Food Chemistry 359 (2021) 129961

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Fruit quality and volatile compound composition of processing tomato as

affected by fertilisation practices and arbuscular mycorrhizal
fungi application
Igor Pasković a, 1, Barbara Soldo b, 1, Smiljana Goreta Ban a, c, *, Tomislav Radić d, Marina Lukić a,
Branimir Urlić d, Matea Mimica b, Karolina Brkić Bubola a, Giuseppe Colla e, Youssef Rouphael f,
Nikola Major a, Maja Šimpraga g, Dean Ban a, c, Igor Palčić a, c, Mario Franić a, c, Kristina Grozić a,
Igor Lukić a, c
a
Institute of Agriculture and Tourism, Deparment of Agriculture and Nutrition, K. Huguesa 8, 52440 Poreč, Croatia
b
University of Split, Faculty of Science, Department of Chemistry, R. Boškovića 33, 21000 Split, Croatia
c
Centre of Excellence for Biodiversity and Molecular Plant Breeding, Svetošimunska 25, 10000 Zagreb, Croatia
d
Institute for Adriatic Crops and Karst Reclamation, Put Duilova 11, 21000 Split, Croatia
e
University of Tuscia, Department of Agricultural and Forestry Sciences, 01100 Viterbo, Italy
f
University of Naples Federico II, Department of Agricultural Sciences, 80055 Portici, Italy
g
Faculty of Bioscience Engineering, Department of Plants and Crops, Valentin Vaerwyckweg 1, 9000 Gent, Belgium

A R T I C L E I N F O A B S T R A C T

Keywords: The effects of different fertilisation treatments with arbuscular mycorrhizal fungi (AMF) inoculation on AMF root
Solanum lycopersicum L. colonisation, fruit yield, nutrient and total phenol contents, volatile compound composition, and sensory attri­
Biofertiliser butes of tomato (Solanum lycopersicum L.) were investigated. Mineral, organic, and mineral + organic fertiliser
Volatiles
application positively affected tomato yield (35%–50%) and phosphorus concentration (24%–29%) compared
Sensory quality
Rhizophagus irregularis
with controls. AMF application had a significant impact on the total nitrogen (+9%), manganese (+12%), and
Funneliformis mosseae hydrophilic phenol (+8%) contents in the fruit. Volatile compounds were affected by the interactive effects of
fertilisation and AMF application. The response of tomato fruit sensory quality indicators was relatively modest,
with only a few sensory characteristics affected to a lesser extent. Although tomato showed susceptibility to field-
native AMF, particular combinations of fertilisation and AMF inoculation were more effective at improving the
quality parameters of tomatoes under field conditions applied in this study.

1. Introduction
Arbuscular mycorrhizae are symbionts that provide a range of ben­
efits to host plants, such as the enhancement of water and nutrient
supply and tolerance to abiotic stress. Tomato (Solanum lycopersicum L.)
is an excellent model plant for observing growth and fruit yield in
response to mycorrhization (Bona et al., 2017). The nutritional and
water status of mycorrhized tomato plants can be enhanced under
drought conditions, and fruit quality is significantly improved (Sub­
ramanian et al., 2006). Tomatoes from mycorrhiza-inoculated plants
have been shown to contain higher lycopene (Giovannetti et al., 2012),
ascorbic acid (vitamin C), total soluble solids (Subramanian et al.,
2006), and citric acid content (Bona et al., 2017). Furthermore,
mycorrhizal tomato plants under low nutrient conditions produced
fruits with nutrient contents similar to those from non-mycorrhized
plants under high fertiliser conditions, suggesting arbuscular mycor­
rhizal fungi (AMF) can help reduce the use of exogenous fertilisers
(Zouari et al., 2014). Enhancements in secondary metabolites related to
taste, nutritional, and functional values have also been documented in
mycorrhized plants (Antunes et al., 2012); moreover, concentrations of
particular tomato volatile compounds increased in AMF-inoculated
plants compared with controls (Hart et al., 2015).
In agro-systems with intensive P mineral fertilisation, such as tomato
cultivation in the processing industry, a negative impact of fertilisation

