Archives of Phytopathology and Plant Protection

ISSN: 0323-5408 (Print) 1477-2906 (Online) Journal homepage: https://www.tandfonline.com/loi/gapp20

Arbuscular mycorrhizal fungi improve mineral

nutrition and tolerance of olive tree to Verticillium
wilt

Hanane Boutaj, Abdelilah Meddich, Abdelghani Chakhchar, Said Wahbi,

Zainab El Alaoui-Talibi, Allal Douira, Abdelkarim Filali-Maltouf & Cherkaoui El
Modafar

To cite this article: Hanane Boutaj, Abdelilah Meddich, Abdelghani Chakhchar, Said Wahbi,
Zainab El Alaoui-Talibi, Allal Douira, Abdelkarim Filali-Maltouf & Cherkaoui El Modafar
(2020) Arbuscular mycorrhizal fungi improve mineral nutrition and tolerance of olive tree to
Verticillium wilt, Archives of Phytopathology and Plant Protection, 53:13-14, 673-689, DOI:
10.1080/03235408.2020.1792603

To link to this article: https://doi.org/10.1080/03235408.2020.1792603

Published online: 11 Jul 2020.

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ARCHIVES OF PHYTOPATHOLOGY AND PLANT PROTECTION
2020, VOL. 53, NOS. 13–14, 673–689
https://doi.org/10.1080/03235408.2020.1792603

Arbuscular mycorrhizal fungi improve mineral

nutrition and tolerance of olive tree to
Verticillium wilt
Hanane Boutaja,b , Abdelilah Meddichb, Abdelghani Chakhchara,c,
Said Wahbib, Zainab El Alaoui-Talibia, Allal Douirad,
Abdelkarim Filali-Maltoufe and Cherkaoui El Modafara
a
Laboratoire d’Agro-Biotechnologie et Bio-ing
enierie, Facult
e des Sciences et Techniques
Gu
eliz, Universit
e Cadi Ayyad, Marrakech, Maroc; bLaboratoire Agro-Alimentaire,
Biotechnologies et Valorisation des Bioressources V
eg
etales, Facult
e des Sciences Semlalia,
Universit
e Cadi Ayyad, Marrakech, Maroc; cFacult
e des Sciences de Rabat, Universit
e
Mohammed V, Rabat, Maroc; dLaboratoire de Botanique, de Biotechnologie et de Protection
des Plantes, UFR de Mycologie, D
epartement de Biologie, Facult
e des sciences B.P. 133,
Universit
e Ibn Tofail, Kenitra, Maroc; eLaboratoire de Microbiologie et Biologie Mol
eculaire,
Facult
e des Sciences, Universit
e Mohammed V, Agdal, Maroc Rabat

ABSTRACT ARTICLE HISTORY

The present study aimed to evaluate the effect of the inter- Received 21 May 2020
action of autochthonous mycorrhizal fungi “Rhizolive con- Revised 24 June 2020
sortium” and the soil-borne Verticillium dahliae on root Accepted 25 June 2020
colonization, plant growth and nutrients accumulation in
KEYWORDS
olive plants. The assays were conducted on a susceptible Arbuscular mycorrhizal
olive cultivar “Picholine Marocaine” inoculated for three fungi; Verticillium dahliae;
months with “Rhizolive consortium” then infected with V. root colonization; plant
dahliae under greenhouse conditions. It was found that growth; bioprotection
root colonization was greatly improved by “Rhizolive con-
sortium” application as well as arbuscular colonization rate
in olive plants infected with V. dahliae after 12 months.
“Rhizolive consortium” enhanced growth and biomass traits
in infected olive plants. Nutrients accumulation has been
significantly improved in mycorrhizal-infected olive plants.
This leads to the conclusion that the beneficial effect of
Rhizolive enhances the tolerance of olive plant disease and
supersedes the pathogenic effect of V. dahliae. The use of
this AMF consortium as a potential biological control agent
could also be critical in cultivation of olive trees in
Mediterranean ecosystems where nutrient deficiency often
occurs and where olive-growing occupied a major place.

