J. Plant Nutr. Soil Sci. 2019, 000, 1–9 DOI: 10.1002/jpln.

201800439 1

Mycorrhizal, nutritional and virgin olive oil parameters affected by

groundcovers
Irene Garcı́a-González1, Andrés Garcı́a-Dı́az2, Blanca Sastre2, Nikola Teutscherova3, Ma Ángeles Pérez2,
Ramón Bienes2, Rafael Espejo1, and Chiquinquirá Hontoria1*
1 Departamento de Producción Agraria, Universidad Politécnica de Madrid. Avda. Puerta de Hierro, n° 2, 28040 Madrid, Spain
2 Applied Research and Agricultural Extension, Madrid Institute for Rural, Agricultural and Food Research and Development—IMIDRA,
Carretera A-2 km 38.2, 28800 Alcalá de Henares, Madrid, Spain
3 Department of Crop Science and Agroforestry, Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague. Kamycka 129,

Prague 6-Suchdol, Prague, Czech Republic

Abstract
Management of olive groves faces the challenge of reconciling yield, soil degradation and virgin
olive oil (VOO) quality. We evaluated the effect of replacing tillage management by vegetal
groundcovers (GC) on the relationships between mycorrhizal symbiosis, olive nutritional status,
and VOO quality under field rainfed conditions. The experiment was set up in 2014 in an existing
Cornicabra olive orchard with a Haplic Gypsisol soil under a Mediterranean semiarid climate.
Four treatments were replicated four times and consisted of: (1) annual cover of bitter vetch, (2)
permanent Brachypodium distachyon, (3) spontaneous vegetation cover (mainly Brassicaceae
species), and (4) tilled soil. The use of bitter vetch GC increased the olive root colonization by
50% compared with the tillage treatment. The effect of tillage on VOO differed from that of GC
use. Tillage treatment decreased maturity index and its VOO had lower polyphenol content and
less luminosity than that from the GC treatments. Olive root colonization, together with changes
in nutrients such as Cu, B and other elements resulting from GC use, seems to play an important
role in explaining the variability of VOO quality parameters. Although tillage may lead to higher
yield by controlling competition for water and nutrients, the introduction of GC in olive groves led
to higher polyphenol contents, enhancing VOO quality and, at the same time, protecting soil from
erosion.

Key words: arbuscular mycorrhizal fungi / micronutrients / tillage, rainfed olive grove / VOO

Accepted June 27, 2019

1 Introduction
Olive orchards cover around 10.7 million ha in the world, with
more than 90% in the Mediterranean basin (FAO, 2016),
where it is one of the most important woody crops. In this
area, traditional olive groves are managed under rainfed con-
ditions and were designed with low plant density due to water
scarcity (Connor and Fereres, 2005). Virgin olive oil (VOO) is
one of the main outcomes of olive growing and plays a key
role in the Mediterranean diet. Virgin olive oil production has
increased in recent decades due to the large interest in its
nutritional and health properties (Bendini et al., 2007), repre-
senting 3% of the world’s production of vegetable fats and
oils (Aparicio and Harwood, 2013).
groundcovers (GC) have been shown to be a good alternative
to continuous tillage (Gonzalez-Sanchez et al., 2015; Sastre
et al., 2017), although their effect on olive tree yield is contro-
versial (Pardini et al., 2002; Sastre et al., 2016).
The quality of the VOO is determined by the environment, cul-
tivation and harvesting and technological factors, and their
interrelationships (Aparicio and Harwood, 2013). The quality
of VOO, therefore, may be affected by GC, but this effect has
seldom been studied (Gucci et al., 2012; Sastre et al., 2016).
Thus, there is a lack of knowledge on the effect of replacing
tillage by GC on VOO quality.

There are several factors triggering soil degradation pro-
cesses in Mediterranean olive groves. Olive trees are fre-
quently planted on slopes and managed with continuous till-
age. Consequently, these soils remain bare most of the year,
suffering from soil erosion and low organic matter content and
making them prone to particle and nutrient detachment
(Gómez et al., 2009; Gucci et al., 2012). In order to reduce
sediment and nutrient loss and to increase soil fertility, vegetal
Olive roots are susceptible to colonization by arbuscular my-
corrhizal fungi (AMF), which may provide important benefits
(Smith and Read, 2008; Meddad-Hamza et al., 2017). The
symbiotic relationship between AMF and olive roots can affect
shoot and root biomass and certain leaf nutrient contents
under controlled conditions (Porras-Soriano et al., 2009;
Jiménez-Moreno et al., 2018), mitigate adverse substrate
conditions (Khabou et al., 2014; Bati et al., 2015), and even

