plant height, fresh and dry weight) demonstrated the increased values in all treatments, compared with the control, except for the Al1 effect on the root parameters (Table 1). The highest values were found in the case of mixed treatments (AM + Al1 and AM + Al2) followed by the mycorrhiza application (AM) alone.

b

b

a C

b

1,0

d

AL

2

AM + AL 1

0,0

AM + AL

2

a

C

AM

AL

1

AL

2

AM + AL 1

AM + AL

2

The levels of soil APA activity in AL1 and AL2 plants were equal to control. The interaction between mycorrhizae and algae (AM + Al1) also increased the function of soil APA, where the values were identical to AM plants. Some differences were noticed in the root APA responses, where the activity levels were highest in AM plants. The Al1 and AM + Al2 treatments were distinguished with higher activity compared with control and AL2, as their values precede those of root APA in AM + Al1 plants (Fig. 1). Compared to controls, inoculation with AMF or microalgae separately increased glomalin in soil (EE-GRSP and TE-GRSP) (Fig. 1). Co-inoculation with both AMF and microalgae further enhanced soil glomalin. Glomalin production (EE-GRSP and TE-GRSP) showed an apparent in-

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Table 2 Gas-exchange parameters (intercellular CO2 concentration—ci CO2, net CO2 assimilation—A, transpiration rate—Tr, stomatal conductance—Gs , water use efficiency—WUE) in the leaves of non-inoculated (control) and inoculated (AMF and/or algae) Ocimum basilicum L. plants (5-months old). Letters in common within a graph indicate no significant differences assessed by Fisher LSD test (P Ä 0.05) after performing ANOVA (n = 9)

C AM Al1 Al2 AM + Al1 AM + Al2

ci CO2 ppm

Tr mmol m–2 s–1

Gs mol m–2 s–1

A µmol m–2 s–1

WUE µmol mmol–1 H2O

277ba 280bc 274b 252a 293c 273b

2.55a 3.29c 3.33c 3.12b 3.50d 3.90d

0.09a 0.17d 0.11b 0.11b 0.15c 0.15c

4.80a 10.67d 6.69b 6.73b 8.25c 8.87c

1.88a 3.24d 2.01b 2.16b 2.36c 2.27c


a

Table 3 Dualex-derived data of leaf chlorophyll content (Simple Fluorescence Ratio, SFR), flavonols (FLAV), anthocyanins (ANTH) and their ratio (Nitrogen Balance Index, NBI) in the leaves of non-inoculated (control) and inoculated (AMF and/or algae) Ocimum basilicum L. plants (5-months old). Letters in common within a graph indicate no significant differences assessed by Fisher LSD test (P Ä 0.05) after performing ANOVA (n = 9) C AM Al1 Al2 AM + Al1 AM + Al2

SFR

FLAV

ANTH

NBI

27.531 cda 27.814d 27.193 cd 25.847ab 25.273a 26.746bc

0.519b 0.537b 0.580c 0.453a 0.539b 0.466a

0.439c 0.435bc 0.551e 0.402a 0.490d 0.413ab

53.699b 53.653b 47.121a 57.665c 49.088a 57.878c


a

crease in the values in the AM + Al2 and AM + Al1, followed by the AM plants (Fig. 1). In Al1 and AL2 variants the glomalin values were relatively lower but higher than in the control plants. Gas exchange parameters in the leaves of basil plants showed sensitive changes in response to treatments with AM fungi and algae, especially photosynthetic rate (A) and stomatal conductance (Gs) (Table 2). The most notable rise of Gs, A and WUE was marked in the plants inoculated with mycorrhizal fungi solely. The cyanobacterium Synechocystis sp. R 10 and green alga Scenedesmus incrassatulus R 83 also have a positive, albeit less pronounced effect on the monitored parameters. Co-inoculation of AM fungi and algae (AM + Al1/AM + Al2) increased the rate of transpiration and photosynthesis, values of stomatal conductance and WUE compared with algae treatments (Table 2). While mycorrhizal plants thus acquire more water and nutrients, hence they would transpire more water too. Leaf chlorophyll did not change significantly as a result of AMF inoculation or application of Scenedesmus as compared with the control (Table 3). A slight reduction of chlorophyll was observed after addition of Synechocystis and treatment with fungi plus algae. Indexes of secondary metabolites such as flavonols and anthocyanins increased after treatment with Scenedesmus (Al1 and AM + Al1) com-

