Mycological Progress (2024) 23:3

https://doi.org/10.1007/s11557-023-01939-9

ORIGINAL ARTICLE

Comprehensive analysis of Nematode–Tomato Plant–Mycorrhizal

Fungus system for bio based product development
Sebastián Andrés Garita1 · Valeria Fernanda Bernardo1 · Matías González1 · María Cecilia Arango1 ·
Marcela Ruscitti1,2

Received: 18 April 2023 / Revised: 26 November 2023 / Accepted: 29 November 2023

© German Mycological Society and Springer-Verlag GmbH Germany, part of Springer Nature 2023

Abstract
Nacobbus aberrans is a plant parasitic nematode that causes significant economic losses in the American Continent and is
considered a quarantine pest in many countries. Some arbuscular mycorrhizal fungi have shown the ability to reduce the
population of this pathogen; however, most studies do not consider other relevant aspects that have to do with the crop or
the fungus. The purpose of this work was to select an isolated mycorrhizal fungus based on a comprehensive analysis of
the Nematode–Plant–Mycorrhizal Fungus system. The test was conducted on Solanum lycopersicum L. grown in pots. The
evaluated isolates were Funneliformis mosseae, Rhizoglomus intraradices A2, and Rhizoglomus intraradices B1. All the
isolates reduced the nematode population significantly; however, differences in the content of photosynthetic pigments,
soluble proteins, and osmoregulatory metabolites were identified. All of these impacted on the photosynthetic rates of the
different treatments. There were also differences in the growth of the fungi within the roots, and in the viability of the fungal
structures. The comprehensive analysis of the plant–nematode–fungus group allows us to conclude that the isolate with the
greatest capacity to compensate for the negative effect of parasitism, and with the greatest possibility of lasting as a biocon-
trol agent is Funneliformis mosseae.

Keywords Nacobbus aberrans · Biocontrol · Funneliformis mosseae · Mycorrhiza

Introduction which can cause yield losses of 50–90% in Latin America

(Manzanilla-López et al. 2008).
Nacobbus aberrans is a parasitic nematode of plants that The juvenile stages of this pathogen are vermiform, have
causes great economic losses in the horticultural crops of a length of 300–400 µm and can pierce the root tissue with
the American continent. The tomato, Solanum lycopersi- their stylets to feed. Young females enter the radicular cor-
cum L, belongs to the Solanaceae family and is the second tex, where they remain as sedentary endoparasites for ovi-
most important vegetable in the world due to its production position. At this stage, they cause hyperplasia and cellular
volume, cultivated area, and the technology and research hypertrophy giving rise to their feeding sites called syncytia
developed around it (FAO 2023). Like 84 other botanical (EPPO 2009). Syncytia cause a disorganization of conduc-
species, the tomato is susceptible to attack by N. aberrans tive tissues, altering the normal flow of water and nutrients
(Garita et al. 2019). The external manifestation of this pro-
cess is the presence of galls on roots. The control of this
nematode is traditionally carried out with highly toxic soil
Section Editor: Claus Baessler fumigants, leading to a constant search of biological tools
to mitigate this adversity.
* Sebastián Andrés Garita
sebastiangarita@gmail.com The symbiosis between mycorrhizal–arbuscular, in which
fungi of the phylum Glomeromycota are associated with
1
Instituto de Fisiología Vegetal, (UNLP–CONICET), most of the roots of higher plant species, has been widely
Diagonal 113, n° 495, (1900), La Plata, Buenos Aires, studied from different scientific approaches. In addition to
Argentina
intraradicular growth, the hyphae extend in the soil several
2
ECANA–UNNOBA, Av. Pte. Dr. Arturo Frondizi n° 2650 tens of centimeters thus expanding the depth and extension
(2900), Pergamino, Buenos Aires, Argentina