* Correspondence: C. Hontoria; e-mail: c.hontoria@upm.es

ª 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plant-soil.com

2 Garcı́a-González, Garcı́a-Dı́az, Sastre, Teutscherova et al.
improve olive yield (Estaún et al., 2003). The use of GC can
stimulate mycorrhizal symbiosis in the main crop since they
provide an additional AMF host and the minimization of tillage
enhances the AMF inocula (Garcı́a-González et al., 2018). In
woody crops, mycorrhizal benefits might be increased by the
use of GC (Cheng and Baumgartner, 2004), however, some
field experiments have not found clear benefits from GC on
mycorrhizal activity in olive trees (Turrini et al., 2017) or other
woody crops, such as grapevines (Baumgartner et al., 2005).
Due to the scarcity of studies, more field experiments are
needed to evaluate the effect of different GC species on AMF
in olive groves.
Given that AMF can increase the uptake of particularly immo-
bile nutrients (Lehman et al., 2012; Lehmann and Rillig,
2015), they contribute to enhance crop nutrition, thus increas-
ing crop yield (Bender and van der Heijden, 2015) with poten-
tial effects on quality (Sbrana et al., 2014). Despite the fact
that olive tree nutrition influences VOO quality (Dag et al.,
2009), no studies have focused specifically on the AMF–VOO
quality relationships to date.
The main objective of this study is to assess the effect of
replacing tillage by GC on the relationships between AMF
parameters, olive nutritional status and VOO quality in a
rainfed olive orchard. Specifically, we examine whether: (1)
the use of GC modifies the AMF activity in olive trees affecting
the nutrient uptake of the olive trees and, correspondingly, (2)
VOO quality parameters are affected. These objectives are
based on the hypotheses that: (1) GC increase AMF activity
in the soil and olive tree roots, (2) the increase of AMF activity
is related to a nutritional variation in olive leaves, and (3) GC
influences VOO quality, either directly or via AMF. As far as
we know, this is the first time that the influence of GC via AMF
on VOO quality is reported under field conditions.
2 Material and methods
2.1 Study site and experimental design
The study area is located in Central Spain (40°4¢21¢¢ N,
3°31¢11¢¢ W) in the Tagus River basin. The average elevation
is 540 m asl, and the slope ranges from 9 to 12%. According
to the Köppen classification, the climate is cold semi-arid
(BSk,) with a mean annual temperature of 14.6°C and an
annual precipitation of 373 mm. The soil has developed over
gypsiferous marls and is classified as Haplic Gypsisol (FAO,
2014). The top layer (0–20 cm) is characterized by a pH of
7.8, electrical conductivity of 2.2 dS m–1, low organic matter
(1.14%), and loam texture.
The experiment was set up in 2014 in an olive plantation
(Olea europaea L. cv. Cornicabra) established in 2004. The
Cornicabra olive variety is the most common in Central Spain
and represents 12% of VOO produced in Spain (Rallo et al.,
2005). Olive trees were spaced 6 m · 7 m with a total area of
3.1 ha. In November 2014, the whole area was tilled with a
chisel to a depth of approximately 0.30 m following the con-
ventional management in the area. The tillage was performed
in 6-m-wide inter-rows, at the center of the rows between the
ª 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Plant Nutr. Soil Sci. 2019, 000, 1–9
olive trees. The area was divided into 16 experimental plots
(42 m · 28 m each), randomly distributed in four blocks with
four treatments within each block. The treatments consisted
of three different GC types, each belonging to the three main
families, and a tillage control applied between olive rows: (1)
an annual cover of bitter vetch (Vicia ervilia Willd.; Fabaceae),
which was seeded annually (75 kg ha–1); (2) a permanent
grass cover with purple false brome (Brachypodium dis-
tachyon L. P. Beauv.; Poaceae) at 40 kg ha–1 only seeded
once in November 2014; (3) a spontaneous vegetation cover
dominated by two species of Brassicaceae (Diplotaxis eru-
coides L. and Descurainia sophia L.); (4) tilled soil with two
chisel passes per year (spring and autumn) at a depth of
0.15–0.20 m. For all treatments, weeds along the olive tree
rows (1 m each side) were controlled with a herbicide (glypho-
sate) twice per year. All the GC were mowed once by the end
of April. Plant residues were left spread on the surface. No
irrigation or fertilization was used during the study period fol-
lowing the low-input management common in the area.
2.2 Root and soil sampling and analyses
Root and soil samples were taken in each plot from a depth of
0–20 cm in spring, just before GC were mowed (26/4/2016).
We randomly selected four olive trees per plot and took two
soil samples close to root system from each with a cylindrical
sampler (8 cm diameter) near the external line of the canopy
projection. Soil from the eight points were homogenized and
combined to obtain a composite sample per plot. In the labo-
ratory, GC roots and only young and active olive roots were
carefully separated and rinsed. Roots were cleared with 10%
KOH and 2% H2O2 and then stained with ink-vinegar solution
(Vierheilig et al., 1998). The magnified intersections method
was used to obtain the percentage of mycorrhizal colonization
of GC and olive roots (McGonigle et al., 1990). The length of
the extraradical hyphae was obtained (Garcı́a-González
et al., 2016) from a suspension with 2 g soil that was sequen-
tially sieved through a 250- and a 50-mm sieve. Once
separated, the hyphae were stained with ink-vinegar solution
and filtered through a membrane filter (Whatman 0.45 mm
diameter pores) under vacuum. The hyphae were counted
under a microscope, and the hyphae length was calculated
according to Tennant (1975). Spores of AMF were extracted
from the soil by wet-sieving and decanting followed by
sucrose centrifugation (Sieverding, 1991). The supernatant
was poured through a 50-mm mesh and AMF spores were
grouped in a Petri dish for counting under a dissecting micro-
scope.
Soil parameters were determined in subsamples previously
air-dried and passed through a 2-mm sieve. Soil organic mat-
ter (SOM) was measured by the Walkley–Black method. Soil
pH and electrical conductivity at 25°C were measured in de-
ionized water (1:2.5 soil:water ratio). Soil N was determined
by the Kjeldahl method. Available soil nutrients were meas-
ured by inductively coupled plasma-optical emission spectro-
scopy (ICP-OES; ICP-ICAP 6500 DUO, Thermo Scientific,
UK) in extracts obtained by shaking 3 g of air-dried soil with
30 mL of Mehlich III solution (Mehlich, 1984).
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J. Plant Nutr. Soil Sci. 2019, 000, 1–9 Virgin olive oil is affected by groundcovers and AMF 3

