Biocontrol Science and Technology

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Arbuscular mycorrhizal fungi against the false

root-knot nematode activity in Capsicum annuum:
physiological responses in plants

Valeria Fernanda Bernardo , Sebastian Andrés Garita , Maria Cecilia Arango ,

Juan Ignacio Ripodas , Mario Carlos Nazareno Saparrat & Marcela Fabiana
Ruscitti

To cite this article: Valeria Fernanda Bernardo , Sebastian Andrés Garita , Maria Cecilia
Arango , Juan Ignacio Ripodas , Mario Carlos Nazareno Saparrat & Marcela Fabiana Ruscitti
(2020): Arbuscular mycorrhizal fungi against the false root-knot nematode activity in Capsicum
annuum: physiological responses in plants, Biocontrol Science and Technology, DOI:
10.1080/09583157.2020.1833304

To link to this article: https://doi.org/10.1080/09583157.2020.1833304

Published online: 21 Oct 2020.

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BIOCONTROL SCIENCE AND TECHNOLOGY
https://doi.org/10.1080/09583157.2020.1833304

RESEARCH ARTICLE

Arbuscular mycorrhizal fungi against the false root-knot

nematode activity in Capsicum annuum: physiological
responses in plants
Valeria Fernanda Bernardoa,b, Sebastian Andrés Garitaa, Maria Cecilia Arangoa, Juan
Ignacio Ripodasa, Mario Carlos Nazareno Saparrata and Marcela Fabiana Ruscittia,c
a
Instituto de Fisiología Vegetal (INFIVE-CONICET-UNLP), La Plata, Buenos Aires, Argentina; bCICBA –
Comisión de Investigaciones Científicas, La Plata, Buenos Aires, Argentina; cDepartamento de Ciencias
Básicas y Experimentales - UNNOBA, Junín, Buenos Aires, Argentina

ABSTRACT ARTICLE HISTORY

Nacobbus aberrans affects plants growth and development, and Received 14 February 2020
decreases the production of numerous horticultural crops. Several Accepted 3 October 2020
investigations have shown that symbiosis with arbuscular
KEYWORDS
mycorrhizae can confer tolerance to pathogens through different Biotic stress; Mycorrhizal
action mechanisms, such as nutrient space competition and plant symbiosis; Nacobbus
resistance induction. The aim of this study was to analyse the aberrans; Pepper; Plant
potential of three arbuscular mycorrhizal fungi (AMF): Rhizophagus protection
intraradices B1, Rhizophagus intraradices A2 and Funneliformis
mosseae in the control of N. aberrans, and to evaluate the
physiological and biochemical responses to inoculation in pepper
plants. The experiment was conducted under controlled
conditions. Mycorrhization percentages, chlorophyll, carotenes,
soluble proteins, proline, phenolic compounds, total and reducing
sugar content, relative conductivity of root cell membranes and
final nematode population were determined. The mycorrhization
percentages were higher in plants parasitised by N. aberrans than
in those not parasitised. The final population of nematodes was
reduced in the presence of mycorrhizae. In the absence of AMF,
parasitism by N. aberrans caused a decrease in the soluble proteins
and chlorophyll content. The loss of functionality of infected roots
in the plants induced water stress, reflected by the accumulation
of proline, sugar and increased the relative conductivity of cell
membranes. At the same time, as a product of pathogenesis,
phenolic compounds accumulated. Inoculation with AMF
decreased the proline, sugars, phenolic compounds content and
relative conductivity. These results show the potential for the use
of AMF in pepper production to reduce the population of
N. aberrans, being the strain R. intraradices B1 as the most
promising candidate.

Introduction
The false root-knot nematode Nacobbus aberrans (Thorne & Allen) is a sedentary endo-
parasite nematode responsible for causing significant losses in numerous crops

CONTACT Valeria Fernanda Bernardo valebernardo35@gmail.com Instituto de Fisiología Vegetal (INFIVE-

CONICET-UNLP), Diagonal 113 n°495, La Plata, CP: 1900 Buenos Aires, Argentina
© 2020 Informa UK Limited, trading as Taylor & Francis Group
2 V. F. BERNARDO ET AL.