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of root exploration which determines various agronomic ben- The isolates of the genus Rhizoglomus come from the
efits. The increase in the volume of soil explored by hyphae “Bank of Glomeromycota in–vitro” (BGIV) Buenos Aires,
and the higher absorption rate per unit area puts mycorrhi- Argentina. And the isolate of the genus Funneliformis comes
zal plants at an advantage over non-mycorrhizal plants. The from the “Instituto Spegazzini” (LPS) La Plata, Argentina.
external mycelium exudes hydrophobic glycoproteins called After 35 days of planting, 5 tomato seedlings were
glomalin-related soil protein, highly stable carbon-rich com- removed from each tray; the roots were washed, and the
pounds that cause drastic increases in soil biological activity technique described by Phillips and Hayman (1970) was
and aggregate stabilization (Rillig 2004; Wang et al. 2021). applied to dye the roots. Preparations were then made with
Although mycorrhization does not cause visible modifica- the roots to confirm and quantify the level of mycorrhization
tions in the root structure, metabolic modifications occur that (Trouvelot et al. 1986).
change its relationship with the environment. The synthesis
of hydrolytic enzymes, such as chitinase and β–1,3–glucanase Transplant
(El–Khallal 2007), and enzymes associated with the synthesis
of phenolic compounds (Guillon et al. 2002) is modified. The At 40 days after sowing, the transplant was carried out to 10
above information about mycorrhizal symbiosis allows us to L pots, using tindalized soil as substrate (Vertic Argiudol,
explain the existence of innumerable investigations interested pH 5.5, 12 mg kg − 1 total P, 3.5% organic matter, 2% total
in evaluating how mycorrhized tomato plants can more easily C, and 0.24% total N). The sterilization was performed to
tolerate or overcome different stress situations: low tempera- eliminate the positive or negative effect of any other soil
tures, flooding, drought (Ruscitti et al. 2015); salinity (Abdel microorganism and to visualize exclusively the action of
Latef and Chaoxing 2011); heavy metal toxicity (Cavagnaro mycorrhizae against N. aberrans. The plants were placed in
et al. 2009); and extreme temperatures (Morales-Guevara a greenhouse with forced ventilation, the temperature during
et al. 2018). There also have been documented the benefits the test was maintained between 22 and 29 °C, natural light-
against fungal pathogens (Pérez Ortega et al. 2015); bacterial ing with average irradiance of 5.75 Kw.h ­m−2, and irrigation
(Abo-Elyousr et al. 2014; Tahat et al. 2012); chewing insects was carried out daily.
(Song et al. 2013); and nematodes (Garita et al. 2019).
This work proposes to evaluate isolates of arbuscular Inoculation with N. aberrans
mycorrhizae-forming fungi for the control of N. aberrans
with a comprehensive analysis, analyzing biochemical and The suspension with eggs and juveniles used as inoculum
physiological parameters of the host plant (Solanum lycoper- was obtained from greenhouse-maintained tomato plants
sicum L.), parameters linked to the population of the patho- whose roots host a monoxenic population of N. aberrans.
gen, and parameters of the mycorrhizae forming fungus. The extraction was carried out using the technique of shak-
ing the roots in a 0.5% NaClO solution (Hussey and Barker
1973) with subsequent purification and concentration by the
Materials and methods centrifugation–flotation technique (Coolen 1979). The day
after the transplant half of the plants were inoculated with
Plant material and inoculation with Arbuscular 5000 eggs and juveniles of N. aberrans, defining the differ-
mycorrhizal fungi (AMF) ent treatments.

To produce the seedlings of the test, tomato seeds var. plat- Non-mycorrhized plants (Control) without N. aberrans
ense were disinfested with NaOCl (10%) for 5 min and were Non-mycorrhized plants (Control) with N. aberrans
planted in 4 seedling trays of 72 cells each, with a capacity Mycorrhized plants with Funneliformis moseeae, without
of 5.5 mL each. The mixture used to complete the cells was N. aberrans
formed by autoclaved perlite, vermiculite, and mycorrhizae Mycorrhized plants with Funneliformis moseeae with N.
inoculum, in a ratio of 1:1:1. The inoculum contains one of aberrans
the following fungi: Funneliformis mosseae (Fm), Rhizo- Mycorrhized plants with Rhizoglomus intraradices A2
glomus intraradices B1 (Ri–B1), or Rhizoglomus intrara- without N. aberrans
dices A2 (Ri–A2), and autoclaved inoculum to be used in Mycorrhized plants with Rhizoglomus intraradices A2
the control treatment. The provision of nutrients was made with N. aberrans
using a complete nutrient Hoagland solution. The inocula Mycorrhized plants with Rhizoglomus intraradices B1
used consist of a mixture of perlite, vermiculite, and ground without N. aberrans
roots, which have a concentration of 100 spores ­g−1. Mycorrhized plants with Rhizoglomus intraradices B1
with N. aberrans