Introduction
Verticillium wilt of olive (VWO) is a relevant economic issue because it
produces considerable yield losses (Sanei and Razavi 2017). The disease

CONTACT Boutaj Hanane hanane.boutaj@edu.uca.ac.ma; Cherkaoui El Modafar elmodafar@uca.ac.ma

Laboratoire d’Agro-Biotechnologie et Bio-ing
enierie, Facult
e des Sciences et Techniques Gu
eliz, Universit
e
Cadi Ayyad, Marrakech, Maroc
ß 2020 Informa UK Limited, trading as Taylor & Francis Group
674 H. BOUTAJ ET AL.

is caused by the soil-borne fungus Verticillium dahliae Kleb and stated as

the most frequently causal agent that produces microsclerotia capable of
surviving for more than 10 years in the soil, which makes extremely diffi-
cult the control of VWO (Wilhelm 1955). The biological control of vas-
cular wilt diseases should be in soil to control and manage the pathogen
with antagonistic microbes, which is an important approach to avoid
applying chemical products (Druvefors 2004). Arbuscular mycorrhizal
fungi (AMF) are known as wide commonest symbiotic organisms spread
in soils associated with high plant roots, increased in contrast to non-
mycorrhizal plants. Their beneficial effect on the vegetative growth and
enhancing soil structure (Pasqualini et al. 2007; Sheng et al. 2009; Nafady
et al. 2018) have been associated largely with high nutrient uptake (Porras-
Soriano et al. 2006; Smith and Read 2008). Furthermore, apart from the
effect of AMF on nutrient accumulation, they facilitate phosphorus (P)
absorption by plants, improve plant tolerance to salinity (Porras-Soriano
et al. 2009) drought (Wu et al. 2013) and enhance resistance to pathogenic
soil microorganisms including fungi (Abdel-Fattah and Shabana 2002;
Chandanie et al. 2006; Scheffknecht et al. 2006; Wehner et al. 2010).
Numerous studies showed that mycorrhization of many plants is essential to
achieve a high growth rate (Ganz et al. 2002; Esta
un et al. 2003; Querejeta
et al. 2003). In eggplant, inoculated plants with Glomus mosseae in the pres-
ence of V. dahliae greatly enhanced the accumulation of Ca, K and available
P (Karagiannidis et al. 2002). Opening of ion channels and depolarization of
the transmembrane electric potential were due to the accumulation of K, Ca
and Na ions in the interfacial apoplast (B€ucking et al. 2012).
Among the higher plants, olive tree constitutes a particular mycotrophic
species (Jim
enez-Moreno et al. 2018). It is widely known that root coloniza-
tion by AMF enhances the growth of olive plants (Porras-Soriano et al. 2006)
and its nutrient uptake (Dag et al. 2009; Chatzistathis et al. 2013). In nursery,
during transplantation olive plants are inoculated with mycorrhizae to
improve root system and enhance growth rate (Meddad-Hamza et al. 2010).
Based on the symbiosis with olive plant root, the study of mycorrhiza-patho-
gen interactions has mainly focused on single AMF species in defense versus a
single pathogen (Karajeh and Al-Raddad 1999; Porras-Soriano et al. 2006;
Kapulnik et al. 2010). Autochthonous mycorrhizal fungi seem to have a great
effect on root colonization and nutrients uptake, which could be explained by
its species diversity (Wehner et al. 2010; Eke et al. 2016). It is well demon-
strated that AMF consortia improved plant shoot height and total dry weight
in comparison to non-mycorrhizal ones (Mwangi et al. 2011). Other beneficial
effects can be observed as the result of significant and positive relationships
between the strains forming the AMF consortia as well as the additional bene-
ficial effect of each individual AMF strain, which therefore leads to increased
 ARCHIVES OF PHYTOPATHOLOGY AND PLANT PROTECTION 675