* Corresponding author.
E-mail address: smilja@iptpo.hr (S. Goreta Ban).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.foodchem.2021.129961
Received 28 July 2020; Received in revised form 20 April 2021; Accepted 24 April 2021
Available online 27 April 2021
0308-8146/© 2021 Elsevier Ltd. All rights reserved.
I. Pasković et al.
on the native AMF soil community can be observed through the reduc­
tion of AMF number or selection of less efficient AMF species for fungi-
plant interactions (Conversa et al., 2013). In addition, frequent and
excessive use of mineral fertilisers can cause eutrophication of aquatic
ecosystems, thereby negatively affecting the environment.
In contrast to mineral fertilisers, organic amendments positively in­
fluence AMF growth by enhancing spore production and hyphal prolif­
eration and improving root colonisation (Bilalis & Karamanos, 2010).
The release of nutrients from organic fertilisers, associated with micro­
bial activity, was shown to support the formation and functioning of
AMF compared with most inorganic fertilisers, except when the P levels
in the latter were low (Linderman & Davis, 2004). Gryndler et al. (2006)
concluded that the development of external mycelium of AMF is
increased by organic fertilisation but decreased by mineral fertilisation.
Organic fertilisers may exhibit positive effects on soil physical proper­
ties; however, their use may also result in low plant nutrient availability
(Linderman & Davis, 2004). Tomato plants may need both fertilisation
and AMF inoculation to achieve optimal growth and yield, and the
application of AMF can compensate for the reduction in chemical fer­
tilisers (Ziane et al., 2017). Carefully adjusting fertilisation with the
application of AMF can enhance the sustainable production of process­
ing tomato.
The influence of AMF on nutrient concentrations in tomato plant
roots and shoots has been well documented (Bona et al., 2017). In
contrast, limited information is available on the effect of AMF applica­
tion on nutritionally and sensorially relevant chemical compounds and
sensory features of tomato fruit (Antunes et al., 2012). Additionally, the
impact of fertilisers of different origins in combination with AMF on
tomato quality and nutritive value has not been thoroughly investigated
to date. Therefore, the aim of this study was to determine the influence
of different fertilisation practices combined with the application of AMF
in open field production conditions on the chemical composition of to­
mato fruit, including the contents of minerals, phenols, and volatile
aroma compounds and sensory quality.
2. Materials and methods
2.1. Plant material, growth conditions, and experimental design
The seeds of tomato hybrid Red Valley H1 (Esasem S.p.A., Casaleone,
Italy) were sown on 24 March 2017 in Longo Nursery, Rovinj, Croatia.
Klasmann BioPot Grond® (Klasmann-Deilmann GmbH, Geeste, Ger­
many) was used as a substrate. The untreated tomato seeds were steri­
lised with 10% H2O2 (VWR International, Radnor, PA, USA) for 10 min
and then sown in pots with a volume of 27 mL (-AMF) or inoculated with
AMF before sowing (+AMF). Aegis microgranule® (Atens, Tarragona,
Spain), which contained 25 spores per gram of Rhizophagus irregularis
(syn. Glomus intraradices) and 25 spores per gram of Funneliformis mos­
seae (syn. Glomus mossease), at a quantity of 1 g per 50 mL of substrate
was used for inoculation.
On May 3, 2017, a field experiment was set in a split-plot design
performed on Terra Rossa soil (Table S1) with two factors: (i) fertilisa­
tion consistent with NPK recommendations in processing tomato pro­
duction (Hartz et al., 2008) based on equal NPK amounts for each of the
different fertiliser sources (four treatments: unfertilised [UNF], mineral
[MIN] [400 kg/ha; NPK 15:15:15] [Petrokemija d.d., Kutina, Croatia],
organic chicken pelleted manure [ORG] [1500 kg/ha; NPK 4:4:4]
[Italpollina, Rivoli Veronese, Italy], and mineral + organic [MIN +
ORG] [200 kg/ha NPK 15:15:15 + 750 kg/ha NPK 4:4:4]), and (ii) AMF
application (two treatments: with [+AMF] or without AMF inoculation
[-AMF]). A total of eight treatments were used. Each treatment was
represented by 200 plants split into four replicates. After 1 week, each
tomato AMF plant (+AMF) was additionally amended with 40 mg of
Aegis Irriga® product (Atens) (700 spores/g of R. irregularis and 700
spores/g of F. mosseae) suspended in 100 mL of water and applied
directly to the plant base according to the manufacturer’s instructions.
2
Food Chemistry 359 (2021) 129961
During the experiment, all tomato plants were fertigated weekly with
urea (Petrokemija d.d.) up to an additional 60 kg N/ha. Fruits were
harvested at full maturity (103 days after transplanting; DAT), after over
90% of fruits reached the pink or red stage of ripening (Lešić et al.,
2016). Fully developed and coloured fruits without physiological
symptoms, diseases, or mechanical disorders were sampled from the
second cluster. A total of 96 fruits per treatment, composed of four
replicates, were collected and equally pooled into sub-samples for sen­
sory, mineral, and quality parameter or volatile compound analysis.
Samples were taken into the laboratory and carefully rinsed with dem­
ineralized water.
2.2. Soil physicochemical properties and fruit micro- and macronutrient
determination
The chemical and physical properties of the selected soil were
determined as described by Pasković et al. (2019) (Table S1).
Tomato fruits were dried in an oven (Memmert GmbH + Co. KG,
Schwabach, Germany) with circulating air at 80 ◦ C until constant mass
and grounded (Model MSM7400, Type CNHR21; Robert Bosch GmbH,
Gerlingen, Germany) for nutrient analysis. Subsequently, powdered
material (0.5 g) was obtained from each sample, subjected to dry ashing
in a muffle furnace (Nabertherm B 180; Nabertherm GmbH, Lilienthal,
Germany) at 550 ◦ C for 5 h, and used to extract P, K, Ca, Mg, Zn, Mn, and
Cu after dissolving in 2 mL of HCl (VWR International) (Hančević et al.,
2018). The P concentration was determined by the vanadate-molybdate
yellow colour method using a spectrophotometer at 420 nm (Varian
Cary 50 Scan; Varian Inc., Harbor City, CA, USA). The K concentration
was measured using a flame photometer (Model 410; Sherwood Scien­
tific Ltd., Cambridge, UK). Furthermore, Ca, Mg, Zn, Mn, and Cu were
determined by atomic absorption spectrometry (SpectrAA 220; Varian
Inc.). The total N concentration was measured using a micro-Kjeldahl
digestion system 1026 (Kjeltec system 1026 distilling unit; Tecator,
Höganäs, Sweden). The repeatability of the methods was checked by
performing three repeated measurements of each of the samples from
the selected treatments. The obtained relative standard deviations of P,
K, Ca, Mg, Zn, Mn, Cu, and N were 5.5%, 3.5%, 5.4%, 5.0%, 5.6%, 4.4%,
4.9%, and 0.5%, respectively. The accuracy of the methods was checked
regularly by analysing samples in the framework of a proficiency testing
scheme (WEPAL/QUASIMEME, Wageningen, The Netherlands), with
satisfactory results within tolerance limits in all cases.
2.3. Root colonization by arbuscular mycorrhizal fungi
Tomato roots were collected for microscopic analysis at 103 DAT.
Each treatment was represented by eight roots split into four replicates.
Sampled tomato roots were thoroughly washed with tap water, cleaned
with 10% KOH (w/v; T.T.T. d.o.o., Sveta Nedjelja, Croatia) at 80 ◦ C,
acidified with 1% HCl w/v (VWR International), stained overnight at
room temperature (20–25 ◦ C) with Trypan blue (Sigma-Aldrich, St.
Louis, MO, USA), and stored in 50% glycerol (Gram-Mol d.o.o., Zagreb,
Croatia). The stained root fragments were then mounted on slides, and
the total (TMC%), arbuscular (AC%), vesicular (VC%), and hyphal-only
(HOC%) colonisation was determined under a light microscope
(magnification 200×; Axio Plus; Carl Zeiss AG, Oberkochen, Germany).
One hundred fifty root intersections per plant were analysed using the
magnified intersections method (McGonigle et al., 1990).
2.4. Determination of yield and quality parameters
Tomato yield is represented as kg/fruits per plant. Basic quality pa­
rameters such as dry matter content (%), juice pH (MP220 Basic pH/
mV/◦ C Meter; Mettler-Toledo GmbH, Giessen, Germany), soluble solids