2.3 Sampling of olive leaves and analyses
Olive leaves were taken on two dates (13/5/2016 and
19/7/2016) from eight random trees in each plot. Leaves were
washed, dried at 60°C and finely ground in a ball mill. Leaf N
was determined by the Kjeldahl method and other elements
were determined by ICP-OES (ICP-ICAP 6500 DUO, Thermo
Scientific, UK).
2.4 Harvest and olive oil quality parameters
Olive trees were harvested on 28th November 2016. Two
healthy olive trees per plot were randomly selected and
fruits were hand-picked. The yield per tree was measured
in the field. Maturity index was determined in the laboratory
for each subsample of fruits, following the method of
Beltrán et al. (2008) based on skin and pulp fruit colors.
Olive oils from each subsample were extracted using the
Abencor system (MC2 Ingenierı́as y Sistemas) (Martı́nez
et al., 1975).
Total phenolic compounds were determined in VOO (after
methanol extraction and subsequent reac-
tion with Folin–Ciocalteu reagent) by meas- (A) 80 GC root colonizaon
uring at a wavelength of 725 nm (Vázquez-
Olive root colonizaon
Roncero et al., 1973). Results are ex-
pressed as mg of caffeic acid equivalents
60
Root colonizaon (%)
used to evaluate relationships between dependent variables
with Statistica 10 software (StatSoft Inc., 2011).
3 Results
3.1 Mycorrhizal and nutritional parameters
Mycorrhizal colonization in GC roots was significantly higher
in bitter vetch (71.5%) and Brachypodium (50.3%) roots
compared with spontaneous vegetation (3%) (Fig. 1A). The
larger mycorrhizal colonization in bitter vetch roots was parti-
ally transferred to the olive roots so that olive root colonization
was higher (p < 0.1) in the bitter vetch treatment (64.4%) than
in the tilled treatment (42%). The number of AMF spores and
the length of hyphae were not affected by the different treat-
ments (Fig. 1B). Tillage gave higher concentrations of K, Co,
Mg, and Mn (and Cu, p < 0.1) and lower concentrations of Ca
and Sr compared with all or some of the GC treatments
(Tab. 1). The treatment with spontaneous vegetation had the
lowest K and had lower Co compared with the tillage and bit-
ter vetch treatments.
a
a
ab ab

per kg of oil. Carotenoids and chlorophylls b

were determined at a wavelength of 470 b
and 670 nm, respectively, in cyclohexane,
according to the method of Minguez- 40
Mosquera et al. (1991). The concentrations
of chlorophyll and carotenoids were ex-
pressed as mg of pheophytin and lutein per
kg, respectively. Chromatic coordinates 20
were measured by the software CINTRAL
to obtain the colour according to the c
c
International Commission on Illumination 0
(CIE) L* a* b* method (Standard UNE
72031:1983). (B) 40 AMF spores Hyphae length 30