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pared to control plants. On the other hand, the addition of Synechocystis alone and in combination with fungi (Al2 and AM + Al2) resulted in the lowest levels of those parameters but positively influenced nitrogen balance index (Table 3). Three enzymes that link nitrogen and carbon metabolism (GOGAT, AAT and ME) were analyzed. Compared with control plants (C), the relative total GOGAT activity did not change markedly subsequent AL1 and AM + AL2 inoculation, while the capacity was about three times higher in plants manipulated with AMF, AL2 and AM + AL1 (Fig. 2). All variants, except for AL2, led to an AAT rise from 154% for AMF + AL2 to 219% for AMF (Fig. 2). The relative total NADP-ME activity was the lowest in control (accepted for 100%) and the highest in AM plants (366%). AL1 and AM + AL2 treatments also markedly elevated the enzyme (by about 100 and 80%, respectively) (Fig. 2).

Discussion AMF are key elements of soil microbiota and a great variety of relations occurs between them and the surrounding organisms (Jansa and Gryndler 2010). The highest biometrical data results were due to mycorrhizal stimulation—alone and in the mixed treatments with both

Author's personal copy Influence of Mycorrhizal Fungi and Microalgae Dual Inoculation on Basil Plants Performance 150

Relative total GOGAT activity

c

b

d

IO D ( a r b itr a r y u n its )

10500 9000 7500 6000

a

70 Relative total NADP-ME activity

120

12000

4500

Relative total AAT activity

b

a

a

e

d c

90

b 60

a

a

60 50 40

c

b

30 20

a

a

a

30

3000

10

1500 0

IOD (arbitrary units)

13500

IOD (arbitrary units)

15000

NM

AM

AL1

AL 2

AM

AM

0

NM

AM

+AL1 + AL2

AL1

AL 2

AM

AM

+AL1 + AL2

0

NM

AM

AL 1

AL2

AM

AM

+AL1 + AL2

Fig. 2 Relative total glutamate synthase (GOGAT), aspartate aminotransferase (AAT) and NADP-malic enzyme (NADP-ME) activities of non-inoculated (control) and inoculated (AMF and/or algae) Ocimum basilicum L. plants (5-months old). Values are means ± SE (n = 3)

microalgae strains. The strengthening effect of mycorrhiza on the O. basilicum L. was described in different publications (Khaosaad et al. 2006; Toussaint et al. 2007; Rouphael et al. 2015). In all cases, the authors indicated the stimulatory effect of AMF on plant growth. Advantages of multiple inoculations with mycorrhiza fungi and algae Nostoc muscorum on plant growth, shoot and root biomass, the nutritional value of faba bean had also been reported (El Gamal et al. 2009). El-Khateeb et al. (2011) underline the higher positive result of mycorrhiza application compared with commercial algae extract over the development of Calia secundiflora plants. According to Welc et al. (2010), the presence of AMF may suppress the growth of different microbial groups under some unclarified circumstances, respectively their functions. It seems that at some point algae are in competition for resources with the plants or with the mycorrhizal fungi. Inoculation of basil plants with AM strain stimulated the acid phosphatase activity (APA) in roots and soil (Fig. 1), which was accompanied by the highest mycorrhiza percentage. The acid phosphatase activity can provide useful information on organic P mineralization potential and biological activity of soils and has been used as an indicator to evaluate P limitation (Krämer and Green 2000). In some cases, there is not a correspondence between soil and root phosphatase activities, based on the fact that plant roots, fungi and other microorganisms into the ground have a different expression of APA (Abd-Alla 1994). APA associated with the phosphorus acquisition in the rhizosphere, growth and development of the fungus within the host tissue and it is managed by an unspecified mechanism controlled by the plants (Prasad et al. 2012). The difference in the glomalin production as a consequence of the function of AMF species and/or interaction with algae has several important implications. Based on the fact that soil algae excrete growth-promoting sub-