content (◦ Brix) (mg/kg fresh weight [FW]) (Artisan, HR200 Portable
refractometer; APT Instruments, Omaha, NE USA), and total acidity (%
citric acid/FW) (Solarus 20 mL burette; Hirschmann Laborgeräte GmbH
I. Pasković et al.
& Co., Eberstadt, Germany) were determined according to the principles
of AOAC International (1980), with minor modifications. The amount of
lycopene (mg/kg dry weight [DW]) in the tomato puree samples was
determined by UV/Vis spectrophotometry as proposed by Fish et al.
(2002) using a Carry UV/Vis 50 spectrophotometer (Varian Inc.). Hy­
drophilic (H) and lipophilic extracts (L) of tomato samples were pre­
pared as proposed by Fanasca et al. (2006) and further subjected to
spectrophotometric determination (Varian Inc.) of the total phenol (TP)
content and antioxidant activity (AA). The TP content was estimated as
gallic acid equivalents (mg GAE/100 g DW) based on the reaction of
phenol coloration with Folin–Ciocalteu reagent (Kemika d.d., Zagreb,
Croatia) and sodium carbonate (Kemika d.d.) as TP_H DW, TP_L DW, and
TP(H + L) DW. AA was determined as Trolox equivalents (mmol TE/100
g DW) using the stable radical DPPH• (Merck KgaA, Darmstadt, Ger­
many) based on the principles of the methods described by Sánchez-
Moreno et al. (1998) as AA_H DW, AA_L DW, and AA_(H + L) DW. The
repeatability of the methods was checked by performing four repeated
measurements of each of the several samples from the selected treat­
ments. The relative standard deviations obtained for the analyses of
lycopene and TP contents and AA were below 5% in all cases. All cali­
bration curves (with seven concentration levels) were constructed and
measurements were performed in the absorbance range of 0.1–0.8. The
linearity was determined based on the analysis of standard solutions and
was satisfactory, with coefficients of determination (R2) of 0.9993 and
0.9971 for the analyses of TP content and AA, respectively.
2.5. Determination of volatile compounds
2.5.1. Sample preparation and extraction of volatile compounds
Before the measurements, tomato fruits were homogenised using a
Polytron PT 1600 E (Kinematica AG, Lucerne, Switzerland) in two cycles
of 30 s at 15,000 rpm with a 1 min pause between the cycles. The
homogenised tissue (4 g) was weighed (Mettler Toledo AB204; Mettler-
Toledo GmbH, Greifensee, Switzerland) in 20 mL tubes (Supelco, Bel­
lefonte, CA, USA), after which a magnetic stirrer was added, and the
tube was then closed with a silicon lid. The samples were then kept at
–20 ◦ C (Liebherr Profi line, Bulle, Switzerland) until analysis.
Volatile compound isolation from tomato fruit was performed using
a method previously described by Marković et al. (2007) with slight
modifications. Headspace solid-phase microextraction (HS-SPME) was
performed using a two cm divinylbenzene/carboxen/
polydimethyiloxane-coated fibre with an absorbent polymer thickness
of 50/30 µm (Supelco). Preequilibration was performed for 15 min at
40 ◦ C in a thermostated water bath (MSH-300; Biosan, Riga, Latvia) with
stirring (400 rpm). Volatile compounds were extracted by inserting the
SPME needle through the silicon lid and exposing it to the sample vapour
for 20 min at 40 ◦ C. The desorption of the extracted compounds was
performed in the injection port of a gas chromatogram for 10 min at
250 ◦ C.
2.5.2. Analysis of volatile compounds
The volatile compounds in the tomato samples were analysed using a
Varian GC 3900 (Varian Inc.) gas chromatograph (GC) equipped with a
split/splitless injector, flame ionisation detector (FID), and capillary
column CP-WAX 57 CB (50 m × 0.25 mm ID × 0.2 µm film thickness)
(Varian Inc.). Nitrogen was used as a carrier gas at a flow rate of 5.0 mL
min− 1 and pressure of 22.6 psi (155821.5 Pa). Injection was performed
in splitless mode by temperature desorption of the SPME fibre for 10 min
at 250 ◦ C. For the next sampling, the SPME needle/fiber was cleaned by
heating in the injector of another GC instrument for 10 min at 250 ◦ C.
The starting temperature of the instrument oven was maintained at
40 ◦ C for 4 min. Thereafter, it was raised to 190 ◦ C at a rate of 5 ◦ C/min
and maintained for 11 min. The temperature was then raised by
applying the same temperature interval up to 200 ◦ C, which was
maintained for 8 min. The temperature of the detector was maintained
at 250 ◦ C. The volatile compounds in the tomato fruit samples were
3
Food Chemistry 359 (2021) 129961
identified by comparing their retention times with those of pure stan­
dards, after spiking with pure standards of each of the investigated
volatile compounds. The standards used were ≥ 97% pure, except for
(Z)-3-hexenal, which was stabilised in a 50% triacetin solution. The
calibration was performed using the external reference method, and the
results were expressed in µg/kg as n-amyl alcohol equivalents (Marković
et al., 2007). The suitability of the method was verified by analysing its
repeatability based on three repeated measurements of a sample from
the same treatment. The obtained relative standard deviations for the
majority of compounds were lower than 10%, except for 2-phenyletha­
nol (17%), ethyl butyrate (22%), β-ionone (30%), (+)-2-carene (31%),
and (-)-caryophyllene oxide (31%), which was considered satisfactory
considering the lower concentrations of these compounds and the nature
of the analysed samples (fruit with strong enzymatic activity). The an­
alyses were performed in triplicates and the obtained data were ana­
lysed using GC Workstation 6.41 software (Varian Inc.). A representative
GC-FID chromatogram with the assigned chromatographic peaks of the
investigated volatile compounds is shown in Fig. S1.
2.6. Sensory analysis of tomato fruits
Quantitative descriptive sensory analysis of tomato fruit samples was
performed by eight assessors, with extensive experience in sensory
analysis of foods and previously trained for tomato descriptors, ac­
cording to the methods proposed by Gajewski et al. (2014) and Auers­
wald et al. (1999). The odour, internal appearance, texture/mouthfeel,
and taste/flavour of tomato fruit were determined according to the
method proposed by Gajewski et al. (2014) using a modified profile
sheet expanded with descriptors for external appearance and firmness by
touch, as proposed by Auerswald et al. (1999). Single sensory attributes
were quantified using a 10 cm unstructured ordinal intensity rating scale
from 0 (no perception) to 10 (highest intensity). For overall quality
evaluation, tomato fruits were graded from zero (the lowest quality) to
10 (the highest quality). All the fertilisation × AMF application in­
teractions were assessed based on four replicates (32 in total) in plastic
containers coded randomly. The sensory analysis was divided into four
sessions with an equal number of samples. Each assessor worked in a
separate booth and received a tomato fruit sample per treatment. An
arithmetic mean of eight scores (one per assessor) for each sensory
attribute was used for further data treatment.
2.7. Statistical analysis
Data were tested for normality of distribution and homogeneity of
variance and transformed when necessary. To establish the main effects
of the investigated factors and to determine possible interactions be­
tween them, the data were subjected to a two-way analysis of variance
(ANOVA), with fertilisation and AMF application as factors. Means were
compared using the least significant difference Tukey’s post hoc test at
different levels (p < 0.05, p < 0.01, and p < 0.001). For significant in­
teractions, simple effects were computed at each level of the other factor
using Tukey’s post hoc test at p < 0.05. Partial least squares (PLS)
analysis was employed to build a linear model based on 15 variables
with the highest variable importance in projection (VIP) values. Statis­
tical analysis was performed using Statistica v. 13.4 software (Tibco®,
Palo Alto, CA, USA).
3. Results
3.1. Arbuscular, vesicular, hyphal-only, and total mycorrhizal
colonization
Inoculation of a substrate with AMF at sowing enabled initial colo­
nisation of up to 12% of inoculated seedlings at the time of trans­
plantation into the field, while at the sampling (103 DAT), inoculated
plants reached 55%–70% colonisation (data not shown). The
I. Pasković et al. Food Chemistry 359 (2021) 129961