2.5 Statistical analyses 25

AMF spores (spores g-1)

30

Hyphae length (cm g-1)

Data were tested for normal distribution with
the Kolmogorov–Smirnov test and for homo- 20
geneity of variances with the Levene test.
Variables were examined using a general lin- 20 15
eal model and Fisher’s Least Significant Dif-
ference test was used to compare means 10
(p < 0.05). When the assumptions of ANOVA
10
were not met, even after variable transforma-
5
tions, then the Kruskall–Wallis test was ap-
plied. The Pearson’s product-moment was
obtained to analyze the correlation between 0 0
variables. We carried out multiple regression TIL SPV BRA VET
analyses to identify the variables that best ex-
Figure 1: (A) Groundcover (GC) and olive root colonization, and (B) arbuscular mycorrhi-
plain VOO parameters. Statistical analyses zal fungi (AMF) spores and hyphae length for the four different treatments: tillage (TIL),
were carried out using the Statgraphics Cen- spontaneous vegetation (SPV), Brachypodium (BRA), and bitter vetch (VET). Columns
turion XVII software (StatPoint Technologies are mean values and bars are standard errors. Different letters indicate statistically signifi-
Inc., Rockville, Maryland, USA). In addition, a cant differences between treatments for GC root colonization (LSD, p < 0.05) and for olive
Principal Component Analysis (PCA) was root colonization (p < 0.1).

ª 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plant-soil.com

4 Garcı́a-González, Garcı́a-Dı́az, Sastre, Teutscherova et al. J. Plant Nutr. Soil Sci. 2019, 000, 1–9

Table 1: Nutrient concentrations in soil (April 2016) and olive leaves (July 2016) for the four treatments: tillage (TIL), spontaneous vegetation
(SPV), Brachypodium (BRA), and bitter vetch (VET).a

Soil Olive leaves

TIL SPV BRA VET TIL SPV BRA VET

N (%) 0.09 (0.03) a 0.08 (0.01) a 0.09 (0.01) a 0.08 (0.01) a 1.60 (0.04) a 1.42 (0.03) b 1.33 (0.07) b 1.44 (0.03) b

K (%) 0.06 (0.006) a 0.03 (0.001) c 0.04 (0.003) b 0.05 (0.004) ab 1.07 (0.05) a 1.18 (0.04) a 1.15 (0.02) a 1.17 (0.07) a

Ca (%) 0.77 (0.18) c 0.81 (0.16) bc 0.87 (0.07) a 0.86 (0.27) ab 1.07 (0.05) a 1.01 (0.04) a 0.99 (0.07) a 1.04 (0.03) a

S (mg kg–1) 2918 (42) a 2915 (44) a 2897 (81) a 2730 (65) a 1225 (63) a 1200 (41) a 1275 (63) a 1225 (103) a

P (mg kg–1) 49.46 (3.8) a 37.33 (2.6) a 49.71 (5.6) a 43.95 (4.4) a 975 (50) a 975 (50) a 950 (30) a 1000 (80) a

B (mg kg–1) 2.53(0.14 ) a 2,38(0.03) a 2.47(0.09 ) a 2.80 (0.16) a 23.50 (1.44) c 31.42 (1.05) a 27.60 (1.25) ab 25.69 (1.78) bc

Co (mg kg–1) 1.24 (0.08) a 0.83 (0.03) c 0.98 (0.07) bc 1.01 (0.04) ab n.d. n.d. n.d. n.d.

