Effectiveness of mycorrhizal fungi on globe artichoke (Cynara cardunculus L. var. scolymus) micropropagation

Abstract

The effectiveness of two arbuscular mycorrhizal (AM) fungal isolates (Glomus intraradices and Glomus viscosum) in sustaining plant growth and the physiological activities of the micropropagated globe artichoke (Cynara cardunculus L. var. scolymus (L.) Fiori) were investigated during acclimatization and 90 days after plant establishment. All the mycorrhizal microplants survived transplant shock thus confirming the positive role of AM fungi colonization on ex vitro establishment. The growth increased in mycorrhizal plants, especially in plants inoculated with Glomus viscosum. Mycorrhizal plantlets showed higher stomatal conductance, which is probably necessary to supply the carbon needs of fungal symbionts. The SPAD (soil plant analysis development) data could be useful for plant management as a predictor for tissue nitrogen levels. The higher SPAD values in mycorrhizal plants are strictly related to a higher photosynthetic potential, and consequently to their better nitrogen nutrient status due to the symbiotic relationship. Regardless of the mycorrhizal performance in the host–fungus combination, the most efficient fungus for the artichoke microplants was Glomus viscosum.

Keywords:

• Glomus viscosum
• Glomus intraradices
• Cynara cardunculus L. var. scolymus (L.) Fiori
• ex vitro establishment

Introduction

The globe artichoke (Cynara cardunculus L. var. scolymus (L.) Fiori), belonging to the family of Asteraceae, is a herbaceous perennial crop, widely cultivated in the Mediterranean (Bianco 2005). Its cultivation is very important for the Italian horticultural economy, Italy being the world's main producer (FAO Statistical database 2010).

Plant propagation of this species usually starts from offshoots (basal shoots) (Cardarelli et al. 2005) or ovules (semi-dormant shoots with a limited root system), which could potentially guarantee high yields of the marketable product. Currently, however, due to the poor quality and lack of uniformity of the planting materials, this kind of propagation has many disadvantages such as the physiological heterogeneity of the propagative material, the low rate of multiplication, and the spread of pathogens (mainly viruses).

This is why many producers must replace the varieties that are more susceptible to diseases, with certified planting materials. The need to increase the production of clean materials has stimulated the study of new propagation techniques. Among these, the in vitro culture of vegetal tissues has been identified as the most suitable for obtaining disease-free plantlets (Acquadro et al. 2010).

In addition, the tissue cultures have been widely used for rapid mass production, resulting in higher multiplication rates than other traditional methods of propagation.

Micropropagation ensures the large-scale production of high-quality commercial plant materials, thus allowing for the easy transfer of healthy material from one country to another (Vaast et al. 1996).

Many studies have tried to identify the most appropriate technique of artichoke micropropagation by defining the correct culture media and evaluating the optimum conditions to increase the multiplication coefficients, in order to facilitate in vitro rooting and acclimatization in the greenhouse (Ancora et al. 1981; Morone-Fortunato et al. 2005; Pacifici et al. 2007; Bedini et al. 2012; Boullani et al. 2012).

In vitro rooting is the phase of micropropagation that leads to the biggest structural changes in the leaf tissues such as the thickening of the cuticle and the cell wall, and an improvement in stomata physiology, thus promoting acclimatization and increasing the ex vitro survival of the microplants (Apòstolo et al. 2005).

The improvement of ex vitro establishment is also favored by the mycorrhization of the micropropagated plantlets (Morone-Fortunato et al. 2005; Ruta et al. 2009). Arbuscular mycorrhizal (AM) fungi are important soil microorganisms that can establish a symbiotic association with the roots of nearly 90% of all plants (Smith et al. 2010). Due to endomycorrhizal inoculation, the micropropagated plants change root morphology (Gutjahr et al. 2009; Derelle et al. 2012; Wu et al. 2012). Many studies have demonstrated that plants inoculated with AM fungi develop a more economical root system (Schellenbaum et al. 1991; Cruz et al. 2004), show higher ex vitro survival (Estrada-Luna et al. 2000; Moraes et al. 2004; Morone-Fortunato et al. 2005) and better physiological activities (Krishna et al. 2005) owing to the possibility of enhancing plant nutrition by symbiosis (Smith & Smith 2012).