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Inoculation was carried out by making three holes in the protein concentration was carried out using a standard
substrate around the plant, depositing 1.5 mL of inoculum curve prepared with different concentrations of bovine
in each hole and then covering the holes with the same serum albumin (BSA, Sigma Chemical Co).
substrate. – Number of eggs housed in the roots. The entire root was
placed in a blender and the agitation extraction technique
Experimental design was applied. (Hussey and Barker 1973; Coolen 1979)
with subsequent purification and concentration by the
The test had a completely randomized experimental design centrifugation–flotation technique (Coolen 1979).
and the treatments a factorial arrangement, being two fac- – Number of mobile forms in soil (Coolen 1979). One hun-
tors: mycorrhization and infestation, with 4 and 2 levels, dred microliters of pot substrate was placed and 5 L of
respectively. Each of the 8 treatments consisted of 8 plants, water was added. It was shaken and the purification and
each of them constituting a repetition. concentration by the centrifugation–flotation technique
(Coolen 1979) was applied. The number of vermiforms
Statistical analysis counted was multiplied by the total volume of the pot.
– Final population of the nematode: Eggs housed in the
The data were analyzed by ANOVA, and the mean compari- root + Vermiforms in soil
son test was performed by Fisher’s LSD test using the SIS- – Reproduction factor: Final Population/Initial population
VAR program. For the statistical analysis of variables whose (Oostenbrink 1966).
results were expressed in percentage, these were previously – Malonyldialdehyde (MDA) content in leaf and root tissue
transformed by the arcsine function. (Heath and Packer 1968). Two hundred micrograms of
tissue was macerated with 1 mL of 0.1% trichloroacetic
Analyzed variables acid and centrifuged for 10 min at 10,000 g. The super-
natant was reacted with 1 mL of the reagent TCA–BHT–
At 90 days after the transplant, non-destructive physiologi- TBA (TCA 20%, thiobarbituric acid (TBA) 0.37% and
cal determinations were made: Net photosynthesis (IRGA: butylhydroxytoluene (BHT) 0.01 g) and the tubes were
CIRAS-2® model, PP Systems), stomatal conductance, and incubated for 30 min at 95 °C. After this period, they
stem diameter measurements. were placed in an ice bath to stop the reaction and centri-
At 100 days, the plants were removed and the leaves, root, fuged at 3000 g for 10 min. Finally, the supernatant was
and soil samples were taken. Material was collected to make separated and the absorbance was read at 532 and 600 nm
the following determinations: on a Shimadzu UV 160 UV/V spectrophotometer. The
MDA concentration was calculated using an extinction
– Fresh root and dry aerial weight coefficient of 155 mM ­cm−1: MDA equivalents (nmol
– Total chlorophyll (Wellburn 1994). A leaf disc 1 cm in ­ml−1) = [(A532 – A600) / 155000].
diameter was placed in 1.5 mL of N–Dimethylforma- – Proline content in leaf and root tissue (Bates et al. 1973).
mide. The absorbances of the solution were measured at One hundred milligrams of fresh tissue was homogenized
wavelengths 646.8, 663.8, and 480 nm, in a Shimadzu with 2 mL of sulfosalicylic acid 3%. It was centrifuged
UV160 A spectrophotometer. at 12,000 g for 15 min and 1 mL of the supernatant was
taken. It was reacted with 1 mL of the acid reagent Nin-
Chlorophyll (µg c­ m−2) = 12 × Ab 663.8 – 3.11 × Ab 646. hydrine and 1 mL of glacial acetic acid in a 15-mL tube,
Chlorophyll b (µg c­m −2 ) = 20.78 × Ab 646.8 – in a water bath at 100 °C for 1 h. The reaction was then
4.88 × Ab 663.8 interrupted by cooling the samples in ice. To the pre-
Total Chlorophyll a+b (µg ­cm −2 ) = 17.67 × Ab vious mixture, 2 mL of toluene was added and stirred
646.8 + 7.12 × Ab 663.8 for 15 s in vortex. The phases were allowed to separate,
and the aqueous phase containing the toluene–proline
– Soluble proteins in leaf and root tissue (Bradford 1976). chromophore was taken. Absorbance was measured at
Two hundred milligrams of tissue was homogeneized 520 nm using toluene as a target. Proline content was cal-
with a 1 mL extraction buffer (TRIS 50 mM, EDTA culated according to µmol proline.g−1FW = [(µg proline/
1 mM, PVPP insoluble 1%, MeSH 0.1% a pH 7.5) at mL × mL toluene)/115.5 µg/µmoles]/[(g FW)/5
4 °C. The resulting homogenate was centrifuged at – Total phenol content in leaf and root tissue (Singleton
10,000 g for 10 min at 4 °C. One hundred microliter and Rossi 1965). Five hundred milligrams of fresh tissue
of the supernatant was taken and 5 mL of Coomassie’s was homogenized in ethanol. The mixture was centri-
Bright Blue reagent was added, stirred in vortex, and the fuged at 10,000 g for 15 min at 4 °C. Forty microliters
absorbance was read at 595 nm. The calculation of the are extracted from the sample and mixed with distilled