Cu (mg kg–1) 3.94 (0.27) a 3.23 (0.53) a 2.58 (0.10) a 2.62 (0.37) a 12.19 (3.05) a 3.54 (0.14) b 3.65 (0.39) b 4.76 (0.9) b

Fe (mg kg–1) 37.69 (3.5) a 41.04 (1.5) a 38.71 (1.6) a 36.60 (1.7) a 48.17 (1.03) a 43.07 (1.62) a 45.89 (3.31) a 47.93 (2.58) a

Mg (mg kg–1) 110.4 (11.9) a 72.9 (6.0) b 80.7 (7.9) b 77.4 (7.7) b 1150 (64.5) a 1050 (64.5) a 1075 (47.9) a 1075 (25) a

Mn (mg kg–1) 68.36 (8.1) a 48.85 (4.1) b 56.06 (5.0) ab 47.29 (3.4) b 35.32 (2.28) a 23.95 (1.14) b 25.93 (1.24) b 27.70 (2.74) b

Mo (mg kg–1) n.d. n.d. n.d. n.d. 0.05 (0.01) a 0.06 (0.01) a 0.05 (0.01) a 0.06 (0.01) a

Na (mg kg–1) 33.59 (10.8) a 29.22 (14.4) a 8.18 (6.6) a 14.89 (5.3) a 27.5 (4.8) a 17.5 (2.5) a 17.5 (2.5) a 20 (0.0) a

Sr (mg kg–1) 57.85 (0.4) b 76.48 (4.8) a 78.14 (2.9) a 73.01 (5.5) a 141.2 (7.11) b 234.8 (10.8) a 227.3 (9.7) a 210.0 (7.4) a

Zn (mg kg–1) 0.63 (0.13) a 0.28 (0.16) a 0.43 (0.1) a 0.20 (0.08) a 13.12 (0.82) a 12.08 (0.32) a 12.52 (0.35) a 12.42 (1.09) a

an.d.:not detectable; nutrients in bold with different letters indicate significant differences between treatments (LSD or Kruskal–Wallis at
p < 0.05). Values shown are means with standard errors in parentheses.

Nutrient concentrations in olive leaves had similar differences Table 2: Correlation of olive root colonization, hyphae length and
between treatments for both May and July samplings. In July arbuscular mycorrhizal fungi (AMF) spores with selected olive leaf
(Tab. 1, May sampling not shown), the tillage treatment had nutrients (July) and VOO quality parameters.a
significantly higher N, Cu, and Mn and lower Sr than GC,
whereas it had lower B than both spontaneous vegetation Olive root Hyphae AMF
and Brachypodium. Mycorrhizal parameters were correlated colonization length spores
with some leaf nutrients analyzed in July, giving positive rela- P ns 0.57* 0.55*
tionships for P, K, S, Fe, and Zn (Tab. 2).
K ns ns 0.79***

Ca 0.54* ns –0.51*
3.2 VOO parameters
S ns 0.49+ ns
Given the effect of fruit yield and maturity index on VOO qual-
ity, they were included in the study together with VOO quality Fe 0.49+ 0.48+ ns
parameters (Tab. 3). Treatments differed considerably in their Zn ns 0.55* ns
effect on olive yield and VOO quality: while the mean yield
was increased by 66% (13.81 kg tree–1 vs. an average of Polyphenols ns ns ns
8.30 kg tree–1 for the GC) in tilled plots, the maturity index, Carotenoids –0.50+ ns ns
polyphenols and L* were reduced when compared to CG
treatments. In general, correlations between AMF parameters Chlorophyll ns ns ns
and VOO quality parameters were weak, except for L* L* 0.58* ns ns
(Tab. 2). Polyphenols were positively correlated with
a* ns ns ns
B (r = 0.58*) and Sr (0.81**) and negatively with N (–0.79**)
and Cu (–0.62*). b* –0.56+ ns ns

The multiple regression analyses showed that approximately ans:not significant. +,*, ***significant at p < 0.1, < 0.05, and < 0.001,
80–85% of the variability of L* and b*chromatic coordinates respectively.

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J. Plant Nutr. Soil Sci. 2019, 000, 1–9 Virgin olive oil is affected by groundcovers and AMF 5

Table 3: Fruit yield (kg tree–1), maturity index, and VOO quality parameters: concentrations of polyphenols (mg kg–1), carotenoids (mg kg–1)
and chlorophylls (mg kg–1), and colour chromatic coordinates (L*, a* and b*) for the four treatments: tillage (TIL), spontaneous vegetation
(SPV), Brachypodium (BRA), and bitter vetch (VET).a