Some research has also shown that different mycorrhizal inoculants induce different plant development and consequently different mycorrhizal dependencies (Sensoy et al. 2007; Sensoy et al. 2011; Jha et al. 2012)

The goal of this investigation was to assess the effectiveness of two arbuscular mycorrhizal species, Glomus intraradices and Glomus viscosum, tested ex vitro on microcuttings of Cynara cardunculus L. var. scolymus (L.) Fiori under glasshouse conditions.

Materials and methods

In vitro culture of artichoke explants

Artichoke plantlets (Cynara cardunculus L. var. scolymus (L.) Fiori of the ‘Catanese type’) were micropropagated following Morone-Fortunato et al. (2005). Excised artichoke shoot tip explants, 5–6 mm in length, were surface sterilized by immersion in a mercuric chloride solution (0.05 g mercury (II) chloride – Panreac Quimica SA/100 ml sterile distilled water) for 15 minutes and washed three times with sterile distilled water. The explants were then multiplied and rooted in vitro, employing the culture media selected by Morone-Fortunato et al. All the cultures were maintained in a growth chamber at 23°C ±1 with a photoperiod of 16-h light and under a light intensity of 50 µE s⁻¹ m⁻².

Ex vitro establishment and mycorrhizal inoculation

The uniform 180 rooted microplants, 6–8 cm long with 2–3 rootlets (4–5 cm long) and 4–6 small leaves, were transferred to a climatic greenhouse at the University of Bari ‘Aldo Moro,’ Apulia, Italy (41°7′31″N, 16°52′0″E).

Acclimatization took place under greenhouse conditions at 18–25°C with mist, reducing the humidity level from 85–90% to 50–60% over 20 days.

The microplants were washed with distillated water to remove the adhering agar from the root systems. They were then transplanted into individual plastic pots with a volume of 0.5 dm³ containing a sterile peat (46% organic carbon, 1–2% organic nitrogen, 80% organic matter) and a perlite mixture (2:1, v/v ratio).

At the time of the transplant, the microplants chosen at random to become mycorrhizal were inoculated with crude AM fungi inocula of G. viscosum H.T. Nicolson strain A6 or G. intraradices Schenck and Smith (1982) (basionym of the actual name Rhizophagus intraradices Schüβler and Walker 2010). G. viscosum was provided by the Department of Crop Plant Biology (University of Pisa, Italy); G. intraradices was multiplied from a commercial mycorrhizal inoculant (Italpollina spa – Verona, Italy).

AM pure cultures were multiplied on strawberry (Fragaria×Ananassa) plants preferred as the host crop for the high mycotrophy according to Dalpé and Monreal's method (2004).

The obtained AM fungal inocula consisted of sand soil which contained spores, external mycelium, and infected strawberry root fragments from the strawberry pot cultures. Approximately 10 g (about 100–120 spores) of each AM fungal inoculum were placed immediately below the roots. Non-mycorrhizal plants (non-AM) were obtained with no added AM fungal inocula and used as controls. In order to verify the effect of the substrate present in the inocula, approximately 10 g of the same sterile sand soil were placed below the root system.

After acclimatization, 40 plantlets for each mycorrhizal treatment were kept for 90 days at 50–60% humidity and a temperature of 18–25°C.

Plant growth responses and mycorrhizal colonization

Ex vitro survival (%) was measured 30 days after mycorrhizal inoculation. After growing the plants in the greenhouse for 90 days, 10 plants for each mycorrhizal treatment were processed. Shoots were separated from the roots at the root collar, and the foliar area was measured by a leaf area meter (Model Li-Cor 3000). Shoots and roots were then weighed to determine their fresh weight, oven-dried (48 h, 60°C), and reweighed to determine the dry weight.

Symbiotic parameters were estimated on artichoke plantlets 30, 60, and 90 days after mycorrhizal inoculation to verify the establishment of symbiosis.