plant growth and productivity (Singh 2015; Eke et al. 2016). Also, the applica-
tion of AMF in form of consortium increased potentially plant protection
compared to a single AMF species (Maherali and Klironomos 2007; Wehner
et al. 2010). AMF consortium contributes in alleviation of vascular wilt symp-
toms of olive plants (Boutaj et al. 2019).
However, only few studies have been conducted on this subject in
olive tree (Karajeh and Al-Raddad 1999; Porras-Soriano et al. 2006;
Kapulnik et al. 2010). Our work aims to assess the role of an indigenous
mycorrhizal inoculum “Rhizolive consortium” both in improving mineral
nutrition and the tolerance of olive tree to Verticillium wilt.

Materials and methods

Biological material
The present study was carried out on susceptible cultivar “Picholine
Marocaine” to V. dahliae. An autochthonous AMF consortium “Rhizolive
consortium” was collected from rhizospheric soil and roots of olive trees in
several Moroccan groves and it was composed of 26 species of
Claroideoglomus etunicatum, Rhizophagus prolifer, R. clarus, R. diaphanum,
R. intraradices, Funneliformis mosseae, F. geosporum, Septoglomus constric-
tum, Diversispora versiformis, Glomus sp1, Glomus sp2, Glomus sp3,
Glomus sp4, Glomus sp5, Acaulospora denticulata, A. spinosa, A. kentinen-
sis, Acaulospora sp1, Acaulospora sp2, Acaulospora sp3, Acaulospora sp4,
Entrophospora sp1, Gigaspora sp1, Gigaspora sp2, Gigaspora sp3 and
Scutellospora sp1 (Kachkouch et al. 2014; Boutaj et al. 2020). This consor-
tium was cultivated in maize roots in plastic pots (13 cm
09 cm) contain-
ing soil already infested with “Rhizolive consortium” for three months
under greenhouse conditions. These plants were irrigated regularly with
distilled water. The infection frequency (F) of maize roots was determined
by the technique described by Trouvelot et al. (1986) (F ¼ 100%).
Regarding the pathogen agent, an isolate OV1 of V. dahliae was pro-
vided by the Laboratory of Botanical, Biotechnology and Plant protec-
tion, Ibn Tofail University, Kenitra, Morocco. This isolate was adjusted
at 2106 conidia/ml at laboratory using Malassez blades under sterile
conditions using laminar flow hood.

Plants inoculation
The experiment was carried out over one year in the Faculty of Sciences
Semlalia greenhouse, Marrakesh, Morocco. Plants at six months were removed
carefully from their plastic pots into others filled with 2 Kg of sterilized mix-
ture of soil and peat 1/1 (v/v). Both soil and peat were properly sterilized in
676 H. BOUTAJ ET AL.

oven at 180  C for 3 h and 1 h, respectively. The characteristics of soil used

were as follows: Sand (50.81%), loam (29.85%), clay (19.33%), pH (8.19), elec-
trical conductivity (73.50 mS/cm), calcite (5.58%), organic matter (0.52%),
available phosphorus (7.72 ppm), calcium (111.30 ppm), potassium
(13.20 ppm) and sodium (29.50 ppm). Before transplantation, cuttings olive
plants were naturally mycorrhized (10% of mycorrhizal frequency). A block of
124 plants was inoculated with 50 g of “Rhizolive consortium” (maize roots
and soil infested with the consortium) deposited directly around the roots of
each plant. A similar number of non-inoculated plants were regarded as a con-
trol block, which received the same quantity of autoclaved inoculum. Plants
were maintained at an average temperature of 19 (during winter) to 30  C
(during summer), 62% relative humidity and 330 m2 s1 of light and irri-
gated twice a week. After 3 months of mycorrhization, 62 plants of each block
were inoculated with 10 ml OV1 isolate of V. dahliae (2106 conidia/ml) with
test tube using a hole of 5 cm depth in the upper part of the stem. However,
10 ml of sterile-distilled water were added to control plants. There were thus
four blocks arranged in a complete randomized design: Non-inoculated plants
(control), inoculated plants with V. dahliae (Vd), inoculated plants with
“Rhizolive consortium” (RC) and inoculated plants with “Rhizolive con-
sortium” and infected with V. dahliae (RCV).