(Manzanilla-López et al., 2002). It is a native organism of South America that has spread to
other geographical areas. It has a broad range of hosts, including 84 species of plants
belonging to several families, with well-known representatives such as tomato (Solanum
lycopersicum L.), pepper (Capsicum annuum L.), potato (Solanum tuberosum L.) and
sugar beet (Beta vulgaris L.) (Manzanilla-López, 2010). Plants affected by N. aberrans
present galls arranged individually in a rosary-like pattern on their roots (Manzanilla-
López et al., 2002). At cellular level, this nematode generates hyperplasia and hypertrophy
responses (Hewezi & Baum, 2013). These alterations lead to the rupture of the xylem tissue
and the phloem. As a consequence, the flow of substances through the conduction tissues is
reduced and the plants suffer wilt, deficiency in the absorption of N, P, K, Ca and Mg, low
stomatic conductance and CO2 assimilation, decrease in growth, yield losses, and even
plant death (Castillo & Marbán-Mendoza, 1984; Inserra et al., 1985). In Argentina,
N. aberrans is the main pest affecting tomato and pepper greenhouse production, possibly
due to its polyphagia, high reproductive potential, and notable adaptive capacity (Doucet
& Lax, 2005). A report by INTA-Mar del Plata indicates that the damage to undercover
tomato and pepper plants reaches 40% (Adlercreutz et al., 2007). Although there are no
official data indicating economic losses in the study area, for a few years it has been the
main phytosanitary adversity (Nico, 2014). Although there are several strategies for con-
trolling this nematode sort, methyl bromide (CH3Br) is the most widely used soil disinfec-
tant (Adlercreutz et al., 2007). However, in 2015, when it was found that this gas affects the
ozone layer, it was withdrawn from the market. This, in addition to the emergence of
certain resistance and social pressure for access to healthier food (Gortari & Hours,
2016) and care for the environment, has led Argentina to search for sustainable alternatives
to methyl bromide, highlighting the importance of redesigning integrated management
programs. A large amount of research has shown that some soil microorganisms have
the potential to act as control agents of phytoparasitic nematodes, both directly and
indirectly, such is the case of arbuscular mycorrhizal fungi (AMF) (Schouteden et al.,
2015). It is known that these mycorrhizae can confer tolerance against pathogens and
pests through different action mechanisms, including mycoparasitism, enzymatic lysis,
antibiosis, competition for space or nutrients, and induction of plant resistance (Baum
et al., 2015). Information about the effect of the interaction between N. aberrans and
AMF in plant physiology and biochemistry is very scarce. Most of the researches evaluated
AMF interaction with root-knot nematode Meloidogyne or Pratylenchus but not with
N. aberrans itself (Bañuelos et al., 2012; Frew et al., 2018; Korayem et al., 2012; Sharma
& Sharma, 2017). Furthermore, little information exists regarding N. aberrans and it is
about its action in tomato plants, not in pepper plants. The mechanisms activated in the
plant-AMF-nematode specific interaction are variable and depend on the species of
fungus, host plant and nematode, and even the results are ambiguous or contradictory.
The aim of this work was to analyse the potential of AMF for the control of N. aberrans,
and evaluate the physiological and biochemical response to inoculation in pepper plants
grown in the presence of the nematode in the soil.