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water, and the Folin–Ciocalteu reagent 1 N. After 3 min nematode, Ri–A2 was the isolate that presented the least
at 25 °C, the Na2CO3 2% (w/v) solution was added in mycorrhization (Table 1). The other isolated showed no sta-
NaOH 0.1 N. It was incubated for 90 min at 25 °C, and tistical differences when the nematode was present.
the absorbance was measured at 760 nm in a spectropho- Regarding the viability of the fungal structures Ri–A2 and
tometer. A standard curve was made based on different Fm maintained values without significant differences in the
concentrations of gallic acid. presence and absence of the nematode, while Ri–B1 had a
– Relative conductivity of leaf and root tissue (Lutts et al. reduction of 17.8 points when the nematode was present. In
1996). Two hundred milligrams of tissue was weighed the parasitized treatments, Ri–B1 was the isolate that pre-
and washed three times with doubly distilled water for sented significantly lower values of fungal viability (Table 1).
15 s. Each sample was immersed in a tube with 10 mL Most research involving AMF and plant parasitic nem-
of doubly distilled water where they remained for 4 h at atodes focuses on parasite control, without studying in
room temperature. Subsequently, the electrical conduc- detail what are the effects on fungi. The results presented
tivity (dS m − 1) was determined using a Jenco conduc- here show that the isolates of the genus Rhizoglomus used
tivity meter model 3173. The tubes were then plugged in this assay have some type of interaction with the nem-
and autoclaved (20 min, 120 °C, 1 atmosphere pres- atode since it impacts the colonization and/or the viabil-
sure). Finally, the tubes were allowed to cool to room ity of the fungal structures. Jung et al. (2012) and Vos
temperature and the electrical conductivity was measured et al. (2014) suggest that photoassimilates are a scarce
again. The relative conductivity of cell membranes was resource and there is competition for them between both
estimated from the following formula: CR (%) = (L1 / populations, which affects the optimal development of
L2) × 100, where L1 and L2 are the electrical conductiv- both. The syncytia of N. aberrrans and the arbuscules of
ity readings before and after autoclaving, respectively. the AMF develop in the root pericycle, so it is possible
– Percent of mycorrhizal colonization (Phillips and Hayman that there is a competition for physical space in addition
1970). Root fragments were immersed in KOH 10% (w/v), to what is hypothesized with respect to photoassimilates.
10 min at 100 °C. They were then washed three times with These considerations suggest that early inoculation with
distilled water and a 0. 1N HCl solution was applied, 5 min AMF at the planting and growth stage of the seedling in
at 20 °C. Structure staining was performed with trypan blue, the tray will have a positive impact on AMF/nematode
5 min at 95 °C. The roots are placed on slides, lactic acid drops competition. Mycorrhizal symbiosis will be established
are added, and observed under an optical microscope. before transplanting, at which time the root will come
– Viability of fungal structures (Smith and Gianinazzi- into contact with the nematodes present in the soil. Myc-
Pearson 1990). Root fragments were placed for 18 h orrhizal symbiosis is a dynamic interaction between the
in a 0.2 M Tris–HCL solution, pH 7; 2.5 M sodium fungus and the plant that undergoes alterations due to the
succinate.6H2O; 5 mM M ­ gCl2 and 4 mg/mL nitroblue influence of various factors. In a test where isolates are
tetrazolium. After that time, they were rinsed with a 75%
chloral hydrate solution. Finally, they were placed in a
0.01% acid-fuchsin solution in lactic acid for 8 min at Table 1  a) Percent of mycorrhizal colonization and b) viability of
90 °C. The root fragments are observed under the micro- the fungal structures of three arbuscular mycorrhizal fungi in tomato
lants grown in the presence and absence of Nacobbus aberrans
scope, and the percent of viable and active fungal struc-
tures that are colored blue is quantified. Treat. Without N. aberrans With N. aberrans

a)
Control 0 ± 0 Ab 0 ± Ac
Results and discussion Ri–A2 49.79 ± 5.9 Aa 30.27 ± 9.1 Bb
Ri–B1 45.63 ± 9.2 Aa 55.78 ± 8.8 Aa
Parameters linked to AMF Fm 62.95 ± 14.3 Aa 46.71 ± 5.4 Aa
b)
The root staining and the quantification of the percent of Control 0 ± 0 Ac 0 ± 0 Ac
mycorrhizal colonization performed prior to transplantation, Ri–A2 31.18 ± 8.2 Aa 37.8 ± 12.6 Aa
confirmed that the roots were colonized by the three isolates Ri–B1 40.16 ± 7.7 Aab 22.3 ± 10.02 Bb
used. The percent of mycorrhization was 85%, 76%, and 72% Fm 43.16 ± 6.7 Ab 43.7 ± 5.73 Aa
for Fm, Ri–B1, and Ri–A2, respectively.
Staining of root samples at the end of the test indicated Capital letters on the line compare the same mycorrhizal condition in
that parasitism of N. aberrans caused an average reduction the absence and presence of the nematode. Lowercase letters in the
circle compare the different AMF under the same nematological con-
of 19 points in the percent of colonization in Ri–A2. In dition. Means accompanied by different letters indicates significant
turn, comparing the different AMF in the presence of the differences according to Fisher’s LSD test (p ≤ 0.05)