Root colonization and plant growth assessment

Shoot height and number of leaves were estimated each month for one
year. The data were collected from ten plants per treatment. Other meas-
urements have been made at four times; 3, 6, 9 and 12 months to determine
root length and branch number. Shoot and root fresh parts were separated,
weighted and oven-dried at 70  C for 48 h to record shoot and root dry
weight. The data were collected from five plants per treatment.
After one year, root colonization was determined by staining according
to the method described by Phillips and Hayman (1970). Mycorrhizal
intensity, vesicular and arbuscular colonization rate were evaluated as
described by Trouvelot et al. (1986), Derkowska et al. (2008) and
Kachkouch et al. (2012).

Mycorrhizal intensity (M %)

M % ¼ ð95 n5 þ 70 n4 þ 30 n3 þ 5 n2 þ nÞ=N

where, n: number of affected fragments roots with note of 0, 1, 2, 3, 4

or 5.
 ARCHIVES OF PHYTOPATHOLOGY AND PLANT PROTECTION 677

Arbuscular content (a %)

A % ¼ ð100 mA3 þ 50 mA2 þ 10 mA1Þ=100

where mA3, mA2 and mA1 are, respectively, the percentages given to
A3, A2 and A1 (Ao: no arbuscules, A1: some arbuscules 10%, A2: mod-
erately abundant arbuscular 50%, A3: arbuscular very abundant 100%),
with, mA3% ¼ ð95 n5 A3 þ 70 n4 A3 þ 30 n3 A3 þ 5 n2 A3 þ
n1 A3Þ=N: The same for A2 and A1. n5 A3 represents the number of
fragments noted 5 with A3; n4 A3 is the number of fragments noted 4
with A3, N: total number of fragments.

Vesicular content (V %)

V % ¼ ð100 mV3 þ 50 mV2 þ 10 mV1Þ=100

where mV3, mV2 and mV1 are, respectively, the percentages given to
V3, V2 and V1 (V0: no vesicles, V1: some vesicles 10%, V2: moderately
abundant vesicles 50%, V3: abundant vesicles 100%), with, mA3 ¼ (95
n5 V3 þ 70 n4 V3 þ 30 n3 V3 þ 5 n2 V3 þ n1 V3)/N. The same for V2
and V1. n5 V3 represents the number of fragments noted 5 with V3; n4
V3 is the number of fragments noted 4 with V3, N: total number
of fragments.

Determination of nutrient accumulation

To assess available phosphorus (P), calcium (Ca), potassium (K) and
sodium (Na) concentrations, fresh leaves and roots of olive plants were
dried in oven for 48 h at 80  C and incinerated for 6 h at 600  C in the
furnace. The incinerated matter was digested in 3 ml of 6 N HCl and K,
Na and Ca concentrations were estimated with a flame photometer
(AFP100 Biotech Engineering Management) according to the method of
Brown and Lilleland (1946), whereas available P was determined as
reported by Olsen et al. (1954). The data were collected from five repli-
cates per treatment.

Statistical analysis
Data were statistically analyzed through the analysis of variance
(ANOVA), using SPSS (20.0 version). The mean values and standard
deviation (SD) were calculated. Significant differences between means
were examined using Tukey’s HSD Post hoc test at p < 0.05.
678 H. BOUTAJ ET AL.