fertilisation treatment had no effect on the percentage of AC, VC, and
HOC; however, a higher TMC percentage was found for the UNF than for
the MIN and ORG treatments and for the MIN + ORG compared with the
MIN treatment. The percentages of AC, VC, and TMC were higher in the
+ AMF treatment than in the -AMF treatment (Table 1).
3.2. Fruit mineral content
A significant effect of fertilisation on the concentration of N and P in
tomato fruit was observed (Table 2). The addition of MIN fertiliser
increased the concentration of N as compared to that found in UNF
plants. Furthermore, the P concentration was higher in fruits from all
fertilisation treatments than in those obtained from UNF plants. No
significant effect of fertilisation was found for the concentrations of K,
Mg, Ca, Mn, Zn, and Cu. Higher concentrations of N and Mn were found
in the tomato fruits of + AMF plants than in those of -AMF plants
(Table 2). The effect of the interaction between fertilisation and AMF
application was not significant for any of the investigated nutrients.
3.3. Fruit yield and quality parameters
Fertilised plants had higher yields than the unfertilised control,
whereas differences between the + AMF and -AMF plants were not
observed for the F × M interactions (Table S2). No differences in lyco­
pene, juice pH, total soluble solids content, or total acidity were found
regardless of the fertilisation or AMF inoculation treatment and their
interaction (Table S2). The combination of mineral and organic fertiliser
(MIN + ORG) increased the AA_H and AA_H + L concentrations when
compared to those of the application of mineral fertiliser (MIN) alone
(Table S3). AMF application influenced TP_H, and a higher concentra­
tion was found in the + AMF treatment than in the -AMF treatment
(Table S3). A significant interaction between fertilisation and AMF
application was observed only in DW. A simple effect test revealed that
the effect of fertilisation on DW was significant only at the -AMF level
(fertilisation × (-AMF), p < 0.05), with higher DW values found in fruits
from MIN×(-AMF) than in those from (MIN + ORG)×(-AMF) treatment
(Table S3).
3.4. Volatile compound profile of tomato fruit
The synthesis of particular volatile compounds in fatty acid catabo­
lism was affected by both factors (Table 3). The concentration of (E,E)-
2,4-decadienal was higher in the ORG treatment than in the UNF con­
trol. Among alcohols, the contents of 1-hexanol and 1-penten-3-ol were
the highest in the UNF with some exceptions, whereas (Z)-3-hexen-1-ol
was more abundant in the UNF and ORG than in the MIN treatment.
AMF application treatment affected both 1-hexanol and 1-penten-3-ol
by strongly decreasing their concentrations in the + AMF compared
with -AMF treatments. AMF application also decreased the total con­
centration of compounds generated during fatty acid catabolism,
although to a lesser extent. The interaction of fertilisation and AMF
application significantly affected fatty acid-derived alcohols as well as
the total concentration of this compound class (Table 3). Simple effect
tests showed that the effect of fertilisation was significant mostly at the
-AMF level (Fig. 1). The concentration of (E)-2-heptenal was higher in
the (MIN + ORG)×(-AMF) and UNF×(-AMF) than in the ORG×(-AMF),
whereas that of (E,E)-2,4-decadienal was the highest in the ORG×
(-AMF) treatment (Fig. 1a and b). 1-Penten-3-one was the only com­
pound from this group that was significantly affected by fertilisation at
the + AMF level, with a higher concentration obtained after ORG×
(+AMF) than in the MIN×(+AMF) and (MIN + ORG)×(+AMF) treat­
ments (Fig. 1d). At the -AMF level, the ORG×(-AMF) treatment was
characterised by the lowest 1-penten-3-one concentration as compared
to the other AMF treatments (Fig. 1d). AMF application exhibited a
negative simple effect at UNF for (E)-2-heptenal and a positive effect at
ORG for (E)-2-heptenal and at MIN for (E,E)-2,4-decadienal (Fig. 1a and
b). The simple effect of AMF application was significant at UNF for 1-
hexanol, MIN + ORG for 1-penten-3-ol, and at UNF, ORG, and ORG
+ MIN for total concentration (Fig. 1c, e, and f). The aforementioned
simple effects had the same sign as the main effects, except for the total
concentration in the ORG treatment, with a higher concentration found
in the ORG×(+AMF) than the ORG×(-AMF) treatment (Fig. 1f). The
simple effect of AMF application on 1-penten-3-one was positive when
interacting with ORG and negative when interacting with MIN + ORG
fertilisation (Fig. 1d).
The main effect of fertilisation was significant only for a single
compound originating from amino acid catabolism, 3-methyl-1-butanol.
The concentration found in the ORG was higher than that found in the
MIN and MIN + ORG treatments (Table 3). The main effect of AMF
application was significant for the majority of C5 compounds from this
group and for the total concentration of amino acid-derived compounds,
with higher levels observed in the -AMF than in the + AMF treatment.
The interaction between fertilisation and AMF inoculation significantly
affected 3-methylbutanal and 1-pentanol and the total concentration of
amino acid-derived compounds (Table 3), with similar simple effects
(Fig. 1g, h, and i). The simple effect of fertilisation was significant only
at the -AMF level in all cases (Fig. 1g, h, and i). Higher concentrations of
3-methylbutanal and total concentrations of amino acid-derived com­
pounds were found in the UNF and MIN + ORG than in the MIN and ORG
treatments (Fig. 1g and i). The concentration of 1-pentanol was higher in
the UNF than in the ORG and MIN treatments, while in the MIN + ORG,
it was also higher than that in the MIN treatment (Fig. 1h). The simple
effect of AMF application on these compounds was significant only at
the UNF level, resulting in higher concentrations of both compounds and
the total sum in the UNF×(-AMF) in relation to the UNF×(+AMF)
treatment (Fig. 1g, h, and i).