Material and methods

The experiments were conducted at INFIVE (Plant Physiology Institute), located in the
City of La Plata (34° SL, 54′ WL) (Argentina). They were carried out in a greenhouse with
 BIOCONTROL SCIENCE AND TECHNOLOGY 3

controlled temperature between 20°C and 28°C, natural photoperiod and periodic irriga-
tion. Pepper plants ‘Paco f1’ cultivar were grown in the greenhouse between August and
December 2017. Seeds of pepper were sown in trays of 72 cells, previously filled with a
substrate composed of a perlite and vermiculite (2:1 v/v) mixture. The substrate had
been tyndallized 60 min at 100°C, for 3 consecutive days, to eliminate native AMF pro-
pagules and pathogenic microorganisms. AMF inoculum, consisting of a mixture of soil,
spores (60 spores·g−1 inoculum), mycelium and Trifolium repens root fragments colo-
nised by each fungus (90% of mycorrhization) was added to the substrate (20% v/v).
The same amount of sterilised inoculum was added to non-inoculated pots to reproduce
the same soil conditions. The AMF inoculum used was Funneliformis mosseae (Schenck
and Smith) isolate GA1 (FM) (Colección Instituto Spegazzini, UNLP [National Univer-
sity of La Plata, for its Spanish acronym], City of La Plata, Argentina) and Rhizophagus
intraradices (Nicolson and Gerdemann) Gerdemann and Trappe isolates B1 (RI B1) and
A2 (RI A2) (In Vitro Glomeromycota Banks, BGIV, Buenos Aires, Argentina). They were
multiplied through culture with Trifolium repens L. for four months in a semi-controlled
growth chamber. Thirty-five days after sowing, five plants per treatment were removed to
confirm inoculation. Mycorrhization percentage was estimated by observation under an
optical microscope after clarifying the roots in KOH 10% and staining them with Trypan
blue 5% (Phillips & Hayman, 1970). When root colonisation was approximately 50%,
pepper plants were transplanted to pots containing 10 kg of sterilised substrate composed
of a mixture of soil (Vertic Argiudol, pH 5.5, 12 mg kg−1 total P, 3.5% organic matter, 2%
total C and 0.24% total N) and sand (2:1 v/v). Three days after the transplant, the pots
were inoculated with 5000 eggs of the phytoparasitic nematode N. aberrans. The eggs
for inoculation were obtained from tomato plants grown in a sterile substrate in a
growth chamber, which were inoculated with a population of N. aberrans from the hor-
ticultural belt of La Plata according to Coolen (1979). The protocol consists in processing
the roots, centrifugation-flotation and then counting under an optical microscope. The
suspension obtained was then diluted to achieve a concentration of 1000 eggs per ml.
For the inoculation, three holes of approximately 2 cm depth were opened next to the
seedlings with the aid of a glass rod. Using an automatic pipette, 5 ml of the egg suspen-
sion were deposited in each hole. Then, the holes were filled back with substrate.
The treatments were:

(a) Pepper plants without N. aberrans (N0): no mycorrhizal inoculation (NI), inocu-
lation with F. mosseae (FM), inoculation with R. intraradices B1 (RI B1), and inocu-
lation with R. intraradices A2 (RI A2)

(b) Pepper plants with N. aberrans (N1): (NI), (FM), (RI B1), and (RI A2)

Ninety days after inoculation, all the plants were removed from the pots and final
nematode population on root (Coolen, 1979) and nematode reproductive factor (RF),
were determined according to Oostenbrink (1966). The RF was calculated from the
expression RF = Fp·Pi−1, where: Fp = final population (number of eggs and larvae
extracted from the roots) and Pi = number of inoculated eggs. Plants with RF = 0 were
considered as immune; with RF < 1 were considered as resistant, and with RF > 1
were considered as susceptible (Oostenbrink, 1966). Root colonisation by AMF and
4 V. F. BERNARDO ET AL.