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compared, the viability test is of paramount importance, An improvement in the nutritional status of the host
since the Tripan blue staining technique does not dif- plant. The association of mycorrhizae allows a greater
ferentiate between active and non-active structures. In absorption of nutrients such as P, N, K, Ca, Mg, Fe,
tests where co–inoculations of mycorrhizae and galling and Mn resulting in more vigorous and more patho-
nematodes have been carried out, it is observed that not gen–tolerant plants (Gianinazzi et al. 2010).
all isolates are affected in the same way and even some Changes in the root system: after colonization, the myc-
seem to be stimulated by the presence of the nematode orrhizal fungus causes morphological and physiologi-
(Pinochet et al. 1998; Cofcewicz et al. 2001). cal changes in the root, which hinder the entry of other
microorganisms, or in the case of endoparasitic nema-
Parameters linked to the population of N. aberrans todes, syncytia of smaller numbers of cells are formed
(Fassuliotis 1970; Pozo et al. 2013).
The quantification of eggs lodged in the roots indicated that Modifications in root exudates: The establishment of sym-
mycorrhization caused a significant reduction in them, with biosis generates qualitative and quantitative changes in root
no significant differences between the 3 isolates of AMF exudates. This results in the appearance of substances that
(Fig. 1a). exert a direct effect on harmful microorganisms or alter the
In the count of mobile forms extracted from 100 mL plant/pathogen signaling, decreasing the entry of juveniles
of soil, non-mycorrhizal treatment presented the highest into the root (Sikora and Fernández 2005).
number of individuals (Fig. 1b). Mycorrhization resulted Synthesis of compounds: Mycorrhiza can stimulate the
in reductions in the Ri–B1 and Fm treatments compared to production of substances involved in the plant’s defense
the uninoculated control. system, such as phytoalexins, phenolic compounds,
Accounting for the final population values of nematodes enzymes (peroxidases, beta–glucanases, ammonioliases
recorded, reductions of 71%, 51%, and 73% were measured for phenylalanine, chitinases, polyphenol oxidases) and other
the Ri–A2, Ri–B1, and Fm treatments, respectively (Fig. 1c). proteins (Solórzano et al. 2001; Vos et al. 2012).
The results allow us to conclude that the symbiosis Classical biological control: some AMF behaves in cer-
between the tomato and any of the 3 isolates of AMF tain circumstances as weak parasites of the eggs of some
had an antagonistic effect over the N. aberrans popu- nematodes (Francl and Dropkin 1985; Tribe 1977), and
lation. For this essay, we can rule out at least one of this phenomenon could be contributing to some extent to
the explanations suggested by Barea et al. (2002) which explain the mechanism of inhibition.
include the possibility that the protective effect comes
from the development of antagonistic microorganisms Parameters linked to plant growth and physiology
growing stimulated by hyphae in the rhizosphere, since
the substrate used in the test was previously sterilized Prior to the completion of the test, with the plants still
by tyndallization. standing, we proceeded to measure the stomatal conduct-
Among the possible justifications for why mycorrhizal ance (CEs) and net photosynthesis (PN). The analysis of
plants cause a decrease in the population of phytoparasitic the variance of the CEs did not show a significant inter-
nematodes, the following can be considered: action between the factors mycorrhization and infestation

Fig. 1  Eggs in roots, number of mobile forms extracted from the soil, mus intraradices A2; Ri–B1, Rhizoglomus intraradices B1; Fm, Fun-
final population, and reproduction factor of Platense tomato plants neliformis moseeae. Stockings accompanied by different letters indi-
inoculated with three arbuscular mycorrhizal fungi. Ri–A2, Rhizoglo- cate significant differences according to Fisher’s LSD test (p ≤ 0.05)

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with the nematode (p = 0.32); however, there were signifi- N. aberrans, Ri–A2 had a similar PN to that of the control
cant differences between the levels of these main factors. treatment (Fig. 2).
The gas exchange of plants inoculated with Ri–B1 and Fm Measurements linked to plant growth, such as dry mass
was greater than that of control plants and those inoculated of the aerial part and stem diameter in the middle part of
with Ri–A2. Plants parasitized by N. aberrans had a lower the plants, showed no significant differences between treat-
stomatal conductance than non-parasitized plants (Table 2). ments. In the case of fresh root weight in the absence of
In the absence of the nematode, the lowest PN was the nematode, the mycorrhizal plants with Fm had a lower
recorded in Ri–A2. Comparing parasitized and non-para- weight than the rest of the treatments, but in the presence of
sitized plants, control plants had a significant reduction in the nematode, the weight remained unaffected. In the control
CO2 fixation; Ri–A2 and Ri–B1 showed no changes and, plants, the attack of the nematode reduced the fresh root
although Fm had a reduction, maintained values significantly weight (Table 3).
higher than the non-mycorrhized control. In the presence of The concentration of chlorophyll in the leaves in the
absence of the nematode was lower in the Ri–A2 treatment.
Comparing parasitized and non-parasitized plants, control
Table 2  Stomatal conductance of Platense tomato plants: a) inocu-
lated with different arbuscular mycorrhizal fungi, b) inoculated and
plants and Fm had a reduction in chlorophyll content as a
not inoculated with Nacobbus aberrans result of parasitism, while Ri–A2 and Ri–B1 plants did not
present significant differences (Table 3).
Treatment Mean
In the absence of the nematode, no advantages were
a) observed in the growth and physiological processes of myc-
Control 90.4 ± 17.9 b orrhizal plants over non-mycorrhizal plants. The fresh root
Ri–A2 92.3 ± 16.2 b weight of the three mycorrhizal treatments was lower than
Fm 113.2 ± 13.2 a that of the controls. It is known that the proliferation of
Ri–B1 120.9 ± 29.2 a hyphae fulfilling absorption functions made plants allocate
b) fewer resources to root growth (Pereira et al. 2001).
Without N. aberrans 116.0 ± 23.8 a In the presence of the nematode, a reduction in PN, total
With N. aberrans 92.4 ± 16.6 b chlorophyll and fresh root weight are observed for control
plants; shrinkage that does not occur in mycorrhizal plants.
Means accompanied by different letters, indicates significant differ-
ences according to Fisher’s LSD test (p ≤ 0.05) Comparing the 3 isolates of mycorrhiza, RI–A2 presented
Ri–A2 Rhizoglomus intraradices A2, Ri–B1 Rhizoglomus intraradi-
PN values and total chlorophyll significantly lower than
ces B1, Fm Funneliformis moseae RI–B1 and Fm.