Table 1
The effect of fertilisation (F) (UNF – unfertilised, MIN – mineral fertiliser, ORG – organic fertiliser, MIN + ORG – mineral + organic fertiliser), AEGIS mycorrhiza
inoculation (M) (-AMF – non-mycorrhized, +AMF – mycorrhized), and their interaction (F × M) on the formation of arbuscular (AC%), vesicular (VC%), hyphal-only
(HOC%), and total mycorrhizal colonisation (TMC%) in tomato (mean ± SE, n = 4).
Treatments AC% VC% HOC% TMC%

F
UNF 38.50 ± 3.47 13.54 ± 3.34 21.93 ± 2.41 62.72 ± 2.62a
MIN 27.84 ± 4.21 9.36 ± 2.30 14.37 ± 1.94 45.96 ± 4.23c
ORG 29.01 ± 5.29 12.66 ± 3.20 17.28 ± 2.39 48.53 ± 6.52bc
MIN + ORG 32.94 ± 3.43 11.49 ± 1.95 21.49 ± 4.18 60.43 ± 4.83ab
M
-AMF 25.36 ± 2.10b 7.85 ± 1.07b 17.69 ± 1.85 45.61 ± 3.19b
+AMF 38.78 ± 2.85a 15.67 ± 2.06a 19.84 ± 2.32 63.21 ± 2.78a
F n.s. n.s. n.s. **
M *** ** n.s. ***
F£M n.s. n.s. n.s. n.s.

Mean values in columns followed by different letters are significantly different at p < 0.05, according to Tukey’s test.
n.s. non significant; *** p < 0.001; **p < 0.01; *p < 0.05. Two-way Analysis of Variance (ANOVA).

4
I. Pasković et al. Food Chemistry 359 (2021) 129961

Table 2
The effect of fertilisation (F) (UNF – unfertilised, MIN – mineral fertiliser, ORG – organic fertiliser, MIN + ORG – mineral + organic fertiliser), AEGIS mycorrhiza
inoculation (M) (-AMF – non-mycorrhized, +AMF – mycorrhized), and their interaction (F × M) on macronutrient and micronutrient contents of tomato fruit (mean ±
SE, n = 4).
Treatments N (g/kg) P (g/kg) K (g/kg) Mg (g/kg) Ca (g/kg) Mn (mg/kg) Zn (mg/kg) Cu (mg/kg)

F
UNF 18.96 ± 0.52b 3.19 ± 0.10b 29.65 ± 0.84 1.41 ± 0.03 1.48 ± 0.16 8.62 ± 0.31 14.98 ± 0.45 8.47 ± 0.18
MIN 22.61 ± 0.90a 3.96 ± 0.22a 34.99 ± 3.47 1.55 ± 0.06 1.37 ± 0.05 9.87 ± 0.51 16.63 ± 0.56 9.40 ± 0.46
ORG 21.01 ± 0.85ab 4.03 ± 0.08a 38.00 ± 2.45 1.56 ± 0.07 1.52 ± 0.04 9.36 ± 0.39 17.30 ± 1.70 8.75 ± 0.52
MIN + ORG 21.31 ± 1.00ab 4.13 ± 0.13a 33.08 ± 1.48 1.43 ± 0.09 1.41 ± 0.04 8.84 ± 0.57 16.06 ± 0.62 8.74 ± 0.48
M
-AMF 20.03 ± 0.58b 3.83 ± 0.11 33.23 ± 1.60 1.44 ± 0.03 1.43 ± 0.03 8.66 ± 0.28b 15.54 ± 0.45 8.72 ± 0.18
+AMF 21.93 ± 0.65a 3.91 ± 0.13 34.63 ± 1.87 1.54 ± 0.06 1.46 ± 0.08 9.69 ± 0.33a 16.94 ± 0.85 8.96 ± 0.40
F * *** n.s n.s. n.s. n.s. n.s. n.s.
M * n.s. n.s. n.s. n.s. ** n.s. n.s.
F£M n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

Mean values in columns followed by different letters are significantly different at p < 0.05, according to Tukey’s test.
n.s. non significant; ***p < 0.001; **p < 0.01; *p < 0.05. Two-way Analysis of Variance (ANOVA).