percentage of arbuscules was estimated according to Phillips and Hayman (1970), and
the viability of the fungal structures inside the root using the succinate dehydrogenase
method based on the percentage of mycorrhization (Smith & Gianinazzi-Pearson,
1990). The percentage of viable hyphae was determined by optical microscopic obser-
vation, using the grid method of Giovanetti and Mosse (1980). The carotenoids and
chlorophyll contents were determined from a 0.5 cm diameter leaf disk. N, N-
Dimethylformamide was used as the extraction solvent, and the absorbance of the sol-
ution was determined at 647, 664 and 480 nm. The calculation of the pigment content
was carried out according to Wellburn (1994). Soluble protein content was determined
from 100 mg of leaf and root fresh weight. The absorbance reading was taken at a wave-
length of 595 nm. The calculation of the protein concentration was carried out using a
standard curve prepared with different concentrations of bovine serum albumin (BSA)
(Sigma Chemical Co. Argentine) according to Bradford (1976). Several parameters,
potentially stress indicators in plants, were evaluated on leaf and root for each treat-
ment, such as the proline content, which was measured from 100 mg of leaf and
root fresh weight. The absorbance reading was taken at a wavelength of 520 nm, and
proline content per unit fresh weight (FW) was calculated as: μmol proline·g−1 FW
= [(μg proline/ml × ml toluene)/115.5 μg/μmol]/[(g FW)/5] according to Bates et al.
(1973). Total and reducing sugar content was measured from 500 mg of fresh material
according to the Somogy method (Cronin & Smith, 1979). For total sugar, acid
hydrolysis was conducted with 0.1 N HCL and a water bath at 100°C, and the sub-
sequent reaction obtained with cupric reactive. The absorbance was measured at a
wavelength of 520 nm. Total phenolic compounds were quantified by a colorimetric
method using the Folin–Ciocalteu reagent. Extraction was made from 500 mg of
fresh material with 96% ethanol. An aliquot of this sample was diluted with distilled
water and mixed with 1 N Folin–Ciocalteu reagent and 2% (w/v) Na2CO3 in 0.1 N
NaOH. The mixture was incubated for 90 min at 25°C and the absorbance was
measured at 760 nm. A standard curve was built using different concentrations of
gallic acid (Singleton & Rossi, 1965). The relative conductivity of cell membranes
was determined according to Lutts et al. (1996), from 200 mg of fresh material from
leaves and roots. Electrical conductivity (dS m−1) was measured using a Jenco 3173
conductivity meter in order to determine the stability of cell membranes.
The study was carried out in a completely randomised design, in a 4 × 2 factorial
scheme of four mycorrhizal conditions (NI, FM, RI B1, RI A2) and two soil infection con-
ditions (N0, N1). Ten replications were performed for each treatment, each one compris-
ing one plant. Normality and homoscedasticity were verified using the Shapiro–Wilk and
Lemens tests, respectively. The results were subjected to analysis of variance (ANOVA)
and mean values were compared by LSD at a significance level of 0.05. For the statistical
analysis, mycorrhizal inoculation percentage and viability percentage values were arcsine
transformed to improve homogeneity. The program used for the statistical analysis was
SISVAR.

Results
RI B1 was the only treatment that showed significant differences in %M between N0 and
N1. In the presence of nematodes, RI B1 presented the highest %M, with significant
 BIOCONTROL SCIENCE AND TECHNOLOGY 5

Table 1. Percentage of mycorrhization (%M), percentage of arbuscules (%A) and mycorrhizal viability
(%SDH) of Capsicum annuum plants inoculated with arbuscular mycorrhizal fungi (AMF): Funneliformis
mosseae (FM), Rhizophagus intraradices B1 (RI B1) and A2 (RI A2); in absence (N0) and presence (N1) of
Nacobbus aberrans in the soil.
N. aberrans AMF %M %A % SDH
N0 FM 34.53 Ab 11.55 Aa 19.97 Aa
RI B1 31.06 Ab 11.58 Ab 9.95 Bb
RI A2 37.70 Ab 12,60 Aa 20.25 Aa
N1 FM 37.80 Bb 9,83 Bb 18.50 Aa
RI B1 47.70 Aa 13,95 Aa 9.00 Bb
RI A2 38.80 Bb 10,09 Bb 7.25 Bb
Note: Different lowercase letters indicate significant differences between the presence and absence of N. aberrans for the
same treatment of AMF inoculation; different capital letters indicate significant differences between AMF for the same
treatment of N. aberrans infection (p < 0.05).

