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Acta Hortic. 1284. ISHS 2020. DOI 10.17660/ActaHortic.2020.1284.11
Proc. X International Symposium on Artichoke, Cardoon and Their Wild Relatives
Eds.: D. Valero and M. Serrano
85
Artichoke inoculation with
Rhizoglomus irregulare,
Funneliformis mosseae,
and
Trichoderma koningii
changes metabolic profile of heads
G. Erice1, F. Colosimo2, L. Lucini3, Y. Rouphael4, G. Colla5, V. Cirino2 and P. Bonini1, a
1NGAlab, La Riera de Gaia, Tarragona, Spain; 2ATENS, La Riera de Gaia, Tarragona, Spain; 3Department for
Sustainable Food Process, Universita Cattolica del Sacro Cuore, Milan, Italy; 4Department of Agricultural Sciences,
University of Naples Federico II, Naples, Italy; 5Department of Agriculture and Forest Sciences, University of Tuscia,
Viterbo, Italy.
Abstract
In many irrigated areas of the southern Mediterranean region, vegetable growers
are forced to use saline water to irrigate their crops due to an inadequate supply of
fresh water with detrimental effects on crop productivity. In the region of La Vega Baja
del Segura, Spain, soil salinity has become an increasing concern for growers producing
traditional and sensitive cultivars (Cynara cardunculus L. subsp. scolymus (L.) Hayek)
such as ‘Blanca de Tudela’. In these areas, salinity stress of artichoke crop is enhanced
by the use of low-quality irrigation water. The aim of the present study was to
investigate the effects of inoculation with AMF (Rhizoglomus irregulare BEG72, and
Funneliformis mosseae BEG234) and Trichoderma koningii TK7 on metabolic profile of
artichoke heads. Seven-week-old artichoke plants grown in a saline soil at Orihuela
(Spain) were inoculated through a drip irrigation system using a water suspension
containing propagules of AMF (7×105 spores ha-1 of each AMF species) and Trichoderma
koningii TK7 (1012 CFU ha-1). The OPLS-DA analysis allowed to discriminate the
microbial-inoculated plants from the untreated plants. A total of 105 metabolites were
detected and significantly changed. Microbial inoculation induced an hormonal
alteration of the plants with a downregulation of cytokinin metabolism, and an up-
accumulation of 5 metabolites involved in the jasmonic acid pathway. A decrease of
ribulose 5-phosphate was also recorded in microbial-inoculated treatment. Artichoke
heads from inoculated plants showed the highest levels of pantothenate (vitamin B5),
aminobenzoate (vitamin B9) and pyridoxine (vitamin B6).
Keywords: saline stress, arbuscular mycorrhizal fungi, Trichoderma spp., metabolomics,
Rhizoglomus irregulare, Funneliformis mosseae, artichoke
INTRODUCTION
Soil salinity is limiting plant productivity worldwide and recent studies suggest that
salinity affects more than 20% of irrigated crops (Gupta and Huang, 2014). Soil salinity
decreases plant yield through the combination of osmotic stress and ion-specific toxicity. In
vegetable crops, NaCl-induced salinity disturbs physiological, biochemical, and metabolic
processes leading to growth inhibition (Colla et al., 2013; Rouphael et al., 2017a, b). According
to the Food and Agriculture Organization (FAO) Spain produces 33% of EU artichoke (Cynara
cardunculus L. subsp. scolymus (L.) Hayek) especially in the region of La Vega Baja. However,
in the past years there has been growing concern among growers about soil salinization and
its negative impact on artichoke growth and productivity. Artichoke has been reported as a
moderately salt-tolerant crop based on a greenhouse study (Graifenberg et al., 1993) and a
field trial conducted in an irrigated desert area (Francois, 1995). Francois (1995) reported a
salinity threshold of 6.1 dS m-1 and a slope of 11.5% for field grown artichoke. Above the
salinity threshold value, artichoke crop showed a decrease of growth, leaf area and head yield.