The symbiotic measurements were taken on the set of 10 randomly chosen plants for each mycorrhizal treatment.

The staining of roots was carried out following Phillips and Hayman's method (1970). A total of 10×1 cm root pieces per plant were selected at random from the staining root fragments and placed on a microscope slide. Ten microscope slides per mycorrhizal treatment were prepared. The root fragments, mounted in a drop of glycerol, were observed using an optical microscope (Leica DMLB100).

The frequency of mycorrhiza in the root system (F%) and the intensity of mycorrhizal colonization in the root system (M%) were evaluated as symbiotic parameters with the following annotations according to Trouvelot et al. (1986).

The intensity of mycorrhizal colonization (M%) in the root system was defined based on the visual characteristics using mycorrhizal infection scores in classes from 0 to 5:

where n5 is the number of fragments rated 5, n4 is the number of fragments rated 4 etc.

The calculations were performed using MYCOCALC software.

Mycorrhizal dependency (MD) was calculated using the individual total dry weight (DW) of mycorrhizal plants (AM) and the mean dry weight of non-mycorrhizal plants (non-AM) for each artichoke hybrid, according to Plenchette et al. (1983) as:

Physiological parameters

Stomatal conductance and SPAD values were detected to investigate the relationship between the AM fungus and plant physiology. These physiological measurements were taken from plantlets at 30, 60, and 90 days after ex vitro transplantation.

Stomatal conductance was measured using a Leaf porometer (Model SC-1, Decagon Device, Washington, DC, USA). The measurements were taken between 10:00 and 12:00, and were carried out on the first pair of individual fully expanded leaves with two repeated readings for 10 sample plants (both mycorrhizal and non-mycorrhizal).

The SPAD values were monitored using a Chlorophyll meter SPAD502 (Minolta Camera Co. Ltd, Japan) and were performed between 8:00 and 10:00 on the first fully expanded leaf with three readings repeated for 10 sample plants (both mycorrhizal and non-mycorrhizal).

Experimental design

At the time of transplantation in the greenhouse, the artichoke microplants were arranged at random with 60 replications for each mycorrhizal treatment (with G. viscosum, with G. intraradices or without mycorrhiza).

In order to assess ex vitro survival (%), the artichoke plantlets were organized in a completely randomized experimental design with 40 replications for each mycorrhizal treatment.

The results were subjected to ANOVA using CoStat software. The SNK test (P ≤0.01) was used to compare the means of morphological, physiological, or symbiotic parameters in mycorrhizal and non-mycorrhizal treatments. Since the data were expressed as percentages, the measured values were transformed according to the angular transformation, in order to overcome any irregularities.

Results and discussion

This research confirms the effectiveness of the culture medium selected by Morone-Fortunato et al. (2005) in sustaining in vitro proliferation and rooting of the artichoke explants. The well-developed microplants represented the first step in acquiring good acclimatization and in achieving good results in the nursery.

During the trials in the greenhouse, a positive response of the micropropagated plants due to AM fungi inoculation was observed.

With respect to ex vitro survival, all the mycorrhizal treatments showed higher ex vitro survival than the control plantlets (Table 1), which is in line with earlier findings (Krishna et al. 2006; Binet et al. 2007; Wu et al. 2011b; Yadav et al. 2012). Furthermore, there was a significant variation with regard to the survival assessment of the two mycorrhizal fungi used. G. viscosum was found to sustain ex vitro establishment more than G. intraradices (Table 1).

Table 1. Ex vitro survival and morphological characteristics of C. cardunculus L. var. scolymus inoculated with G. viscosum (GV) or with G. intraradices (GI) or non inoculated (C) 90 days after mycorrhizal inoculation.

		Shoot				Root
Treatment	Ex vitro survival	Leaves	FW	DW	Leaf area	FW	DW	Density
	%	n	g	g	cm^2	g	g	cm cm^−3
GV	91.7 a	6.8 a	11.9 a	1.6 a	378.5 a	10.8 a	1.7 a	0.3 a
GI	83.3 b	6.8 a	8.7 b	1.3 b	322.9 b	7.6 b	1.2 b	0.2 b
C	75.0 c	6.0 b	4.5 c	0.5 c	215.6 c	3.3 c	0.5 c	0.1 c

Note: The values are means of ten independent samples. Different letters within each column indicate significant differences between treatments (Test SNK – P ≤ 0.01).