differences with respect to the other strains tested. In the absence of nematodes there
were no significant differences between the AMF (Table 1).
The %A was significantly higher in RI B1 in N1 compared to N0. In the presence of
nematodes, RI B1 presented the highest %A, with significant differences with respect to
the other strains tested. The %A was significantly reduced in FM and RI A2 in the pres-
ence of N. aberrans compared to that of obtained without the nematode. In the absence of
nematodes, there were no significant differences between the AMF (Table 1).
On the other hand, the viability of fungal structures was only significantly reduced in
RI A2 in the presence of N. aberrans, which caused a decrease of 64.2% compared to that
of obtained without the nematode. FM presented high viability in the presence and
absence of N. aberrans, compared to the other inoculums, while for RI B1 the viability
was significantly lower, both in N0 and N1 (Table 1).
With the inoculation of each AMF a significant reduction was observed both in the
number of nematode eggs (expressed as total eggs and number of eggs per gram of
fresh root) and in the reproductive factor (RF). However, there were no significant differ-
ences regarding the type of isolate used (Table 2).
In the plants parasitised by N. aberrans the contents of chlorophyll, carotenoids and
soluble proteins were significantly lower than in non-infected ones (Table 3). Conversely,
no significant differences were observed between AMF treatments for the same infection
condition with the nematode. In N0, the FM, RI B1 and RI A2 isolates caused a differential
effect in leaf protein content relative to that from non-mycorrhizal treatment. In N1, only
RI A2 presented protein content values significantly lower than the other treatments.
Accumulation of plant proline, an indicator of the stress caused by N. aberrans, was
higher in leaves than in roots (Figure 1). In leaves, proline content was significantly

Table 2. Reproductive factor (RF) of Nacobbus aberrans, in Capsicum annuum plants not inoculated
(NI) or inoculated with arbuscular mycorrhizal fungi (AMF): Funneliformis mosseae (FM),
Rhizophagus intraradices B1 (RI B1) and A2 (RI A2).
AMF RF
NI 36.40 a
FM 4.85 b
RI B1 2.57 b
RI A2 4.40 b
Note: Different letters indicate significant differences between AMF (p < 0.05).
6 V. F. BERNARDO ET AL.

Table 3. Chlorophyll (Chl), carotenoids (Ca) and soluble proteins (Pr) content in leaves of Capsicum
annuum plants not inoculated (NI) or inoculated with arbuscular mycorrhizal fungi (AMF):
Funneliformis mosseae (FM), Rhizophagus intraradices B1 (RI B1) and A2 (RI A2); in absence (N0) and
presence (N1) of Nacobbus aberrans in the soil.
N. aberrans AMF Chl (µl·cm−2) Ca (µl·cm−2) Pr (µg·mg−1 FW)
N0 NI 51.97 Aa 8.66 Aa 7.96 Aa
FM 61.98 Aa 9.73 Aa 5.05 Ba
RI B1 48.88 Aa 7.75 Aa 4.86 Ba
RI A2 55.07 Aa 9.16 Aa 5.27 Ba

N1 NI 15.32 Ab 4.40 Ab 3.78 Ab

FM 17.79 Ab 4.35 Ab 3.65 ABb
RI B1 20.09 Ab 5.26 Ab 3.33 ABb
RI A2 16.33 Ab 4.59 Ab 3.02 Bb
Note: Different lowercase letters indicate significant differences between the presence and absence of N. aberrans for the
same treatment of AMF inoculation; different capital letters indicate significant differences between AMF for the same
treatment of N. aberrans infection (p < 0.05).

lower in N0 than in N1. RI B1 was the only tested fungus that kept basal proline content
in the plants infected with N. aberrans. Proline content in non-mycorrhizal plants
infected with N. aberrans was lower than in plants inoculated with FM. In N0 no signifi-
cant differences were observed between the tested fungi for leaves (Figure 1(a)). In roots,
in N0 no significant differences were observed between the different fungi used. Between
N0 and N1 significant differences were observed only in NI and FM. Plants inoculated
with the two strains of RI presented lower proline content in the presence of
N. aberrans (Figure 1(b)) than non-inoculated plants.
The presence of nematodes in soil significantly increased total and reducing sugar
contents in roots and leaves of the plants not inoculated with AMF. Since this was
more evident using leaf data, root data is not shown for this analysis. Total and reducing
sugar contents in leaves for the plants grown in soil in the presence of the nematode were