Moreover, the increase of salinity enhanced the incidence and severity of the physiological
aE-mail: pb@ngalab.com
86
disorder ‘internal browing’ of artichoke heads reducing the number of marketable heads
(Shannon and Grieve, 1998). Several attempts have been made to develop salt tolerant
genotypes in vegetable crops, but the commercial success was very limited due to the
complexity of the salinity trait (Timmusk and Behers, 2012). A promising strategy to mitigate
the detrimental effects of salinity on crops could be the inoculation with beneficial
microorganisms (Azcon et al., 2013). Among these, arbuscular mycorrhizae fungi (AMF), and
plant growth promoting bacteria (PGPB) have been used successfully alone or in combination
under different environmental stress conditions (Colla et al., 2008; Cardarelli et al., 2010;
Rouphael et al., 2010; Zoppellari et al., 2014). Synergistic actions have been often reported on
stimulating crop growth and productivity with combined application of several microbial
species (microbial consortium) (Gamalero et al., 2004; Colla et al., 2015; Rouphael et al., 2015;
Fiorentino et al., 2018). Mycorrhizal fungi are capable to colonize most of the plant species
providing mutual benefit for both plant host and fungus (Smith and Read, 2008). The most
common group is the AMF, capable to colonize up to 80% of plants, including artichoke. The
first benefit for host plants is the enhancement of phosphorus and nitrogen uptake (Jakobsen
et al., 1994; Colla et al., 2008) usually considered as limiting factors for plant growth.
Moreover, AMF can enhance plant micronutrient uptake such as iron, copper and zinc through
the increase of soil exploitation and the release of micronutrient-solubilizing compounds by
the extraradical-hyphae network (Rouphael et al., 2015). This better nutritional status in
inoculated plants has been often associated to increased chlorophyll fluorescence and
improved photosynthetic rate (Yooyongwech et al., 2016). AMF can also modify the plant
secondary metabolism resulting in enhancement of antioxidants and phytochemicals in edible
product (Avio et al., 2018; Rouphael et al., 2018; Kyriacou and Rouphael, 2018). Moreover,
AMF symbiosis can alter the hormonal balance of plants boosting growth and crop
productivity (Aroca et al., 2008). Additionally, Trichoderma is an endophytic fungus with
biocontrol and plant biostimulant properties. Trichoderma enhances crop growth and
productivity by increasing plant uptake of micronutrients (Fe, Zn, Cu and Mn) and releasing
auxin-like compounds in the rhizosphere (Colla et al., 2015; Rouphael et al., 2017a, b).
Trichoderma can also increase crop growth and productivity by reducing the plant diseases;
the biocontrol activity of Trichoderma is due to several mechanisms such as competition for
nutrients and space, mycoparasitism, antibiosis, and induction of systemic resistance (ISR)
(Howell, 1998; Mathys et al., 2012). Colla et al. (2015) reported that combined application of
AMF and Trichoderma spp. can stimulate growth and productivity of several vegetable crops.
Based on the above consideration, the aim of this study was to assess the influence of co-
inoculation of artichoke plants with AMF and Trichoderma koningii on metabolic profile of
heads.
MATERIAL AND METHODS
Plant growth conditions, soil conductivity and experimental design
Artichoke transplants (Cynara cardunculus L. var. scolymus (L.) Hayek ‘Blanca de
Tudela’) were planted on August 10 in a field located at Orihuela (Spain) having a silty clay
soil with low organic matter (1.8%), alkaline pH (8.1) and high salinity level (EC1:1=2.4 dS m-1
corresponding to an ECe=7.2). Plants were grown in single rows at a plant density of 8,000
plants ha-1. A randomized-block design with five blocks per treatment was used to compare
the microbial inoculated and un-inoculated plants. Seven-week-old plants (September 28)
were inoculated with a water suspension containing propagules of mycorrhizal fungi (AEGIS
Sym Irriga commercialized by Atens, Tarragona, Spain) and Trichoderma fungus (CONDOR
Shield, commercialized by Atens, Tarragona, Spain). AEGIS was a spore mixture of
Rhizoglomus irregulare BEG72 and Funneliformis mosseae BEG234 having 700 spores of each
species g-1. CONDOR Shield contained Trichoderma koningii TK7 at a concentration of 109 CFU
g-1. AEGIS and CONDOR were applied at a rate of 2 and 1 kg ha-1, respectively. Both products
were dissolved in water and delivered to the plants with a drip irrigation system composed
by drip lines (Aqua-TraXX®, Toro, US) having an emitter flow rate of 1.14 L h-1 and emitters 20
cm apart. The drip lines were laid along the plant rows and slightly buried (5-10 cm) during
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previous inter-row cultivations made for weed control. Management practices were those
commonly adopted in the artichoke production area of Orihuela. Measurements of soil
electrical conductivity were performed at microbial inoculation and at the sampling time in
soil/water extracts (mass soil/water ratio 1:1) using a HI 98194 Multiparameter Meter
(Hanna Instruments, Woonsocket, RI, USA). Artichoke heads were harvested 11 weeks after
inoculation (December 12). Samples were collected to perform metabolomic analysis through
ultra-high- pressure liquid chromatography coupled with quadrupole-time-of-flight mass
spectrometry.