TIL SPV BRA VET

Yield 13.81 (2.2) a 8.25 (0.9) b 8.53 (0.6) b 8.13 (1.2) b

Maturity index 2.01 (0.12) c 2.77 (0.12) a 2.42 (0.03) b 2.54 (0.03) ab

Polyphenols 976.5 (24.6) c 1118 (38.4) ab 1193 (20.1) a 1011 (28.4) bc

Carotenoids 11.45 (0.5) a 11.09 (1.0) a 11.49 (0.3) a 9.97 (1.2) a

Chlorophyll 19.72 (1.1) a 17.9 (2.3) a 18.04 (0.3) a 16.79 (3.1) a

Carotene/Chlorophyll 0.58 (0.01) a 0.63 (0.03) a 0.64 (0.01) a 0.61 (0.04) a

L* 79.46 (0.24) c 82.72 (1.16) ab 82.47 (0.19) b 84.09 (0.27) a

a* –14.57 (0.3) a –14.37 (0.6) a –12.94 (0.3) a –14.73 (0.2) a

b* 106.55 (1.7) a 99.76 (4.2) a 104.8 (0.6) a 98.16 (2.0) a

aParameters in bold with different letters indicate differences between treatments (LSD or Kruskal–Wallis at p < 0.05). Values shown are means
with standard errors in parentheses.

were explained by only three variables (Tab. 4). By contrast, more by the variables represented in factor 1 (Fig. 2b). Tillage
polyphenols, carotenes and the a* chromatic coordinate scores are concentrated in section 3, close to yield, N and
needed five variables to explain 85% of their variability Cu, while GC are mainly in sections 2 and 3 with scores close
(Tab. 4). Olive root colonization contributed to the explanation to SOM, polyphenols, olive root colonization, B, and Sr.
of all studied VOO parameters. Leaf Cu concentration played Brachypodium and spontaneous vegetation have similar
a relevant role in explaining the variability of polyphenols. In scores but bitter vetch is closer to tillage.
addition, B contributed to explain polyphenols, carotenes and
chlorophyll, whereas Fe partially ex-
plained variability of polyphenols.
Soil organic matter was relevant for Table 4: Multiple regressions for VOO quality parameters (dependent variables).a
carotenes and chlorophyll. Maturity
index contributed to the explanation Number Explanatory variables R2 adj Explanatory variables R2 adj
of all chromatic coordinates togeth-
Polyphenols Carotenoids
er with SOM and elements such as
Sr, Fe, and Mn. 2 Cu, SOM 36.7 Olive RC, SOM 65.4

3 Cu, Fe, Olive RC 56.7 Olive RC, SOM, B 75.3

To facilitate the interpretation of the
PCA, only selected variables were 4 Cu, Fe, Olive RC, P 72.9 Olive RC, SOM, B, K 79.7
included in the analysis based on 5 Cu, Fe, Olive RC, P, B 84.3 Olive RC, SOM, B, K, Mo 85.7
the above results. The first two PCA
axes extracted 37% and 20% of the Chlorophyll L* coordinate
variability (Factors 1 and 2, respec- 2 Olive RC, SOM 49.3 EC, MI 74.4
tively). Copper, yield, N, Sr, B, and
polyphenols had the highest scores 3 Olive RC, SOM, B 72.6 SOM, Olive RC, Cu 80
in Factor 1 (Fig. 2a). By contrast, P, 4 Olive RC, SOM, B, K 80.7 SOM, Olive RC, N, Mn 92.9
b*, and chlorophyll had the highest
5 Olive RC, SOM, K, P, Sr 92.7 SOM, Olive RC, N, Mn, EC 94.2
influence in Factor 2. Yield was
strongly and positively correlated a* coordinate b* coordinate
with N and Cu and negatively with
2 Sr, Olive RC 26 SOM, MI 63.3
Sr, B, polyphenols, and olive root
colonization. Among AMF parame- 3 Sr, MI, Fe 54 SOM, MI, Olive RC 84.3
ters, olive root colonization had
4 Sr, MI, Fe, SOM 73.9 Olive RC, Hyphae L, P, Fe 90.2
similar scores to B, Sr, and polyphe-
nols. There was another group of 5 Sr, MI, SOM, Olive RC, P 86.2 Olive RC, Hyphae L, P, Fe, Mn 95.5
related variables composed of
chlorophyll, b* color, hyphae length, aExplanatory variables: maturity index (MI), olive root colonization (Olive RC), hyphae length
and Fe. The different managements (Hyphae L), leaf nutrients in July, soil organic matter (SOM), soil pH, and soil electrical conductivity
(tillage vs. GC) can be differentiated (EC).