Figure 1. Proline content in leaves (a) and roots (b) of Capsicum annuum plants not inoculated (NI)
and inoculated with arbuscular mycorrhizal fungi (AMF): Funneliformis mosseae (FM), Rhizophagus
intraradices B1 (RI B1) and A2 (RI A2); in absence (N0) and presence (N1) Nacobbus aberrans in the soil.
Note: Different lowercase letters indicate significant differences between the presence and absence of N. aberrans for the
same treatment of AMF inoculation; different capital letters indicate significant differences between AMF for the same
treatment of N. aberrans infection (p < 0.05).
 BIOCONTROL SCIENCE AND TECHNOLOGY 7

Figure 2. Total (a) and reducing (b) sugars content in leaves of Capsicum annuum plants not inocu-
lated (NI) and inoculated with arbuscular mycorrhizal fungi (AMF): Funneliformis mosseae (FM), Rhizo-
phagus intraradices B1 (RI B1) and A2 (RI A2); in absence (N0) and presence (N1) of Nacobbus aberrans
in the soil.
Note: Different lowercase letters indicate significant differences between the presence and absence of N. aberrans for the
same treatment of AMF inoculation; different capital letters indicate significant differences between AMF for the same
treatment of N. aberrans infection (p < 0.05).

significantly lower when inoculated with AMF compared to those not mycorrhized. In
N0, the total sugar content did not present significant differences between the different
fungi tested (Figure 2(a,b)).
Phenolic compounds content was significantly higher in leaves than in roots. The
accumulation of phenolic compounds increased significantly the in plants NI parasitised
with the nematode, in comparison with those not parasitised, both in leaves and roots.
Mycorrhized plants presented significantly lower phenolic content in the presence of
N. aberrans, regardless of the fungal inoculum used. In N0, no significant differences
were observed between the AMF tested, both in leaves and roots (Figure 3(a,b)).
Relative conductivity in roots increased significantly in the presence of N. aberrans as a
result of damage to cell membranes caused by the entry and exit of juveniles. Inoculation
with two strains of the RI fungi showed significantly lower values regarding control
without inoculation with AMF and FM in the nematode presence. In the plants infected
with the nematode and inoculated with FM the relative conductivity was the highest
observed, reaching values up to 40%. On the other hand, it was also observed that in the
absence of infection with N. aberrans, plants inoculated with AMF had lower values for
this parameter compared to non-inoculated plants, so mycorrhizal fungi provided a pro-
tective effect to cell membranes (Figure 4). No significant differences between treatments
were observed in the leaves, therefore data is not shown.

Discussion
When AMF are used as tools in the management of biotic adversities, evaluations should
not only be considered as what happens to the pathogen population, but also the per-
formance of mycorrhizal inoculation against plant stress. Arbuscular mycorrhizal sym-
biosis can provide the plant with several benefits such as improving water absorption
(Barea et al., 2016), nutrients (Azcón-Aguilar & Barea, 2015), and providing hormones
8 V. F. BERNARDO ET AL.