Metabolomic analysis
Frozen leaf samples (0.5 g) were extracted in 5 mL of cold (-20°C) acidified (0.1%
HCOOH) 80% methanol using an Ultra-Turrax (Ika T-25, Staufen, Germany), filtered through
a 0.2 μm cellulose membrane and transferred to amber glass vials for analysis. The screening
of plant metabolites was carried out using a liquid chromatography coupled to quadrupole-
time-of-flight mass spectrometer through a JetStream Electrospray ionization system
(UHPLC-ESI/QTOF-MS) as previously reported (Lucini et al., 2016). A 1290 series liquid
chromatograph was interfaced to a G6550 iFunnel mass spectrometer (all from Agilent
technologies Santa Clara, CA, USA). Chromatographic separation was carried out following the
previously described procedure in (Nakabayashi et al., 2014) and in Riken Plasma website
(http://plasma.riken.jp/). The injection volume was 2 μL, whereas QTOF was operated in
positive SCAN mode (100-1700 m/z+ range) and extended dynamic range mode. MS and
MS/MS acquisition parameters were settled as described in (Showalter et al., 2018).
Deconvolution, mass and retention time alignment, normalization and blank filtering were
carried out using the software MS-DIAL ver 3.40 (Tsugawa et al., 2015; Lai et al., 2018).
Compound identification, for those molecules with MSMS data, was achieved using MS-
FINDER 3.16 (Tsugawa et al., 2016) following the presented workflow in Blazenovic et al.
(2019). Only molecules with match in PlantCyc v13.0 (Schla pfer et al., 2017) was retained for
further analysis, in total 168 metabolites.
Statistical analysis
Orthogonal projections to latent structures discriminant analysis (OPLS-DA) was used
as powerful statistical modeling tool to provide insights into separations between
experimental groups. This analysis is capable to discriminate between groups and it is
adequate to highlight biomarker candidates.
RESULTS AND DISCUSSION
Soil electrical conductivity (EC1:1) at the beginning of the trial (28 September) was 2.22
dS m-1 (ECe=6.7 dS m-1) while at the end of the trial the EC1:1 value was 2.13 (ECe=6.4 dS m-1).
The EC values were always above the salinity threshold reported by Francois (1995) for globe
artichoke (ECe=6.1 dS m-1). After single inoculation of commercial inoculum containing a
mixture of arbuscular mycorrhizal fungi (Rhizoglomus irregulare BEG72 and Funneliformis
mosseae BEG234) and Trichoderma koningii TK7, artichoke plants were harvested and
metabolomic profile of heads was analyzed. OPLS-DA analysis showed significant differences
for 105 metabolites (Figure 1). The goodness of prediction for this model was Q2(cum)=0.983.
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Figure 1. Score plot of OPLS-DA modeling for non-inoculated artichoke head metabolome
group (left) and the AMF and Trichoderma inoculated artichoke head metabolome
group (right).
Metabolites of artichoke head in inoculated and non-inoculated plants were compared
using the ChemRICH analysis (Barupal and Fiehn, 2017). Scatter plot is shown in Figure 2.
Triterpenes as well as epoxyeicosatrienoic acids (EpETrE), and indoles were significantly up-
accumulated in artichoke heads after microbial inoculation whereas other compound groups
such as flavonols, coumarins, umbeliferones and caffeic acids were downregulated.
ChemRICH analysis also showed that large number of flavonoids, glucosides, cinnamates,
methionine, purine nucleosides, aldehydes and apigenin were also altered in heads from
inoculated plants compared to control.
In order to have a deep understanding of the microbial inoculation effects on
metabolome, the significantly altered compounds were annotated using Omics dashboard
(Caspi et al., 2013) available on the PlantCyc website (https://pmn.plantcyc.org/
dashboard/dashboard-intro.shtml) for pathway annotation. The microbial inoculation
enhanced proline synthesis through the increase of γ-L-glutamyl 5-phosphate. Indeed, proline
is a known plant osmolyte, known to play a major role in preserving membrane and solutes,
as well as against redox imbalance damages (Rouphael et al., 2016). The metabolomic analysis
also revealed that microbial inoculation enhanced methionine content (S-methyl-5’-
thioadenosine and S-methyl-L-methionine) which may have implications in ethylene
biosynthesis. This latter is known because the related signaling is indispensable for triggering
plant response and tolerance to salinity stress (Tao et al., 2015). AMF and Trichoderma
inoculation also led to further hormonal alterations in the metabolic profile of artichoke
heads. N6-dimethylallyladenine was up-accumulated in artichoke heads of microbial-
inoculated plants; this compound is involved both in cytokinin synthesis and degradation but
with more implications in this latter process. The downregulation of cytokinin of artichoke
heads in microbial-inoculated plants may be due to the advanced phenological stage reached
by microbial inoculated plants in comparison with control plants (Jin et al., 2015).