Table 1. Mycorrhizal intensity, arbuscular and vesicular content after one year in non-
mycorrhizal and mycorrhizal olive plants inoculated or not with V. dahliae.
Mycorrhizal intensity (%) Arbuscular colonization (%) Vesicular colonization (%)
Control 33.00 ± 4.00c 12.00 ± 1.00c 7.00 ± 1.00b
Rhizolive consortium 71.00 ± 1.80b 46.00 ± 0.92b 37.00 ± 2.40a
V. dahliae 33.00 ± 3.12c 22.00 ± 0.94c 8.00 ± 0.76b
Rhizolive consortium þ 82.00 ± 2.84a 74.00 ± 1.13a 34.00 ± 1.26a
V. dahliae
p-value < 0.0001 < 0.0001 < 0.0001
F 141.622 162.806 138.290
Means values (n ¼ 4) ± SD were compared between treatments using Tukey-HSD test at 5%. Lower-case
letters within the same column do not differ significantly, P < 0.001.

Results
Mycorrhizal root colonization of olive plants
One year after inoculation with “Rhizolive consortium”, high mycorrhizal
intensity percentage (82%) (with a two-and-a-half-fold increase
compared to control) was observed in infected olive plants with
V. dahliae (Table 1). The highest percentage of arbuscular colonization
was observed in mycorrhizal-infected olive plants (RCV) (74%), whereas
that of vesicles colonization was recorded in both RCV and RC
with 34% and 37%, respectively. Compared to control plants, the roots
of RCV treatment exhibited a significant increase in arbuscular and
vesicular colonization rate of about 6 and 5 times, respectively. Under all
treatments, the percentage of arbuscular colonization was higher than
that of vesicles (Table 1).

Plant growth and biomass traits in olive plants

The results of growth and biomass traits measured in “Picholine
Marocaine” cultivar are presented in Figures 1–5. The evolution of shoot
height and number of leaves (Figures 1 and 2), during the experiment
period, was almost similar with a quantitative difference between mycor-
rhizal and non-mycorrhizal olive plants. After infection with V. dahliae,
we observed a positive effect of “Rhizolive consortium” on these both
growth traits, which remains maintained during 9 months. At the end of
the experiment and in comparison with infected plants (Vd), the olive
plants grown under RC and RCV treatments showed an increase of 17%
and 14%, respectively, for shoot height and 46% and 34%, respectively,
for number of leaves. Regarding branch number (Figure 3), the RC treat-
ment enhanced significantly branch growth compared to other treat-
ments including control plants. The highest branch number values were
recorded at 12 months (increase of about 42% in RC inoculated plants
compared to RCV treatment). However, RCV and Vd treatments did not
differ statistically, during all the experimental period. Moreover, after
 ARCHIVES OF PHYTOPATHOLOGY AND PLANT PROTECTION 679

Figure 1. Kinetic of shoot height (cm) under different treatments. Olive plants were
inoculated three months with Rhizolive consortium then infected with V. dahliae. Mean
values (n ¼ 10) ± SD.

Figure 2. Kinetic of number of leaves under different treatments. Olive plants were
inoculated three months with Rhizolive consortium then infected with V. dahliae. Mean
values (n ¼ 10) ± SD.

infection, RCV and RC inoculated plants maintained higher root length

in comparison with other treatments, especially at 9 and 12 months
(Figure 4). At the end of experimental period and compared to Vd ino-
culated plants, both RCV and RC treatments exhibited a significant
increase of root length by 19% and 17%, respectively. “Rhizolive con-
sortium” was most effective in increasing root length compared to con-
trol plants. As for shoot and root dry weights (SDW and RDW),
“Rhizolive consortium” significantly increased the dry biomass in olive
plants (Figure 5(A, B)). Indeed, inoculation only by RC exhibited higher
values of SDW and RDW, throughout the experience period, in compari-
son to all treatment including control plants. On the other hand, the
680 H. BOUTAJ ET AL.

Figure 3. Kinetic of Branch number under different treatments. Olive plants were inoculated
three months with Rhizolive consortium then infected with V. dahliae. Means values (n ¼ 5)
± SD were compared between treatments using Tukey-HSD test at 5%. Same letters do not
differ significantly at 5% level.