Figure 3. Phenolic compounds content in leaves (a) and root (b) of Capsicum annuum plants not
inoculated (NI) and inoculated with arbuscular mycorrhizal fungi (AMF): Funneliformis mosseae
(FM), Rhizophagus intraradices B1 (RI B1) and A2 (RI A2); in absence (N0) and presence (N1) of Nacob-
bus aberrans in the soil.
Note: Different lowercase letters indicate significant differences between the presence and absence of N. aberrans for the
same treatment of AMF inoculation; different capital letters indicate significant differences between AMF for the same
treatment of N. aberrans infection (p < 0.05).

that stimulate plant growth (Ruiz-Lozano et al., 2016), improving their physiological
condition despite not modifying the final pathogen population. Another aspect to be
considered is the performance of the AMF against the nematode with which they
compete for the same establishment site: the root cortex. In this study, plants growing
in soil containing N. aberrans eggs presented a higher mycorrhization compared with
those growing in soil without nematodes. According to Azcón-Aguilar and Barea
(1997), high mycorrhization in the presence of phytoparasitic nematodes may be due
to compensation for the damage caused by nematodes in the roots, increasing the for-
mation of hyphae and other fungal structures.
Several works have reported that some AMF strains cause reductions in the nodular
nematodes final population, however there are reports in which this symbiosis has not
had satisfactory results (Odeyemi et al., 2010). Our results show that inoculation with

Figure 4. Relative conductivity (RC) of root membranes of Capsicum annuum plants not inoculated
(NI) and inoculated with arbuscular mycorrhizal fungi (AMF): Funneliformis mosseae (FM), Rhizophagus
intraradices B1 (RI B1) and A2 (RI A2); in absence (N0) and presence (N1) of Nacobbus aberrans in the
soil.
Note: Different lowercase letters indicate significant differences between the presence and absence of N. aberrans for the
same treatment of AMF inoculation; different capital letters indicate significant differences between AMF for the same
treatment of N. aberrans infection (p < 0.05).
 BIOCONTROL SCIENCE AND TECHNOLOGY 9

AMF at sowing time significantly reduced the final amount of N. aberrans eggs, showing a
bioprotective effect of AMF on the roots against the nematode by reducing the number of
galls and, ultimately, the final population of the nematode. This result is relevant since in
other investigations, where the same species of AMF was used, specific reductions in the
nematode population were not obtained (Alarcón et al., 2013; Cristóbal Alejo et al.,
2010). This highlights the need for further research evaluating AMF strains in symbiosis
with common regional crops against local pathogens. Early establishment of mycorrhizal
symbiosis is considered to delay the penetration of juveniles and/or act as a barrier against
nematodes. Some authors indicate that exudate production by AMF has an adverse effect
on nematodes preventing root penetration (Vos et al., 2012a). These differences might be
due to the fact that AMF inoculation during the sowing of the plants creates an advantage
in favour of the AMF for the occupation of the physical space and production of exudates,
improving root mycorrhization. In the analysis of different physiological responses of
pepper plants growing in the presence of N. aberrans to AMF inoculation, the content
of chlorophyll and soluble proteins in leaves decreased in the plants infected with nema-
todes, although there were no significant differences between mycorrhizae treatments. Al-
Yahya et al. (1998) showed that there was a reduction in the chlorophyll content in wheat
infected with Heterodera avenae, and in tomatoes infected with Meloydogine sp., together
with a reduction in nutritional status (López-Gómez et al., 2015). Alterations in the physi-
ology of plant cells, such as hyperplasia and hypertrophy as a response to nematode infec-
tion, have been recorded, which leads to the rupture of the xylem and phloem (Hewezi &
Baum, 2013). Consequently, the flow of substances through the conduction tissues is
reduced, and a systemic increase of substances is detected, which can be used as
‘markers’ of water stress (Castillo & Marbán-Mendoza, 1984; Inserra et al., 1985).
Under these limiting conditions, proline and sugar are accumulated since the production
of key enzymes in the synthesis pathway of osmolites is triggered as a response to water
stress (Masoudi-Sadaghiani et al., 2011). Ruscitti et al. (2015) showed how uninoculated
tomato plants were more affected by water stress than those inoculated with F. mosseae
and R. intraradices, as it is evidenced from their higher proline values, which coincides
with what was observed in this work. Since the accumulation of proline was lower in
leaves and roots of the plants inoculated with Rhizophagus intraradices B1 and in the pres-
ence of N. aberrans, which possibly suggests a lower stress condition, this strain seems to
have a higher potential for mitigating the effects of N. aberrans on pepper plants with
respect to the other fungi tested. The increase in sugar content can also be a consequence
of osmotic stress, caused either by water deficit or salinity (Yang & Guo, 2018). The
accumulation of total and reducing sugars decreased similarly both in the plants inocu-
lated with the three tested mycorrhizal fungi and in the presence of the nematode with
respect to the infected plants without inoculation. Therefore, the proline content proved
to be a better marker than the sugar level to select the better fungal strain. Phenolic com-
pounds play a key role in the mechanisms involved in plant-nematode interaction, given
that an increase in their content is associated with systemic resistance to migratory and
sedentary nematodes (Arias et al., 2009). These compounds accumulate at nematode local-
isation sites, and the content in plant organs not affected by infection can be increased
several times (systemic effect) as they are able to translocate through tissues mainly via
phloem (Arias et al., 2009). In this work, a lower accumulation of these compounds was
observed in mycorrhized plants growing in the presence of the nematode when compared
10 V. F. BERNARDO ET AL.