Fig. 2  Net photosynthesis in mycorrhizal tomato plants with 3 arbus- nematodes. Lowercase letters compare different mycorrhizae within
cular mycorrhizal fungi in the presence or absence of Nacobbus aber- the same nematological condition. Means accompanied by different
rans. Ri–A2, Rhizoglomus intraradices A2; Ri–B1, Rhizoglomus letters indicate significant differences according to Fisher’s LSD test
intraradices B1; Fm, Funneliformis mosseae. Capital letters com- (p ≤ 0.05)
pare the same mycorrhizal condition in the absence and presence of

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Table 3  Parameters linked to the growth of tomato plants inoculated with 3 mycorrhizal forming fungi in the presence or absence of Nacobbus
aberrans
Without Nacobbus aberrans With Nacobbus aberrans
Control Ri–A2 Ri–B1 Fm Control Ri–A2 Ri–B1 Fm

Fresh root weight (g) 163.7 Aa 132.2 Aab 135.2 Aab 111 Ab 130.7 Ba 108.2 Aa 143.8 Aa 124.7 Aa
Aerial dry weight (g) 60.04 Aa 58.18 Aa 62.02 Aa 57.16 Aa 60.58 Aa 61.3 Aa 62.0 Aa 58.44 Aa
Total chlorophyll (μg.cm−2) 10.28 Aa 7.23 Ab 9.93 Aa 11.9 Aa 7.3 Bb 7.42 Ab 10.1 Aa 10.2 Ba
Stem diameter (cm) 1.2 Aa 1.2 A a 1.16 Aa 1.21 Aa 1.06 Aa 1.1 Aa 1.19 Aa 1.1 Aa

Capital letters compare the same mycorrhizal condition in the absence and presence of the nematode. Lowercase letters compare different myc-
orrhizae within the same nematological condition. Means accompanied by different letters indicate significant differences according to Fisher’s
LSD test (p ≤ 0.05)
Ri–A2 Rhizoglomus intraradices A2, Ri–B1 Rhizoglomus intraradices B1, Fm Funneliformis mosseae

Under controlled conditions, in the absence of stressors, be the most beneficial, but not in jobs where the priority is
mycorrhization may not present quantifiable productive placed on the productivity of the host.
benefits. The mycorrhizal fungus can become an important The content of soluble proteins in the leaf tissue of
destination for photoassimilates that cause a reduction in mycorrhizal plants ranged from 19.9 to 28.03 µg of pro-
growth in the plant. On the other hand, the extensive growth tein.mg−1, both in the absence and in the presence of the
of hyphae in the substrate that is one of the main benefits nematode, without significant differences between the dif-
that the fungus gives to the plant could be limited because ferent isolates. The control treatment plants + N. aberrans
it is a pot test. had a significantly lower protein content than the controls
Mycorrhizal symbiosis, in addition to making signifi- without nematode and lower than the mycorrhized with
cant contributions to the host, fulfills important ecological nematode (Fig. 3a).
functions. The carbon supplied by the plant to the AMF is The soluble proteins of the root tissue presented a differ-
not exclusively for consumption, but through the hyphae, ent behavior. In the absence of the nematode, mycorrhizal
a flow of carbon from the atmosphere to the soil is estab- plants had a lower concentration of proteins compared to the
lished that causes significant increases in biological activ- control treatment. In the presence of the nematode, mycor-
ity (Finlay and Söderström 1992). Not all isolates mani- rhizal plants with Ri–B1 and Fm had a higher protein con-
fest the same efficiency to perform this task. It could be tent than control plants and mycorrhizal plants with Ri–A2.
expected that for cases where what is sought is to remedy Comparing parasitized plants with non-parasitized plants,
a degraded or contaminated soil, this type of isolate would it was verified that in mycorrhizal roots, the protein content

Fig. 3  Soluble protein content in a leaf tissue and b root tissue of rhizal condition in the absence and presence of nematode. Lowercase
mycorrhized Platense tomato plants with 3 arbuscular mycorrhi- letters compare different mycorrhizae within the same nematological
zal fungi in the presence or absence of Nacobbus aberrans. Ri–A2, condition. Columns accompanied by different letters indicate signifi-
Rhizoglomus intraradices A2; Ri–B1, Rhizoglomus intraradices B1; cant differences in the Fisher LSD test (p ≤ 0.05)
Fm, Funneliformis moseae. Capital letters compare the same mycor-