Significant differences were observed between mycorrhizal treatments in their morphological responses to inoculation (Table 1), which is in agreement with previous research (Campanelli et al. 2011; Cartmill et al. 2012; Mohandas 2012; Sink & Gogoi 2012). The positive effect of the mycorrhizal inoculations on the epigeal and hypogeal growth of the plantlets was evident. Mycorrhizal symbiosis stimulated leaf area, fresh and dry shoot weight, fresh and dry root weight, and root density. The growth responses of the host plants were useful in evaluating mycorrhizal efficiency. Mycorrhizal fungi improve the plant uptake of water and mineral nutrients, making the plants highly dependent on mycorrhizal symbionts for their nutrient supply.

G. viscosum was more able to sustain plantlet growth than G. intraradices.

These morphological results were confirmed by symbiotic data (Table 2). Mycorrhizal dependency, evaluated on the basis of plant dry weight, was higher in the G. viscosum-inoculated plants than in G. intraradices-inoculated plants. This was probably due to the greatest increase in the surface area of the host root systems (Allen 1982; Marschener 1998; Wu et al. 2011a) induced in this study by G. viscosum.

Table 2. Mycorrhizal colonization (F%), intensity of mycorrhizal colonization (M%) and mycorrhizal dependency (MD%) of C. cardunculus L. var. scolymus inoculated with G. viscosum (GV) or with G. intraradices (GI), 30, 60 and 90 days after mycorrhizal inoculation.

Days after inoculation	AM treatment	F (%)	M (%)	MD (%)
30	GV	6.0 a	1.4 a	46.4 a
	GI	4.0 b	0.6 b	35.1 b
60	GV	56.0 a	17.4 a	76.4 a
	GI	44.0 b	11.5 b	71.3 b
90	GV	84.0 a	47.9 a	70.4 a
	GI	70.0 b	35.1 b	61.0 b

Note: The values are means of ten independent samples. Different letters within each column indicate significant differences between treatments (Test SNK – P ≤ 0.01).

If mycorrhizal dependency is defined as the degree to which a plant is dependent on mycorrhizal conditions to produce its maximum growth at a given level of soil fertility (Gendermann 1975), it is clear that a specific plant–fungus interaction is key to understanding the beneficial effects of the symbiosis (Jeong et al. 2006).

There is a tendency for plants with less mycorrhizal dependence to have lower rates of root colonization than plants with more mycorrhizal dependence (Graham et al. 1997). In fact, the artichoke plantlets inoculated with G. viscosum showed significantly higher root mycorrhizal colonization compared to those inoculated with G. intraradices, while no mycorrhizal structures were observed in the root systems of the non-inoculated plants.

The artichoke plantlets used in the trial showed a rapid increase in mycorrhizal infection (F%), which was especially widespread in the root system 90 days after mycorrhizal inoculation. Thirty days after inoculation, the artichoke plantlets showed low values of infection, 6% for G. viscosum and 4% for G. intraradices, which increased in subsequent sampling dates, reaching 84% and 70%, respectively, at the end of the experiment (Table 2).

During the trial, the intensity of the mycorrhizal colonization of the roots (M%) also differed between the two mycorrhizal treatments. G. viscosum promoted a higher colonization intensity compared to G. intraradices. A considerable number of studies indicate that the intensity of mycorrhizal colonization can be influenced by various environmental factors such as the availability of light, temperature, moisture, soil pH, and availability of nitrogen (Apple et al. 2005; Tahat & Kamaruzaman 2012). In this study, under the same environmental conditions, the specific differences in symbiotic parameters could be a result of host–fungus specificity because certain plant species may prefer specific fungi (Tahat et al. 2008).

Although mycorrhizal fungi do not have a strict host specificity, it has recently been discovered that both the fungal symbionts and the host plants show a functional specificity, which directly influences the success of the symbiontic interaction (Finlay 2008). A better plant–fungus affinity results in a higher productivity, leading to more efficient resource utilization.