Figure 4. Kinetic of root length under different treatments. Olive plants were inoculated
three months with Rhizolive consortium then infected with V. dahliae. Means values (n ¼ 5)
± SD were compared between treatments using Tukey-HSD test at 5%. Same letters do not
differ significantly at 5% level.

infection with V. dahliae significantly decreased dry shoot and root bio-
mass of olive plants compared to uninfected ones (control plants).
However, the inoculation of “Rhizolive consortium” during transplant-
ation reduced the negative effect of V. dahliae and significantly improved
the dry biomass, especially during three months after infection.
 ARCHIVES OF PHYTOPATHOLOGY AND PLANT PROTECTION 681

Figure 5. Kinetic of shoot (A) and root dry weight (B) under different treatments. Olive
plants were inoculated three months with Rhizolive consortium then infected with V. dahliae.
Means values (n ¼ 5) ± SD were compared between treatments using Tukey-HSD test at 5%.
Same letters do not differ significantly at 5% level.

Nutrient elements accumulation

Determination of the main nutrients in the shoots and roots of olive
plants revealed that RCV treatment significantly increased the four
nutrients studied K, Ca, Na, and available P compared to all other treat-
ments (Figure 6). After infection, these nutrients increased over time
under RCV treatment to reach high values at the end of the experimental
period, in both shoots and roots. The accumulation of Ca, K and avail-
able P in shoots was more enhanced than in roots of olive plants, while
Na content did not differ in both parts. At 9 months of infection (12th
month of the experience period) and in the respect of comparison
between infected plants (Vd) and mycorrhizal-infected olive plants
682 H. BOUTAJ ET AL.

Figure 6. Nutrients content (%) in the shoots and roots under different treatments. Olive
plants were inoculated three months with Rhizolive consortium then infected with V. dahliae.
Means values (n ¼ 5) ± SD were compared between treatments using Tukey-HSD test at 5%.
Same letters do not differ significantly at 5% level.

(RCV), we recorded an increase in RCV treatment by 23% and 58% for

K, 33% and 21% for Ca, 31% and 44% for Na and 65% and 33% for
available P in the shoots and roots, respectively.

Discussion
“Picholine Marocaine” cultivar is one of the most widely olive cultivars
grown in Morocco and developed in different geographic areas. Olive
 ARCHIVES OF PHYTOPATHOLOGY AND PLANT PROTECTION 683