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was increased by the presence of the nematode, while in In the root tissue, the concentration of this metabolite was
non-mycorrhizal roots, it was reduced (Fig. 3b). higher than that quantified in the leaves. In the absence of N.
The protein content in the aerial parts reflects what has aberrans, the phenol content of mycorrhized roots was sig-
been observed in other growth parameters. Control plants are nificantly higher than that of non-mycorrhized roots. In the
affected by the nematode and mycorrhized plants to a greater presence of the nematode, the phenol content of the mycor-
or lesser extent manage to mitigate the negative impact of rhized roots remained unchanged, while in the control plants
parasitism. In the case of the root, the results are not so clear there was a significant increase (Table 4).
and other types of studies should be carried out to know how The concentration of proline in leaves was increased in
the protein pattern is modified in each case. The modifica- all parasitized treatments compared to non-parasitized treat-
tions that occur in the root by the presence of a symbiont ments. The Ri–A2 treatment, in the presence of the nema-
require important changes in the plasma and vacuolar mem- tode, showed the highest amount of proline compared to the
branes that alter the expression of genes that encode proteins other treatments inoculated with AMF (Fig. 4a).
(Krajinski et al. 2000). It has also been well documented that The concentration of proline in roots was increased in
nematode attack stimulates the synthesis of defense proteins control, RI–A2 and RI–B1 parasitized treatments compared
(Mejias et al. 2019). It remains to be studied in greater depth to non-parasitized treatments, but not in those inoculated
how the protein metabolism of the root is modified in the with FM where the presence of the nematode did not cause
presence of a symbiont and a pathogen. changes in proline levels. Control and Fm treatments had
The quantification of phenols in leaf tissue showed no the highest levels of proline in the absence of the nematode.
differences between treatments. In all cases, the concentra- In the presence of the nematode, all mycorrhizal treatments
tion was between 1.35 and 1.40 µg mg of fresh tissue weight. had lower proline levels than control (Fig. 4b).
The mallonyldialdehyde (MDA) content and relative tis-
sue conductivity (CR) of the leaves were not modified by
Table 4  Total phenol content expressed in µg mg fresh weight of root nematode parasitism. Both in the absence and in the pres-
tissue of mycorrhized tomato plants with 3 arbuscular mycorrhizal ence of the nematode, the Ri–A2 leaves presented higher
fungi in the presence and absence of Nacobbus aberrans
MDA values than the control (Table 5).
Treatments Without N. aberrans With N. aberrans In root tissue, N. aberrans caused an increase in MDA
Control 3.41 ± 1.3 Bc 5.42 ± 0.68 Aa
content and %CR in non-mycorrhizal plants. Not so in myc-
Ri–A2 5.74 ± 0.46 Aa 4.90 ± 1.2 Aa
orrhized plants, whose values were not altered, indicating a
Ri–B1 5.27 ± 0.58 Aab 4.78 ± 0.28 Aa
better condition of cell membranes.
Fm 4.75 ± 0.37 Ab 5.27 ± 0.34 Aa
The parasitism of N. aberrans causes mechanical damage
to the membranes and disorders in the absorption of water
Capital letters on the line compare the same mycorrhizal condition in and nutrients that mainly trigger water and nutritional stress.
the absence and presence of the nematode. Lowercase letters in the In this section, it is observed how mycorrhizal symbiosis
column compare the different AMF under the same nematological
condition. Means accompanied by different letters indicate significant acts attenuating at least partially, such damages.
differences according to Fisher’s LSD test (p ≤ 0.05)

Fig. 4  Proline concentration in a leaf tissue b root tissue of mycor- nematode. Lowercase letters compare different mycorrhizae within
rhizal tomato plants with 3 arbuscular mycorrhizal fungi in the pres- the same nematological condition. Columns accompanied by different
ence and absence of Nacobbus aberrans. Capital letters compare letters indicate significant differences according to Fisher’s LSD test
the same mycorrhizal condition in the absence and presence of the (p ≤ 0.05)

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Mycological Progress (2024) 23:3 Page 9 of 11 3

Table 5  Content of mallonyldialdehyde and relative tissue conductivity, foliar, and root of mycorrhized tomato plants with 3 arbuscular mycor-
rhizal fungi in the presence and absence of Nacobbus aberrans

CV Without Nacobbus aberrans With Nacobbus aberrans

Control Ri–A2 Ri–B1 Fm Control Ri–A2 Ri–B1 Fm

­ W−1)
Foliar MDA (nmoles.gr F 22% 7.75 Ab 11.94 Aa 10.4 Aab 8.46 Ab 7.24 Ab 11.4 Aa 9.26 Aab 9.45 Aab
­ W−1)
Radicular MDA (nmoles.gr F 15.1% 2.01 Ba 2.36 Aa 2.20 Aa 2.22 Aa 3.05 Aa 2.31 Ab 2.06 Ab 2.21 Ab
Foliar CR (%) 23% 16.98 Aa 16.22 Aa 19.57 Aa 18.10 Aa 19.14 Aa 14.85 Aa 19.14 Aa 21.19 Aa
Radicular CR (%) 26.3% 23.77 Ba 20.35 Aa 22.88 Aa 23.71 Aa 32.01 Aa 25.33 Aab 27.49 Aab 23.22 Ab