This is consistent with the idea emerging from many physiological studies, that the degree of mycorrhizal specificity may also influence the physiological activities of plants (Ruiz-Lozano et al. 1995).

Measurements of the physiological parameters (Table 3) showed the positive influence of mycorrhizal symbiosis on sustaining the metabolic activities of the mesophyll.

Table 3. SPAD values of C. cardunculus L. var. scolymus inoculated with G. viscosum (GV) or with G. intraradices (GI) or non inoculated (C), 30, 60 and 90 days after mycorrhizal inoculation.

	SPAD values
Treatment	Days after inoculation
	30	60	90
GV	22.0	36.3 a	42.2 a
GI	21.4	35.6 a	40.6 a
C	20.5	27.5 b	35.3 b

Note: The values are means of ten independent samples. Different letters within each column indicate significant differences between treatments (Test SNK – P ≤ 0.01).

During the trial, the SPAD values gradually increased and 60 days after inoculation, were significantly higher for the mycorrhizal plants than the non-inoculated controls, in particular for the symbiosis with G. viscosum (Table 3).

The SPAD values indirectly quantify the greenness or the relative chlorophyll content of the leaves, which is closely linked to the leaf nitrogen content. The higher SPAD values may be associated with the better nitrogen (N) nutrient status (Chang & Robison 2003; Gáborčík 2003; Percival et al. 2008), suggesting that the mobilization of N by mycorrhizal fungi was associated with an increase in the N concentration of the leaves.

The rates of stomatal conductance increased during post-acclimatization (Table 4). Mycorrhizal plants showed a higher stomatal conductance than non-mycorrhizal plants, in accordance with previous studies (Augé et al. 2008). The comparison between the plants inoculated with the two AM fungi species confirmed the greatest affinity with G. viscosum.

Table 4. Stomatal conductance in C. cardunculus L. var. scolymus inoculated with G. viscosum (GV) or with G. intraradices (GI) or non inoculated (C), 30, 60 and 90 days after mycorrhizal inoculation.

	Stomatal conductance (mmol m^−2 s^−1)
Treatment	Days after inoculation
	30	60	90
GV	86.3 a	241.3 a	536.1 a
GI	76.5 a	235.1 a	486.1 a
C	66.5 b	157.9 b	332.1 b

Note: The values are means of ten independent samples. Different letters within each column indicate significant differences between treatments (Test SNK – P ≤ 0.01).

A higher stomatal conductance may be associated with the higher CO₂ influx into the mesophyll tissues, which consequently suggests that the mycorrhizal symbiosis improved the rate of CO₂ assimilation (Syvertsen & Graham 1999; Loewe et al. 2000).

In summary, mycorrhizal inoculation favored the ex vitro survival of the artichoke microplants, averting transplantation shock (Krishna et al. 2005; Krishna et al. 2006). During post-acclimatization, when nutritional stress became the growth-limiting factor, mycorrhizal plants maintained a higher growth performance, which was indirectly linked to higher chlorophyll levels and stomatal conductance.

In addition, our results support the hypothesis that there are differences in the symbiotic performance of different host–endophyte associations, and that G. viscosum shows a better affinity with artichoke plantlets than G. intraradices symbiont.

Conclusion

We believe that this study provides a valid protocol for the production of artichoke plantlets, using two biotechnological approaches: micropropagation and mycorrhizal symbiosis. In addition, the inoculation of plantlets with more effective AM fungi improves the quality of nursery-produced plants.

The results obtained confirm the effectiveness of our methodologies as new tools to obtain high-quality commercial plant materials, ensuring, in addition, a more sustainable horticultural production.

Acknowledgments

The authors are grateful to the Italian ‘Ministero delle Politiche Agricole, Alimentari e Forestali’ (Ministry of Agricultural, Food and Forestry Policies) for providing financial support ‘Project CAR VARVI.’ The authors thank Prof. Manuela Giovannetti of the Department of Crop Plants, University of Pisa (Italy) for providing Glomus viscosum inoculum.