Table 3
The effect of fertilisation (F) (UNF – unfertilised, MIN – mineral fertiliser, ORG – organic fertiliser, MIN + ORG – mineral + organic fertiliser), AEGIS mycorrhiza
inoculation (M) (-AMF – non-mycorrhized, +AMF – mycorrhized), and their interaction (F × M) on the concentrations of volatile aroma compounds in tomato fruit
(mean ± SE, n = 4).
Volatile compounds (µg/kg FW) F M F M F£M

UNF MIN ORG MIN + ORG − AMF +AMF

Fatty acids catabolism

hexanal 14.53 ± 0.91 17.69 ± 1.81 25.41 ± 4.27 22.09 ± 4.35 22.14 ± 2.53 17.72 ± 2.21
(Z)-3-hexenal+(E)-2-hexenal 18.16 ± 2.01 20.63 ± 5.42 16.77 ± 1.53 17.19 ± 2.49 17.97 ± 1.15 18.4 ± 2.96
(E)-2-heptanal 164.65 ± 10.81 188.02 ± 11.82 155.47 ± 17.04 197.13 ± 17.85 175.98 ± 11.74 176.65 ± 10.11 ***
(E,E)-2,4-decadienal 33.79 ± 0.70b 40.31 ± 4.25ab 49.10 ± 3.21a 41.15 ± 3.53ab 38.34 ± 2.57 43.84 ± 2.45 ** *
1-hexanol 63.11 ± 20.48a 33.55 ± 3.90b 29.68 ± 3.92b 37.12 ± 6.10ab 53.29 ± 10.24a 28.44 ± 3.35b * ** ***
(Z)-3-hexen-1-ol 35.08 ± 4.54a 15.42 ± 1.23b 36.16 ± 5.72a 21.79 ± 2.87ab 30.59 ± 3.26 23.64 ± 3.58 **
1-penten-3-one 113.00 ± 8.59 117.08 ± 12.39 103.31 ± 17.04 108.94 ± 13.46 117.47 ± 9.61 103.7 ± 8.24 ***
1-penten-3-ol 19.25 ± 3.58a 11.26 ± 0.87b 13.02 ± 2.59ab 12.51 ± 1.64b 17.41 ± 2.04a 10.61 ± 0.96b * *** **
Total (fatty acids catabolism) 461.55 ± 37.92 443.96 ± 17.56 431.19 ± 31.74 457.92 ± 37.03 474.30 ± 21.90a 423.01 ± 20.36b * ***
Amino acids catabolism
pentanal (valeraldehyde) 141.82 ± 16.62 140.67 ± 8.26 146.59 ± 14.79 149.39 ± 10.48 161.13 ± 6.45a 128.1 ± 8.99b **
3-methylbutanal 187.40 ± 9.10 160.86 ± 11.20 172.62 ± 10.5 189.74 ± 17.85 183.93 ± 10.00 171.38 ± 7.85 *
(isovaleraldehyde)
a b
1-pentanol (n-amyl alcohol) 104.80 ± 17.14 85.33 ± 5.82 99.17 ± 6.80 112.8 ± 14.26 112.16 ± 7.60 88.89 ± 8.50 * *
3-methyl-2-butanol 6.56 ± 1.21 5.49 ± 0.78 7.56 ± 0.76 6.18 ± 1.17 7.49 ± 0.61a 5.41 ± 0.71b *
3-methyl-1-butanol 20.68 ± 2.84ab 15.27 ± 2.01b 26.67 ± 4.42a 12.50 ± 1.32b 21.72 ± 2.97a 15.84 ± 1.33b ** *
4-methyl-2-pentanol 0.83 ± 0.46 1.84 ± 1.68 0.00 ± 0.00 0.00 ± 0.00 1.21 ± 0.86 0.13 ± 0.09
2-phenylethanol 10.11 ± 0.93 9.49 ± 1.59 9.65 ± 0.80 7.94 ± 1.40 8.67 ± 0.61 9.92 ± 1.03
Total (amino acids catabolism) 472.2 ± 40.61 418.94 ± 14.91 462.26 ± 19.78 478.54 ± 40.09 496.31 ± 18.41a 419.67 ± 20.99b ** **
Esters
ab b a b
ethyl butyrate 2.96 ± 0.91 2.28 ± 0.62 4.86 ± 1.38 1.40 ± 0.43 3.34 ± 0.80 2.41 ± 0.56 ** ***
Terpenoid metabolism
(+)-2-carene 1.59 ± 0.45 2.35 ± 0.28 2.31 ± 0.54 1.54 ± 0.51 2.28 ± 0.35 1.61 ± 0.28
(R)-(-) α-phellandrene 6.60 ± 0.68 7.22 ± 0.53 7.03 ± 0.71 7.18 ± 0.64 7.03 ± 0.43 6.99 ± 0.46
(-)-(E)-caryophyllene 4.47 ± 0.71ab 3.14 ± 0.65b 5.27 ± 0.53ab 6.45 ± 1.00a 5.22 ± 0.59 4.45 ± 0.77 * *
α-humulene 4.81 ± 0.86 3.50 ± 1.26 1.87 ± 0.75 6.67 ± 2.01 3.87 ± 1.19 4.55 ± 0.98
(-)-caryophyllene oxide 2.60 ± 0.30b 7.51 ± 2.04a 4.72 ± 0.95b 3.67 ± 1.17b 3.99 ± 0.76 5.26 ± 1.16 *** ***
ß-ionone 1.25 ± 0.21 1.36 ± 0.36 1.54 ± 0.41 1.35 ± 0.26 1.24 ± 0.18 1.51 ± 0.24
Total (terpenoid metabolism) 21.33 ± 1.66 25.08 ± 3.7 22.73 ± 1.23 26.87 ± 4.07 23.64 ± 2.11 24.37 ± 2.05 *

Mean values in columns followed by different letters are significantly different at p < 0.05, according to Tukey’s test; n.s. non-significant; *** p < 0.001; **p < 0.01; *p
< 0.05. Two-way analysis of variance (ANOVA).

Ethyl butyrate was affected by fertilisation, and the concentration
found in the ORG treatment was higher than that in the MIN and MIN +
ORG treatments (Table 3). The source of a significant interaction effect
was by far the highest concentration found in the ORG×(− AMF) treat­
ment, compared with all the other treatments of fertilisation and AMF
inoculation, as observed by the simple effects test (Fig. 1j).
Among the volatile aroma compounds generated in terpenoid
metabolism and related compounds, a significant effect of fertilisation
on (-)-(E)-caryophyllene and its oxide was determined (Table 3). The
combination of MIN and ORG fertilisation resulted in higher (-)-(E)-
caryophyllene concentrations than the mineral fertilisation treatment
(MIN), whereas MIN showed significantly higher (–)-caryophyllene
oxide levels than all the other fertilisation treatments. AMF inoculation
was the main factor that did not affect terpenoids; however, the inter­
action between fertilisation and mycorrhization significantly affected
both caryophyllene derivatives and total terpenoids (Fig. 1k, l, and m).
Simple effects tests revealed that for (− )-(E)-caryophyllene and total
concentration of terpenoids, the effect of fertilisation was significant
only at the -AMF level, with the MIN×(− AMF) treatment having the
lowest concentration with respect to the other -AMF treatments (Fig. 1k
and m). Moreover, the effect of fertilisation on (–)-caryophyllene oxide
was significant only when mycorrhizae were inoculated (+AMF), and
the highest concentration was found in the MIN×(+AMF) treatment
(Fig. 1l). The positive effects of AMF application on (–)-caryophyllene