tree exhibits great adaptability to adverse soil conditions and is treated as

a model plant system for plant-mycorrhizal interactions (Kapulnik et al.
2010). Olive tree growing in symbiosis with AMF can alleviate the nega-
tive effects induced by fungal phytopathogens and help to enhance its
productivity and sustainable use (Kapulnik et al. 2010; Tekaya et al.
2017). However, V. dahliae is one such soil-borne pathogens studied for
causing Verticillium wilt disease in olive trees. Our study highlighted the
positive effect of AMF consortium on growth and nutrients accumula-
tion in olive plants infected with V. dahliae. In previous study, we dem-
onstrated and confirmed, under the same conditions and treatments, the
presence of V. dahliae through its re-isolation and its quantification in
roots and stems of olive tree (“Picholine Marocaine” cultivar) (Boutaj
et al. 2020). The use of “Rhizolive consortium” ensured a high percent-
age of mycorrhizal intensity in mycorrhizal-pathogen-inoculated olive
plants (RCV), with a dominance of arbuscules. This important mycorrh-
ization, taking into account the colonization rate and diversity of
“Rhizolive consortium”, significantly enhanced growth development of
“Picholine Marocaine” cultivar. Hu et al. (2010) demonstrated that a
mixture of several AMF species could significantly suppress cucumber
Fusarium wilt in greenhouse agroecosystems, compared to a single AMF
species. Indeed, an inoculum rich in AMF diversity is more agriculturally
relevant and beneficial. Several studies reported that the intimate inter-
action between AMF and their host plants is able to promote plant
growth as well as to suppress pathogens (Berg 2009; Shokri and Maadi
2009). Our results showed that “Rhizolive consortium” significantly
improved vegetative growth traits (branch and leaves number, dry weight
and plant height) and increased root development (dry root weight and
root length) in mycorrhizal olive plants. Improving the studied growth
and biomass traits in RCV treatment could be attributed to the reduction
in the deleterious effect of V. dahliae on the development of both shoots
and roots of olive plants. Also, the inoculation with “Rhizolive con-
sortium” before infection seems to nullify or attenuate the detrimental
effects of V. dahliae on olive plant growth (Garmendia et al. 2004;
Boutaj et al. 2019). Corroborating our results, Kapulnik et al. (2010)
reported that AMF enhanced vegetative growth and development in dif-
ferent infected olive cultivars with V. dahliae. The deleterious effect of V.
dahliae on pepper growth and yield has been reported to be reduced in
mycorrhizal pepper (Garmendia et al. 2004). Previous studies reported
that mycorrhizal colonization improves plant rooting and establishment,
enhance vegetative growth, accelerate budding and flowering (Smith and
Read 2008) and induce plant resistance to environmental adversities
(Jung et al. 2012). In addition, we demonstrated that “Rhizolive
684 H. BOUTAJ ET AL.

consortium” promoted olive trees ability to increase nutrients accumula-

tion. It is well known that AMF symbiosis plays a major role in ecosys-
tems through its hyphae, which is able to facilitate nutrients cycling by
expanding beyond the rhizosphere to take up and provide plants with
essential nutrients. Indeed, an improved nutrients acquisition is involved
in direct growth promotion. Mycorrhizal olive plants exhibited high
accumulation of K, Ca, Na, and available P in infected olive plants (Vd).
The mycorrhizal olive plants showed a relationship between the increased
accumulation of studied nutrients and vegetative growth. The increased cap-
acity for nutrients uptake by AMF may allow host plants to become more
vigorous and enhance resistance or tolerance to pathogen attack (Azc
on-
Aguilar et al. 2002; Berg 2009). Plant-associated AMF can supply macronu-
trients and micronutrients from the soil which are essential for growth and
development. According to our study, the inoculation with “Rhizolive con-
sortium” provides an alternative pathway - AMF for the uptake of essential
nutrients, especially P, which is one of the most difficult nutrients for plants
to acquire in a phosphorus-poor soil, as in our case. Plants have opted for
the AMF symbiosis strategy to increase the P uptake capacity or its availabil-
ity in soil (Smith et al. 2011). The increase in the content of available P in
RCV treatment may be due to the increase of microbial biomass in mycor-
rhizal rhizosphere soil. Microbial biomass, especially actinomycetes (Mechri
et al. 2014), increases when mycorrhizal fungi are added to the soil, increas-
ing H2CO3 in the soil solution and this acid can dissolve primary P-contain-
ing minerals, thereby increasing P availability (Gholamhoseini et al. 2013).
By excretion of chelating substances that form stable complexes with P, acti-
nomycetes can increase phosphate solubilization in rhizosphere of mycor-
rhizal olive trees (Mechri et al. 2014). The improvement of P uptake could
also be facilitated by the extensive hyphae of AMF, allowing mycorrhizal
olive plants to explore more soil volume than non-mycorrhizal ones. In add-
ition, as our results showed, other studies revealed that mycorrhizal coloniza-
tion can improve K (Gholamhoseini et al. 2013) and Ca absorption by
plants (Liu et al. 2002). In olive tree, Tekaya et al. (2017) reported a signifi-
cant increase in P, K, Ca, Zn, Mn, Fe and Mg contents in the leaves of
mycorrhizal plants. P, K and Ca are essential plant nutrients and play key
roles in plant metabolism (Gholamhoseini et al. 2013; Tekaya et al. 2017),
while Na can be beneficial to plants in many conditions, as a functional
nutrient, particularly when K is deficient (Maathuis 2014). Regarding K, Ca
and Na in RCV treatment, the increased concentrations may be explained
by direct enhance uptake through the presence of widespread extraradical
hyphae networks, which helps to expand the root surface absorption area
and therefore shortens the distance that the nutrients must travel in the soil
before reaching the roots as well as the nutrients that the roots cannot reach
 ARCHIVES OF PHYTOPATHOLOGY AND PLANT PROTECTION 685