Capital letters on the line compare the same mycorrhizal condition in the absence and presence of the nematode. Lowercase letters in the column
compare the different AMF under the same nematological condition. Different letters indicate significant differences according to Fisher’s LSD
mean comparison test (p ≤ 0.05)

In this work, mycorrhization caused a significant increase suggests that symbiosis stimulates the production of antioxi-
in the content of phenolic compounds of root tissue, a fact that dant enzymes that allow plants a greater ability to respond to
was also documented by other authors (Soares et al. 2005). The and neutralize reactive oxygen molecules.
functionality of phenolic compounds in plant defense is broad
and often difficult to determine. Some compounds such as chlo-
rogenic acid and hydroxycinnamic acids can be, in themselves, Conclusions
toxic to pathogens; other can rapidly develop into reactive oxy-
gen species that act in response to attack (Hartleb et al. 1997). Mycorrhizal association is beneficial in conditions where
The higher phenol content of mycorrhized roots in the absence “inverted” photoassimilates are used to reverse or compen-
of the nematode suggests some kind of resistance induction or sate for a stressful situation. The crops, unlike the condi-
priming. This cannot be affirmed, since in order to do so, it is tions given in this test, are continuously exposed to various
necessary to verify that some metabolic pathway linked to plant adverse situations of different magnitude. The possibility
defense has been stimulated or that some group of genes is being of mycorrhiza becoming a symbiont that does not offset
overexpressed (Madriz Ordeñana 2002). “the costs with its benefits is unlikely.”
Proline is a metabolite that accumulates in plant tissues When N. aberrans and AMF are living together in the same
in response to water deficits. Its accumulation decreases the host, there is an interaction between them, either directly or
osmotic potential of the cells, favoring the absorption and indirectly, that causes modifications in their respective popu-
movement of water in the plant. Proline, in addition to acting lations, as well as in plant growth and physiology. The three
as an osmoprotective, plays an important role in blocking reac- isolates used here managed to reduce the population of N.
tive oxygen species (Hossain et al. 2014). aberrans in tomato plants. However, in some parameters such
The exploration of the substrate by the hyphae facilitating as stomatal conductance, net photosynthesis, and chlorophyll
the absorption of water explains the lower levels of proline content RI–A2 showed inferior performance than the other
in situations where, as a result of parasitism, the functionality isolates. The evaluation of the colonization of the fungus in
of the root is affected. In addition, some authors indicate that the root and the survival of its structures shows that Funneli-
mycorrhization stimulates, at the root, the expression of genes formis mosseae had a better performance. This last character-
that encode aquaporins (membrane proteins involved in water istic is important if the use of this isolate as a bioinput for the
absorption) (Ruiz Sánchez et al 2011). integrated management of nematodes is planned.
Malondialdehyde is the main product of the peroxidation of According to the results obtained, it is recommended
cell membrane lipids by the action of reactive oxygen species that inoculation with AMF be carried out at planting so
(Bhattacharjee et al. 2014). The measurement of the relative that the symbiosis is established early before the plants are
electrical conductivity of tissues reflects the ability of mem- exposed to stress situations. The inoculum of AMF that
branes to retain electrolytes (Cseresnyés et al. 2016). These was elaborated for the realization of this work does not
indicators allow the evaluation of oxidative stress and damage present difficulties for its management, conservation, and
to plasma membranes. The less damage to the membranes application in plants, promising the use of this biotechnol-
of mycorrhizal plants may be due to a lower attack by the ogy in productive regions affected by N. aberrans.
nematode in the plants or to a higher antioxidant activity. Sev-
eral authors agree that mycorrhization stimulates the activity
Author contribution All authors contributed to the study conception
of some enzymes such as superoxide dismutase, peroxidase, and design. Material preparation, data collection, and analysis were also
and catalase (Lambais et al. 2003). Borde et al. (2011) also

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performed by all authors. The first draft of the manuscript was written by EPPO Bull 39:376–381. https://​doi.​org/​10.​1111/j.​1365-​2338.​
the first author, and all authors commented on previous versions of the 2009.​02325.x
manuscript. All authors read and approved the final manuscript. Fassuliotis G (1970) Resistance in Cucumis spp. to rootknot nematode
Meloidogyne incognita acrita. J Nematol 2:174
Funding The study was completely funded by the authors. Finlay R, Söderström B (1992) Mycorrhiza and carbon flow to the soil.
En: Allen, M. F. Mycorrhizal functioning: an integrative plant–
Data availability The datasets generated and analyzed during the cur- fungal process. Chapman & Hall, New York, pp 134–160
rent study are available from the corresponding author on reasonable Food and Agriculture Organization of the United Nations (2023)
request. Food and agriculture data. https://​www.​fao.​org/​faost​at/​en/#​data.
Accessed 2 Nov 2023
Declarations Francl LJ, Dropkin VH (1985) Glomus fasciculatum, a weak pathogen
of Heterodera glycines. J Nematol 17:470–475
Competing interests The authors declare no competing interests. Garita S, Bernardo V, De Almeida GM, Arango MC, Ruscitti M (2019)
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Gianinazzi S, Gollotte A, Binet M, van Tuinen D, Redecker D, Wipf
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