with non-mycorrhized ones. This is related to the ability of mycorrhizal fungi to increase
host tolerance to the attack of phytopathogens and pests mainly due to the maintenance of
root cell activity through arbuscular formation (Azcón-Aguilar & Barea, 1997). As a result
of these mechanisms, the absorption of water and nutrients and, consequently, the
normal functioning of plant physiological processes are ensured. Another stress par-
ameter analysed in this work was the relative conductivity of plant cell membranes,
which can be affected by various environmental factors and stresses. This relates to
the altering of cellular integrity and the retention capacity of intracellular substances,
which leads to an increase in relative conductivity of plant tissues due to the release of
electrolytes (Beltrano et al., 2013). In this study, the relative conductivity was higher
in non-mycorrhized plants infected with N. aberrans, which may result from injuries
caused by nematode juveniles and the formation of galls. Relative conductivity of root
cellular membranes was significantly lower for plants in the absence of the nematode
and for those infected with N. aberrans and inoculated with RI B1 and RI A2. This
response could be, at least in part, either due to early root colonisation by AMF,
which interfered with nematode penetration, or to the action of mycorrhizal exudates.
Reduction in the penetration of Meloidogyne incognita was found in mycorrhized
tomato plants, suggesting that mycorrhizal root exudates contributed to this process,
and had a negative effect on the nematode and its motility in the soil (Vos et al.,
2012b). Various authors have reported physiological and biochemical changes in mycor-
rhized plants infected with nematodes, such as production of phenols, sugars or phytoa-
lexins (Hajra et al., 2013; Morandi, 1996). This could explain the resistance or tolerance
conferred by different strains of mycorrhizae to infected plants (Udo et al., 2013). As the
results of this study suggest, pepper plants inoculated with AMF during sowing are more
tolerant to Nacobbus aberrans, which is reflected in a decrease of its population. While
this also suggests that AMF is an efficient biological tool for the control of phytoparasitic
nematodes in horticultural species such as pepper, especially for its production in green-
houses, our work provides further information about a promising ability of fungus
R. intraradices B1 against N. aberrans infection when compared to the other fungi
tested, as proline accumulation in plants reveals. This work demonstrates that mycorrhi-
zal association between pepper plants and tested strains not only reduces the nematode
population but also increases mineral nutrition and plant defense. Although in this work
the plants yield was not determined, it is expected that the benefit granted by the inocu-
lation will cause an increase in the size and quantity of fruits, enhancing the yield of the
pepper plants. Inoculation with AMF is a tool with proven efficiency as biocontroller and
mitigator of losses caused by N. aberrans that could be easily adopted by horticultural
producers.

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

Funding
This work was supported by the Universidad Nacional del Noroeste de la provincia de Buenos
Aires and Universidad Nacional de La Plata.
 BIOCONTROL SCIENCE AND TECHNOLOGY 11

ORCID
Mario Carlos Nazareno Saparrat http://orcid.org/0000-0001-7403-1713

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