stances that affect other organisms and has a tremendous potential to supply them with nitrogen and carbon (Wilson 2006), we assumed that soil algae stimulated the function of mycorrhizal structures. In this relation, Van Aarle et al. (2002) emphasized the importance of studying not only the amount of fungal mycelium but also the proportion of active mycelium. The amount of biomass produced by plants is intimately related to photosynthesis. Plants absorb solar energy and assimilate CO2 and the products of carbon fixation are then further turned into organic materials, which are stored as the plant biomass (Zhu and Long 2008). Several studies have also reported a mycorrhiza-induced increase in plant water use efficiency (WUE) (Kaya et al. 2003; Ruiz-Lozano and Aroca 2010). In agreement with our results, enhanced photosynthetic rates due to mycorrhiza have been revealed in various plant species (Kaschuk et al. 2009; Zhang et al. 2012). Plants have a photosynthetic capacity larger than their C requirements but are usually limited by soil nutrients. Therefore, the high photosynthetic performance of AM plants may be interpreted either as a result of the improved nutrient supply, as an adaptation to compensate for the additional fungal C demand, or both (Kaschuk et al. 2009). AMF inoculation or application of microalgae did not change significantly leaf chlorophyll indexes compared with the control. In the study of Paradi et al. (2003), no differences were found regarding the chlorophyll content between mycorrhizal and nonmycorrhizal plants. It is ordinarily accepted that the therapeutic properties of Origanum sp. are due to secondary metabolites such as phenolic compounds as well as anthocyanins. Zeng et al. (2013) established that mycorrhizal fungi had differential effects on accumulation and composition of plant secondary metabolites (phenolic compounds, terpenes and nitrogenous compounds). In our case, AM had no actual effect on the

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content of flavonols and anthocyanins in contrast to the addition of the green microalga in the soil. Treatments with Scenedesmus (Al1 and AM + Al1) increased flavonols and anthocyanins. The explanation of the results may be hidden in a higher phosphate production followed by increased activity of APA in root and soil. The other strain of microalgae (Synechocystis) contribute to a nitrogen nutrition of the plants resulted in positively influenced nitrogen balance index. To further investigate the influence of functional interactions between AMF and soil microalgae over O. basilicum L. we evaluated the plant carbon-nitrogen metabolic status. Carbon and nitrogen metabolisms are regulated to bring about the coordination essential for plant growth and development. Inorganic nitrogen assimilation, in the form of ammonium, onto carbon skeletons for the generation of amino acids, is one of the most vital biochemical processes in plants (Hodges 2002). Compared with control variants, AMF plants exhibited higher metabolic potential as was indicated by the enhanced functions of all studied enzymes (from 2.3- to 3-fold rises). The inoculation with algae (alone and in combination with AMF) was outlined with differential effects on enzymes levels. AL1 addition did not affect GOGAT capacity but raised the performances of AAT and NADP-ME by about two times. GOGAT was the only active responder to AL2 treatment. The reply of plant enzymes to a dual addition of AM + AL1 was similar to that of the AMF-treated plants, although the AAT and ME levels were increased to a lesser degree. The combination between AM and AL2 stimulated both AAT and ME, whereas GOGAT function remained unchanged compared with the control. The results demonstrated the close relationships between the activity levels of nitrogen-carbon metabolizing enzymes and the plant growth phenotype.

Conclusions The data in present research show that the tested green alga Scenedesmus incrassatulus R83 and blue-green alga Synechocystis sp. R10 added to the soil separately or in combination with AMF improve plant performance both directly and indirectly through mycorrhizal stimulation. Since algae were not in direct physical contact with plant roots, it was expected that the algal metabolic exudates would affect mainly mycorrhizal development, which was refuted by the obtained results. Different modes of manipulation lead to an increase of gas-exchange parameters, as the most significant were distinguished in mycorrhizal plants. Dual inoculation stimulated mycorrhizal function (glomalin-related proteins) and plant metabolism (anthocyanins contents and nitrogen balance index).

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The mycorrhizal root colonization of basil may bear considerable economic importance. Thus, the addition of suitable AMF to the rhizosphere would significantly improve the growth and productivity of commercial Ocimum spp. cultivation. The synergistic effects of AMF and microalgae may be attributed to the nature and compatibility of the used strains, as well as the interactions that take place between algae/fungi and plant species. The proposed applications of different bio-stimulants combination showed that the targeted AMF and algae treatment affect plant metabolism and this may be the way of manipulation aimed at the synthesis of valuable metabolites. Therefore, understanding which factors modulate the performance and interactions of these bio-inoculants will be very useful for improving the efficiency of this inoculum pool in future. The inoculation with AMF and microalgae may be very beneficial in agroecosystem applications as a result of their primary functions to enhance soil porosity, aggregation, and water-retention capacity and to improve nutrient cycling. Conflict of interest M. Hristozkova,L Gigova, M. Geneva,I. Stancheva, V. Velikova and G Marinova declare that they have no competing interests.

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