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6 Garcı́a-González, Garcı́a-Dı́az, Sastre, Teutscherova et al.
Figure 2: Ordination of variables (a) and cases (b) on biplots resulting
from the Principal Component Analysis; (a) Chlorophyll (Chloro),
Electrical conductivity (EC), Hyphae length (Hyph L), soil organic
matter (SOM), olive root colonization (Olive RC), polyphenols (Pol-
yph), (b) tillage (Til), spontaneous vegetation (Spv), bitter vetch (Vet),
and Brachypodium distachyon groundcover (Bra).
4 Discussion
In the climate and soil conditions of our study, the introduction
of a legume GC such as bitter vetch increased AMF coloniza-
tion in the olive roots by 50% with respect to the tillage treat-
ment. This disagrees with other studies that did not find any
AMF stimulation from the use of GC in olive groves (Turrini
et al., 2017) or vineyards (Baumgartner et al., 2005). The dif-
ference between those studies and ours could be the result of
the adverse growing conditions: a gypsic soil with low SOM
under semiarid climate, without irrigation and fertilizers. Since
Brachypodium is a non-tilled permanent GC, we should have
expected a larger AMF stimulation in olive roots than the
yearly bitter vetch cover. We attribute its poor response to an
incomplete plant establishment and, perhaps, factors related
to AMF affinity given the lower root colonization shown by
Brachypodium roots (50%) compared with bitter vetch roots
(72%). In any case, we cannot discard a positive effect of
Brachypodium on olive mycorrhizal symbiosis once this per-
manent GC has been fully established. As expected, the
ª 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Plant Nutr. Soil Sci. 2019, 000, 1–9
spontaneous GC did not increase olive root colonization
because it is mainly composed by Brassicaceae species,
which are not AMF hosts. Even more, Brassicaceae are
known to inhibit fungi through isothiocyanates formation with
potential AMF reduction (Fahey et al., 2001). However, we
did not find a negative effect of the spontaneous Brassica-
ceae GC on olive AMF colonization, in agreement with Peller-
in et al. (2007).
Overall, the introduction of GC seems to affect VOO quality,
either due to changes in native mycorrhizal activity and the
corresponding effect on host nutrition, or directly by changes
in olive nutritional status as a result of GC use, although other
processes not considered in this study could also play a role
such as gene activation or enzymatic activities (Velázquez-
Palmero et al., 2017; Hernández et al., 2018). The fact that
AMF root colonization explained the variability of the majority
of the studied VOO parameters, such as polyphenols, carote-
noids, chlorophyll and two color parameters, suggests that
mycorrhizal symbiosis may affect VOO quality, at least in the
conditions of our study. Our results are in agreement with the
idea that mycorrhizal symbiosis induces modifications in plant
metabolism, resulting in enhanced production of health-
promoting phytochemicals, such as polyphenols, carotenoids,
flavonoids, etc. (Sbrana et al., 2014). In this line with Kara
et al. (2015) who found that inoculation of olive trees with
AMF (together with application of micronized calcite) affected
olive oil properties such as certain phenolic compounds.
Other studies have reported that symbiosis with AMF affected
essential oil quality of several aromatic plants (Kapoor et al.,
2002; Chaudhary et al., 2008). This effect may be in relation
with nutritional changes due to mycorrhizal symbiosis. For
example, in our case some nutrients had linear relationships
with AMF parameters (P, Ca, S, Fe, and Zn), which in turn
may induce the accumulation of secondary metabolites
(Sbrana et al., 2014).
Apart from AMF stimulation, several studies reported benefits
from using GC in olive groves (Palese et al., 2014), but very
few have analyzed the connection with VOO parameters
(Gucci et al., 2012; Sastre et al., 2016). In this study, the
effect of tillage on VOO clearly differed from that of GC use.
Maturity index was lower in the tillage compared with the GC
treatments in accordance with the higher yield (66% more) of
the tilled plots compared with GC plots. These results agree
with Gucci et al. (2012), who also found greater fruit yield
under tillage than under a permanent natural cover with
maturity index values tending to be lower under tillage.
Compared with GC plots (spontaneous vegetation and
Brachypodium), polyphenols were also lower in tilled plots in
line with Gucci et al. (2012). Importantly, GC seem to promote
polyphenols which are considered the main antioxidant com-
pounds in VOO (Morelló et al., 2004). Regarding color, tillage
treatment also differed from GC with lower values of luminos-
ity (L*), which may be related to a higher content of pigments
such as chlorophyll. In contrast to our results, in a similar
study area, Sastre et al. (2016) did not find such marked dif-
ferences between tillage (only one chisel pass) and GC.
Instead, they found more differences between GC. They
found that the legume GC had lower pigment content and
higher L* value, which is in line with the tendency observed in