5
I. Pasković et al. Food Chemistry 359 (2021) 129961

Fig. 1. Simple main effects of two

factors, fertilisation (UNF – unfertil­
ised, MIN – mineral fertiliser, ORG –
organic fertiliser, MIN + ORG – min­
eral + organic fertiliser) and AEGIS
mycorrhiza inoculation (-AMF – non-
mycorrhized, +AMF – mycorrhized)
on the concentration (μg/kg) of volatile
compounds in tomato fruit. Asterisks
designate statistically significant dif­
ferences (n = 4, t-test, p < 0.05) be­
tween the treatments with and without
mycorrhiza separately at each fertil­
isation level, black lower-case letters
designate statistically significant dif­
ferences between different fertilisation
treatments without mycorrhiza, and
white upper-case letters designate sta­
tistically significant differences be­
tween different fertilisation treatments
with mycorrhiza. Identification: a) (E)-
2-heptenal (UNF p < 0.05; ORG p <
0.05; -AMF p < 0.05); b) (E,E)-2,4-
decadienal (MIN p < 0.05; -AMF p <
0.01); c) 1-hexanol (UNF p < 0.05;
-AMF p < 0.01); d) 1-penten-3-one
(ORG p < 0.01; MIN + ORG p < 0.05;
-AMF p < 0.001; +AMF p < 0.01); e) 1-
penten-3-ol (UNF p < 0.05; MIN + ORG
p < 0.05; -AMF p < 0.001); f) total
compounds from fatty acid catabolism
(UNF p < 0.01; ORG p < 0.05; MIN +
ORG p < 0.05; -AMF p < 0.01); g) 3-
methylbutanal (isovaleraldehyde)
(UNF p < 0.01; -AMF p < 0.001); h) 1-
pentanol (n-amyl alcohol) (UNF p <
0.01; -AMF p < 0.01); i) total com­
pounds from amino acid catabolism
(UNF p < 0.01; -AMF p < 0.001); j)
ethyl butyrate (ORG p < 0.001; -AMF p
< 0.001); k) (-)-(E)-caryophyllene
(-AMF p < 0.001); l) (-)-caryophyllene
oxide (UNF p < 0.05; MIN p < 0.01;
+AMF p < 0.001; -AMF p < 0.05); m)
total terpenoids (MIN p < 0.05; -AMF p
< 0.05).

6
I. Pasković et al. Food Chemistry 359 (2021) 129961

oxide were significant when applied without fertilisation (UNF) or with

mineral fertilisation (MIN) (Fig. 1l). A similar simple effect of AMF
application on the total terpenoid concentration was observed only at
the MIN level (Fig. 1m).

3.5. Sensory analysis of tomato fruit

The results of the sensory analysis of the tomato fruit are presented in
Table 4. Only a few sensory attributes were affected by the applied
treatments. The intensities of tomato external colour and sourness were
rated higher in fruits from ORG fertilisation than in UNF plants. AMF
application improved the external colour of tomato fruit; however, green
grass odour and skin thickness obtained higher scores in fruits from the
-AMF treatment than in those from the + AMF treatment (Table 4). A
significant effect of interaction between fertilisation and mycorrhization
was observed for flesh juiciness and tomato flavour (Table 4; Fig. S2a and
b). Simple effects tests showed that the effect of fertilisation on flesh
juiciness was significant at the -AMF level, with higher intensity observed
in the UNF and MIN treatments than in the ORG treatment, and at the +
AMF level for tomato flavour, with higher intensity observed in the MIN
Fig. 2. Partial Least Squares Discriminant Analysis (PLS-DA) model for the
and MIN + ORG treatments than in the UNF treatment fruits (Table 4; differentiation between fertilisation treatments (UNF – unfertilised, MIN –
Fig. S2a and b). No foreign odour or taste was found in the analysed mineral fertiliser, ORG – organic fertiliser, MIN + ORG – mineral + organic
fruits regardless of fertilisation, mycorrhization, or their interactions fertiliser) and AEGIS mycorrhiza inoculation (AMF – non-mycorrhized, +AMF –
(Table 4). mycorrhized) based on selected tomato fruit volatile compound contents (3-
methyl-2-butanol μg/kg FW, 1-penten-3-ol μg/kg FW, 1-hexanol μg/kg FW, (E,
3.6. PLS data analysis E)-2,4-decadienal μg/kg FW, (Z)-3-hexen-1-ol μg/kg FW), tomato sensory
properties (Skin thickness, Sweet, Fruit colour), tomato macronutrient content
Fifteen variables with the highest VIP scores were selected to build a (N g/kg DW, P g/kg DW), arbuscular (AC%), vesicular (VC%), and total
mycorrhizal colonisation (TMC%), tomato yield (kg/plant), and total hydro­
PLS model to further explore the differences between the treatments.
philic phenolic content (TP_H mg GAE/100 g DW).
The variables that successfully discriminated between the mycorrhiza-
inoculated and non-inoculated plants were TMC%, AC%, VC%, TP_H,
1-penten-3-ol, colour of the fruit, 3-methyl-2-butanol, N, skin thickness, 1- 4. Discussion
hexanol, and sweet (Fig. 2). The variables that separated the non-
fertilised plants from the other fertilisation treatment plants, regard­ Tomatoes have high natural symbiotic preferences for AMF (Smith
less of the AMF inoculation, were (E,E)-2,4-decadienal, P, yield, and (Z)- et al., 2011); therefore, the roots of tomato plants established from
3-hexen-1-ol (Fig. 2). The obtained PLS model could not distinguish noninoculated plantlets also showed a substantial root mycorrhization
among the MIN, ORG, and MIN + ORG fertilisation treatment plants. rate (TMC%) (Table 1). Nevertheless, native AMF alone did not reach
the level of TMC% colonisation that was obtained in the inoculated
seedlings (45.61% vs. 63.21%, respectively) or the level of AC% and VC

Table 4
The effect of fertilisation (F) (UNF – unfertilised, MIN – mineral fertiliser, ORG – organic fertiliser, MIN + ORG – mineral + organic fertiliser), AEGIS mycorrhiza
inoculation (M) (-AMF – non-mycorrhized, +AMF – mycorrhized), and their interaction (F × M) on the sensory characteristics of tomato fruit (mean ± SE, n = 4).
Sensory characteristics F M F M F£M

UNF MIN ORG MIN + ORG − AMF +AMF

External appearance
Fruit shape 7.33 ± 0.05 7.25 ± 0.03 7.36 ± 0.11 7.28 ± 0.09 7.24 ± 0.04 7.37 ± 0.06
Fruit colour 6.83 ± 0.11b 7.02 ± 0.10ab 7.31 ± 0.12a 7.11 ± 0.15ab 6.92 ± 0.09b 7.22 ± 0.08a ** **
Firmness by touch 7.14 ± 0.09 7.07 ± 0.15 7.18 ± 0.10 7.01 ± 0.08 7.15 ± 0.07 7.05 ± 0.08
Internal appearance
Flesh colour intensity 6.64 ± 0.14 6.67 ± 0.14 6.76 ± 0.18 6.94 ± 0.15 6.76 ± 0.11 6.75 ± 0.11
Odour of the cut fruit
Tomato odour 6.05 ± 0.11 6.11 ± 0.11 5.93 ± 0.15 6.15 ± 0.10 6.06 ± 0.09 6.06 ± 0.08
Green/ grass odour 3.27 ± 0.19 3.25 ± 0.09 3.14 ± 0.08 3.22 ± 0.10 3.33 ± 0.10a 3.11 ± 0.05b *
Fruity odour 1.23 ± 0.20 1.33 ± 0.16 1.02 ± 0.08 1.13 ± 0.11 1.21 ± 0.11 1.14 ± 0.09
Mouthfeel/texture
Skin thickness 7.17 ± 0.09 7.05 ± 0.08 7.28 ± 0.08 7.08 ± 0.09 7.25 ± 0.06a 7.04 ± 0.05b *
Flesh firmness 6.08 ± 0.15 6.01 ± 0.06 6.02 ± 0.05 6.02 ± 0.08 6.12 ± 0.06 5.95 ± 0.06
Flesh mealiness 4.71 ± 0.13 4.68 ± 0.14 4.77 ± 0.11 4.65 ± 0.16 4.62 ± 0.10 4.79 ± 0.08
Flesh juiciness 5.12 ± 0.1 5.4 ± 0.11 5.13 ± 0.12 5.29 ± 0.09 5.18 ± 0.06 5.29 ± 0.09 *
Taste
Tomato flavour 5.74 ± 0.15 5.95 ± 0.11 5.69 ± 0.10 5.91 ± 0.06 5.83 ± 0.08 5.82 ± 0.08 *
Sour 2.56 ± 0.09b 2.79 ± 0.10ab 2.92 ± 0.10a 2.86 ± 0.08ab 2.73 ± 0.07 2.84 ± 0.07 *
Sweet 3.65 ± 0.16 3.68 ± 0.11 3.30 ± 0.11 3.59 ± 0.13 3.64 ± 0.09 3.47 ± 0.10
Bitter 0.27 ± 0.06 0.39 ± 0.11 0.30 ± 0.06 0.32 ± 0 0.10 0.32 ± 0.06 0.32 ± 0.05
OVERALL QUALITY 7.04 ± 0.13 7.38 ± 0.16 7.13 ± 0.14 7.38 ± 0.17 7.16 ± 0.10 7.30 ± 0.12