(Gholamhoseini et al. 2013; Tekaya et al. 2017). Furthermore, a synergistic

relationship between P and K has been reported by Cardoso et al. (2006).
Increased P availability due to mycorrhizal fungal activity can be responsible
of increased K concentration (Gholamhoseini et al. 2013). Besides decreasing
the abundance of the pathogen in both stems and roots, the inoculation by
“Rhizolive consortium” increased the plants olive biomass, revealing a nutri-
tional mycorrhizal effect and suggesting a direct interaction between the
mutualist and the pathogen. Similar results have been reported in other
plant species, including peanut (Abdalla and Abdel-Fattah 2000), pepper
(Garmendia et al. 2004) and cowpea (Abdel-Fattah and Shabana 2002).
Colonization of root by AMF enhances nutrients uptake and translocation
before pathogen invades, which reduces disease development on plants
(Feddermann et al. 2010; Olowe et al. 2018). Knowing that V. dahliae had
some negative impact on growth and productivity of crops, olive plants
colonized with “Rhizolive consortium” for three months were protected
against the phytopathogenic fungus (V. dahliae) (Boutaj et al. 2020). This
can be due to both the effect of improved nutritional status and the direct
interaction between “Rhizolive consortium” and V. dahliae, possibly compe-
tition for infection sites. Thus, the effectiveness of prior mycorrhization may
be related to colonization and protection of the root system by AMF prior
to penetration by the pathogen. Ismail and Hijri (2012) have shown that
potato inoculation by G. irregulare significantly reduces the negative effects
of Fusarium sambucinum on biomass and tuber production. These authors
attribute these effects to the role of AMF in the positive regulation of the
expression of the majority of the defense genes (ChtA3, gluB, CEVI16 and
PR-1) in the host plant roots. It seems that AMF can induce some structural
and physiological changes in plants in response to pathogen attacks. Positive
mycorrhizal growth response (MGR) arises largely from increased uptake of
growth-limiting nutrients via AMF. This MGR is influenced and depends on
many factors at scales from molecular (e.g., transporter gene expression) to
ecological (e.g., plant and fungal composition, density, competition and
environmental conditions) (Smith and Smith 2011).

Conclusion
Our results clearly illustrate that “Rhizolive consortium” (combination of
26 AMF species) is more efficient against V. dahliae and better supports
nutritional uptake and growth of olive plants. The application of this
AMF consortium induced olive tolerance to Verticillium wilt, by main-
taining development of both stems and roots and uptake from soil of
limiting and regulating growth nutrients (P, K and Ca). The present
study demonstrated that mycorrhization of olive plants (“Picholine
686 H. BOUTAJ ET AL.

Marocaine” cultivar), during its transplantation (for 3 months), appears

to improve its tolerance to V. dahliae and be more beneficial under
Mediterranean conditions, where soils are naturally deficiency in
nutrients. The improvement of the nutrient status of olive tree is an
important factor in determining its growth and yield. Therefore, the use
of selective AMF consortium in olive-growing systems could be an effect-
ive alternative for chemical fertilizers.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was supported by the project ArimNet “Pestolive” and the project “Rhizolive”
funded by the “Acad
emie Hassan II des Sciences et Techniques”, Morocco.

ORCID
Hanane Boutaj http://orcid.org/0000-0001-7201-9489

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