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J. Plant Nutr. Soil Sci. 2019, 000, 1–9
our data (no significant differences), although in contrast to
their study we did not find a relationship with the maturity
index. Results of the PCA reflect the disparity between tillage
and GC well since all GC treatments scored similarly in the
PCA, differentiated from the tillage treatment by the variables
in Factor 1. Bitter vetch was closer to tillage than the other
GC probably because, unlike them, the legume had similar
values for B concentration and polyphenols as tillage.
The use of GC affected the nutrient uptake by the olive trees.
Most differences were between the tillage treatment and the
GC, whereas nutritional differences between GC treatments
were hardly noticeable. Rodrigues et al. (2015) also reported
no nutritional difference in olive leaves between GC treat-
ments except for N (tillage treatment was not included). In our
case, tillage increased leaf concentrations of N, Cu and Mn,
but decreased B and Sr. For Mn, Cu, and Sr, the higher or
lower nutrient availability in soil seems to be related to the
higher or lower nutrient concentrations, respectively, in olive
leaves (Erel et al., 2008). Despite similarities in soil N, tillage
enhanced N uptake by the olive trees, increasing yield by
66%, which is probably due to the lack of competition with
GC. The study area with degraded gypsic soils and a precipi-
tation of only 320 mm (average 2007–2017) favors a strong
competition for water and nutrients between olive trees and
GC. Therefore, in these limited conditions, GC may decrease
fruit yield in olive groves, at least in the initial years of GC
establishment. However, yield reduction depends on the par-
ticular conditions of each year since GC had no impact on
fruit yield in a 3-year study under similar conditions (Sastre
et al., 2016).
Our results indicate that olive nutritional status and harvest
parameters are important for oil quality in agreement with Erel
et al. (2013). Olive trees with heavy loads slow down the
ripening process (Trentacoste et al., 2010). Regarding VOO
quality, high N fertilization reduces polyphenol content
(Tekaya et al., 2014), which is in agreement with our results.
Maturity index influences polyphenols (Gómez-Rico et al.,
2006), but in our case, elements such as Cu and B also play
a negative and positive role, respectively, in its content
(Tab. 4, Fig. 2). Copper is a very potent oxidation catalyst and
can promote the formation of reactive oxygen species, which
are neutralized by the polyphenolic compounds, resulting in
lower polyphenol contents. This is in agreement with Ferreira
et al. (2007) who reported that phytosanitary copper applica-
tion decreased total phenols in olive leaves, hence reducing
their antioxidant properties. By contrast, boron foliar ferti-
lization promoted phenol content of olive fruits, which was
attributed to the role of boron in the metabolism of phenolic
compounds (Saadati et al., 2013). With regard to Sr, this ele-
ment has been shown to be related to the accumulation of Ca
and S in plants grown in soils with gypsum in Australia (Reid
et al., 2016), but further investigation is required to elucidate
the relationships found here.
5 Conclusions
Root colonization was stimulated in 12-year old olive trees
after only 2 years of cover cropping with an AMF-host GC,
such as bitter vetch. The poor establishment of Brachypodi-
ª 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Virgin olive oil is affected by groundcovers and AMF 7
um GC could explain the lack of positive effects on the mycor-
rhizal symbiosis in the olive crop, whereas the dominance of
Brassicaceae species, a non AMF-host, can explain the lack
of effects with the spontaneous vegetation GC.
Given that AMF parameters were positively related to certain
nutrients in the olive leaves, we cannot discard the fact that
part of the management effect on olive nutrition was via AMF.
In fact, AMF parameters, mainly olive root colonization,
together with changes in nutritional status due to GC use,
seem to play an important role in explaining VOO quality
parameters.
In this study, soil management clearly affected VOO quality.
The tillage treatment differed from the GC treatments, result-
ing in less polyphenols and less luminosity. These differences
cannot be fully explained by the lower maturity index of the till-
age treatment. Nutrients, such as Cu, B and other elements,
also seem to explain changes in VOO parameters.
Tillage may control competition for water and nutrients, result-
ing in higher fruit yields under certain conditions. However,
the introduction of GC in olive groves promotes polyphenol
contents, hence enhancing VOO quality with respect to health
and sensory characteristics.
Acknowledgments
This work was supported by the Spanish Ministry of Economy
and Competitiveness (AGL201452310R) and the Comunidad
de Madrid (project AGRISOST-II, S2013/AB1-2717). We
acknowledge the Centro de Edafologı́a y Biologı́a Aplicada
del Segura (CEBAS-CSIC) in Murcia for the nutritional
analyses and personnel of Finca La Chimenea (IMIDRA) for
their work.
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