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Figure 2. Node color scale showing the proportion of increased (red) or decreased (blue)
compounds in artichoke heads of inoculated plants compared to non-inoculated
ones. Purple-color nodes have both increased and decreased metabolites. Each
node reflects a significantly altered cluster of metabolites. Node sizes represent the
total number of metabolites in each cluster set.
In this sense, it is noteworthy the decrease of D-ribulose 5-phosphate may be
consequence of the downregulation of pentose phosphate pathway. Interestingly, jasmonic
acid (JA) biosynthesis pathway was promoted in microbial-inoculated heads by the significant
accumulation of 13(S)-HPOTE, 3-oxo-2-(cis-2’-pentenyl)-cyclopentane-1-octanoate and 12-
oxo-cis-10,15-phytodienoate. Moreover, among fatty acid derivatives biosynthesis, microbial
inoculation enhanced 9,10-epoxy-10,12Z,15Z-octadecatrienoate and (Z)-3-hexanal in
artichoke heads. These compounds are resulted from the oxygenation of α-linoleic acid, the
initial step in JA synthesis (Wasternack and Hause, 2013). It is well known that beneficial root-
associated microbes, including mycorrhizal fungi and Trichoderma, induce systemic
resistance (IRS) in plants. IRS is characterized by plant priming for more efficient activation
of defense responses (Conrath et al., 2015) and it is regulated by JA/ethylene signaling (Van
Wees et al., 2008). Among others, jasmonate has also been found to enhance water stress
tolerance in plants (Li et al., 2018). This phytohormone is involved in stress perception and
signal transduction in plants, thus regulating gene or protein expression and leading to
metabolic and physiological responses (Riemann et al., 2015). Microbial inoculation reduced
the coumaric acid metabolism. This plant response to inoculation with both AMF or
Trichoderma has been previously described by Lucini et al. (2016); these authors suggested
that carbon flux may be modified in the phenylpropanoid pathway, resulting in the increase
in phenolic compounds downstream. Noteworthy, coumaric acid and its derivatives have been
reported to be promising agents to modulate the detrimental effects of salinity stress in rice
(Thu Ha et al., 2016). One of the most interesting aspects of microbial inoculation is the
enhancement of nutritional value of artichoke heads. This study highlighted that co-
inoculation of plants with AMF and Trichoderma koningii increased significantly the content
of various compounds having implication in nutritional quality of artichoke heads. For
instance, microbial inoculation increased the (R)-pantothenate, precursor of the synthesis of
4’-phosphopantetheine moiety of coenzyme A. Also, folate biosynthesis has been promoted in
inoculated plants through the enhancement of 4-aminobenzoate, precursor of
tetrahydrofolate (vitamin B9) and its derivatives. These compounds referred as folates are
90
involved in photorespiration, amino acid metabolism and chloroplastic protein biosynthesis
(Hanson and Gregory, 2002; Jabrin et al., 2003). The results of this study showed a significant
increase of pyridoxal and pyridoxine in artichoke heads after microbial inoculation. Vitamin
B6 is group of chemically similar compounds which can be interconverted and includes
pyridoxal, pyridoxine, pyridoxamine and their phosphorylated derivatives.
CONCLUSIONS
To summarize, the inoculation with AMF (Rhizoglomus irregulare BEG72 and
Funneliformis mosseae BEG234) and Trichoderma koningii TK7 of artichoke plants grown
under saline conditions led to significant changes in head metabolome. The OPLS-DA analysis
was capable to discriminate the microbial-based treatment from the untreated plants. A total
of 168 metabolites were detected and significantly altered by microbial inoculation. Hormonal
alteration of the plants comprised the downregulation of cytokinin metabolism and together
with the decrease of Ribulose 5-phosphate, suggest the advanced phenology of inoculated
plants. Enhancement of JA pathway was featured by the upregulation of 5 metabolites
pointing out the priming effect of arbuscular mycorrhiza inoculation and Trichoderma
rhizosphere colonization. Besides, treated artichoke heads featured higher levels of
pantothenate (vitamin B5), aminobenzoate (vitamin B9) and pyridoxine (vitamin B6).
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