Mean values in columns followed by different letters are significantly different at p < 0.05, according to Tukey’s test.
n.s. non significant; *** p < 0.001; **p < 0.01; *p < 0.05. Three-way Analysis of Variance (ANOVA).

7
I. Pasković et al.
% (Table 1). The TMC% value found in the non-inoculated plants in this
study was much higher than that found in the study by Cavagnaro and
Martin (2011) involving 15 different Australian farms, where native
mycorrhizal colonisation of processing tomato roots was generally lower
than 16%. Conversa et al. (2013) reported that native AMF inoculum
positively influenced the percentage of mycorrhization of previously
non-inoculated processing tomato plants at harvest time. In the afore­
mentioned experiment, the mycorrhization rate varied depending on P
fertilisation or the growing season up to 29%.
In this study, the effect of fertilisation was significant only for TMC%,
which was higher in the UNF than in the MIN and ORG treatments
(Table 1). A similar trend was observed for other mycorrhizal coloni­
sation parameters, although the difference was not statistically signifi­
cant (Table 1). Higher P uptake as a result of fertilisation may inhibit the
colonisation of roots to a certain degree, which occurs under conditions
of high P concentrations (Linderman & Davis, 2004; Conversa et al.,
2013). This presumption was corroborated by the higher P concentra­
tions found in the tomato fruits obtained from the treatments involving
fertilisation in relation to those from the UNF treatment (Table 2).
In a long-term field experiment, Gryndler et al. (2006) found that the
development of external mycelium on AMF was increased by organic
fertilisation but decreased by mineral fertilisation. Radić et al. (2014)
stated that fungal endophyte colonization tends to be higher under
organic than conventional soil management practices. Similar findings
were obtained by Linderman and Davis (2004). In this study, a higher
TMC% value (Table 1) was observed in the tomato roots in the MIN +
ORG treatment than in the MIN treatment, suggesting a possible
reduction in the negative effect of mineral fertilisation on root coloni­
sation when applied in combination with organic fertiliser. The lack of
the effect of interaction between fertilisation and AMF application on
the percentages of AC, VC, and TMC suggests that the main factors had a
strong effect (Table 1).
Colonisation with different AMF may result in divergent growth re­
sponses, implying AMF functional diversity (Rivero et al., 2015). In this
study, tomato fruit yield did not differ between -AMF and + AMF plants,
while all fertilised plants (3.52–3.92 kg/plant) had higher yields than
UNF plants (2.61 kg/plant) (Table S2). However, this study revealed the
influence of fertilisation and AMF application on other important to­
mato fruit quality parameters, such as minerals, AA, and TP content
(Tables 2 and S3). The P fruit concentration was higher in all fertilisation
treatments than in the UNF treatment (Table 2). However, no significant
differences in P fruit content were found between the -AMF and + AMF
tomato plants. In addition, the interaction of fertilisation and AMF
application (F × M) also did not significantly affect this parameter
(Table 2). Phosphorous soil acquisition occurs using two methods: direct
uptake by the root epidermis, including the root hairs or through AMF
pathways engaged in the root apex where a well-developed hyphal
network is included. These two pathways have a complex interplay,
which leads to highly variable contributions to P plant uptake (Smith
et al., 2011). In this study, the available soil P was at an adequate level
(Table S1), and the acquired mycorrhizal colonisation rate, either
inoculated or native, was sufficient to achieve a similar P fruit
concentration.
Organic fertilisers release nutrients more slowly than mineral fer­
tilisers, thus acting as slow-release fertilisers. In this experiment, only
mineral fertilisation (MIN) had a significant impact on fruit N concen­
tration as compared to the that of UNF treatment (Table 2), which could
be related to the slow release of nutrients after the ORG and MIN + ORG
treatments. AMF inoculation increased the N and Mn concentrations in
the tomato fruits (+AMF) compared to those in non-inoculated (-AMF)
plants (Table 2). Nitrogen uptake may be possible through the mycor­
rhizal pathway in mycorrhized plants (Smith & Smith, 2011), which
could be related to the observed increase. However, the results of this
study are in contrast to the findings of Kothari et al. (1991), who re­
ported lower acquisition of Mn in mycorrhizal plants. Different pro­
portions of Mn-reducing or Mn-oxidizing microbial populations and root
8
Food Chemistry 359 (2021) 129961
exudates with contrasting capacities for Mn reduction can result in
diverse plant Mn uptake (Nogueira et al., 2007). Therefore, nutrient
uptake is related to nutrient availability in the soil solution but also and
to the absorption capability of root systems, which is increased in
mycorrhizal plants (Liu et al., 2000).
Phenolic compounds are essential in the symbiotic interactions be­
tween plants and microorganisms owing to their ability to act as sig­
nalling molecules at the beginning of the association and establishment
of mycorrhizal colonisation and as defense compounds in the plant
(Mandal et al., 2010). The total concentration of phenols in the hydro­
philic phase (TP_H) was higher in fruits of the inoculated (+AMF) plants
(Table S3), which is consistent with published results reporting the
induced accumulation of phenols in mycorrhizal plants compared to
non-mycorrhizal plants (Baslam et al., 2011) and increased plant
biosynthesis of secondary metabolites in mycorrhizal plants (Ceccarelli
et al., 2010). The AA of tomato fruit derived from the hydrophilic
(AA_H) and from both the hydrophilic and lipophilic fractions (AA_(H +
L)) was higher when the combination of MIN + ORG fertilisers was
applied as compared to MIN fertilisation alone (Table S3). Moreover,
Omar et al. (2012) reported different antioxidant activities in cassava
tubers after the application of organic and mineral fertilisation.
The influence of fertilisation and AMF application and their inter­
active effects were established for volatile compounds in tomato fruits
(Table 3, Fig. 1). However, the observed effects on particular groups of
compounds did not follow uniform patterns, suggesting their
complexity. The differences in the composition of fruit volatiles
observed between different fertilisation treatments could not be attrib­
uted to the variability in their mineral composition because only the
control treatment UNF differed from the others by lower concentrations
of macronutrients such as P and N in some cases (Table 2). Plant lip­
oxygenases are among the key defense-related enzymes involved in both
processes associated with development and response to biotic and
abiotic stresses (da Trindade et al., 2019). Although not deficient, the
lower content of macronutrients found in the UNF plants could have
induced a somewhat higher expression of the genes involved in fatty
acid catabolism through the lipoxygenase (LOX) pathway, which
possibly resulted in higher concentrations of particular volatiles in the
UNF plants, such as 1-hexanol and 1-penten-3-ol (Table 3; Fig. 1e and f).
Organic fertilisation (ORG) resulted in the highest concentration of 3-
methyl-l-butanol, a volatile compound formed during amino acid
catabolism (Table 3). A supposedly high proportion of amino acids,
naturally present in dried poultry manure (Hacking et al., 1977) which
was used as fertiliser in the ORG treatment, may have contributed.
Mycorrhizal colonisation may lead to an increase in the activity of
LOX enzymes (da Trindade et al., 2019). A recent study hypothesised
that AMF symbiosis can help plants react against water stress by
inducing a LOXD gene-mediated pathway that contributes to the
biosynthesis of both terpenoids and “green leaf” volatiles in vegetative
tissue (Chitarra et al., 2016). Furthermore, plants synthetize and emit
specific monoterpenes and sesquiterpenes as important protective and
signaling molecules under stress conditions, and their release can be
influenced and enhanced by mycorrhizae inoculation (Dong et al.,
2016). In this study, AMF inoculation mostly negatively affected the
LOX volatiles (Table 3); however, an increase in the concentrations of
(E)-2-heptenal and 1-penten-3-one in the ORG×(+AMF) treatment and
of (E,E)-2,4-decadienal in the MIN×(+AMF) treatment was observed
(Fig. 1). A negative effect of AMF inoculation on the activity of
phenylalanine ammonia-lyase (PAL), another enzyme involved in plant
protection against stress, was observed in a previous study on alfalfa
roots (da Trindade et al., 2019). During the first stages of AMF inocu­
lation, PAL activity increased as a defence response; however, it sub­
sequently reduced to levels below that of the control group, presumably
to facilitate colonisation in the plant tissues (da Trindade et al., 2019).
An analogous effect of AMF inoculation on the activity of the enzymes
involved in the broader LOX pathway may have resulted in the dual
response of LOX volatiles in this study. A recent study demonstrated that
I. Pasković et al.
AMF inoculation alone did not influence the transcription of genes
involved in the encoding of pathogenesis-related proteins in tomatoes.
However, pathogen attacks on AMF-inoculated plants initiated much
stronger and more rapid defense responses of these plants and higher
expression of defense-related genes related to LOX, AOC, and PAL than
in non-inoculated plants (Song et al., 2015). If this was the case, the
differences in the production of LOX and other volatiles between the
treatments investigated in this study may have been much more
pronounced.
AMF inoculation has previously been shown to affect the volatile
compound composition of tomato fruits, including of fatty and amino
acid-derived volatiles; however, it was not significant in many cases.
Volatile compounds derived from phenylalanine were the most affected,
with mostly positive effects (Hart et al., 2015), which was not the case in
this study, where significant effects were mostly negative (Table 3).
Other studies found no significant differences between the levels of
linoleic acid LOX products in mycorrhizal and non-mycorrhizal roots
(Stumpe et al., 2005). Mycorrhization was previously shown to alter the
volatile terpene composition and emission (Hart et al., 2015; da Trin­
dade et al., 2019) but also with limited statistical significance and
without a predominant pattern.
Particular compounds derived from fatty acid catabolism through
the LOX pathway play an important role in the formation of typical
tomato aroma and flavour. They are commonly described as “green”
owing to their odours similar to green grass, leaves and herbs, and also
of tomato itself (Rambla et al., 2014). In this study, the concentrations of
key tomato “green” odorants, such as (Z)-3- and (E)-2-hexenal, were not
affected by fertilisation or AMF application. Although alcohols, such as
1-hexanol and 1-penten-3-ol, are less odoriferous than the above­
mentioned aldehydes, they are also LOX-generated compounds that
contribute “green” notes. They were found at higher concentrations in
fruits obtained from plants without AMF colonisation (Table 3), indi­
cating the possible pronounced green/grass odour found in these samples
during sensory analysis (Table 4).
The PLS analysis mostly confirmed the univariate statistical results,
and the values of TMC%, AC%, VC%, TP_H, 1-penten-3-ol, colour of the
fruit, 3-methyl-2-butanol, N, skin thickness, 1-hexanol, and sweet were
mainly affected by AMF inoculation, whereas fertilisation principally
influenced tomato fruit yield and P, (Z)-3-hexen-1-ol, and (E,E)-2,4-
decadienal concentrations.
The effects of fertilisation and AMF application on tomato fruit
composition and quality were quite complex. Different fertilisation
strategies influenced tomato fruit yield (Table S2), N and P (Table 2)
concentrations, AA (Table S3), and volatile compound concentrations
(Table 3; Fig. 1). AMF application was found to affect the concentrations
of N (Table 2), Mn (Table 2), TP (Table S3), and volatile compounds
(Table 3, Fig. 1). Volatile compounds were the most affected by the
interaction of the two main factors. They did not show a prevailing
pattern that would allow unambiguous conclusions to be drawn
(Table 3; Fig. 1). Only a few sensory features responded to different
fertilisation treatments combined with AMF application (Table 4;
Fig. S2), without substantial differences in overall tomato fruit quality
among the treatments. Despite the magnitudes of most effects being
relatively small, particular combinations of fertilisation and AMF
application resulted in specific improvements in chemical composition
and sensory properties, thus confirming the existing potential, which
requires further investigation. Such work should include at least a more
comprehensive range of AMF tested in combination with different fer­
tilisation regimes.
The open-field production conditions applied in this study for
obtaining processing tomato fruits were far from those achievable in
controlled greenhouse experiments, which was demonstrated by the
inability to obtain mycorrhiza-free plants in the field because of the
presence of native AMF. This was possibly a reason why some of the
observed effects were not more pronounced and not comparable to those
of previous studies. This finding also suggests that the potential of
9
Food Chemistry 359 (2021) 129961
natural AMF colonisation in a particular field/site could be tested and
used as a decision criterion before designing targeted AMF inoculation
treatments. The results of this study contribute to obtaining more real­
istic insights into the use of AMF as biostimulants capable of improving
the yield, quality, and flavour of commercial tomato.
CRediT authorship contribution statement
Igor Pasković: Conceptualization, Methodology, Formal analysis,
Investigation, Writing - original draft, Writing - review & editing,
Visualization, Supervision, Project administration, Funding acquisition.
Barbara Soldo: Validation, Investigation, Writing - original draft.
Smiljana Goreta Ban: Conceptualization, Methodology, Formal anal­
ysis, Writing - original draft, Writing - review & editing, Visualization,
Supervision. Tomislav Radić: Validation, Investigation, Writing - re­
view & editing. Marina Lukić: Validation, Investigation. Branimir
Urlić: Validation, Investigation. Matea Mimica: Validation, Investiga­
tion. Karolina Brkić Bubola: Validation, Investigation. Giuseppe
Colla: Writing - review & editing. Youssef Rouphael: Writing - review
& editing. Nikola Major: Formal analysis. Maja Šimpraga: Writing -
review & editing. Dean Ban: Writing - review & editing, Supervision.
Igor Palčić: Investigation. Mario Franić: Formal analysis. Kristina
Grozić: Writing - review & editing, Visualization. Igor Lukić: Concep­
tualization, Methodology, Formal analysis, Writing - original draft,
Writing - review & editing, Visualization.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The authors would like to acknowledge the support of the Croatian
Ministry of Agriculture (VIP project no. 2016-14-45 titled: Fertilisation
optimisation in processing tomato cultivation by mycorrhizae application)
and Istria County, Croatia. We would also like to thank Nevija Longo,
MEng for technical support and assistance in the nursery, Silvia Milišić,
Ing. for technical support in conducting mineral analysis, Danko Cvitan,
MEng for technical support in conducting field experiments, Atens,
Tarragona, Spain for Aegis donation, and Mr. Alessandro Belardinelli
(Esasem) for Red Valley seed donation.
The work of doctoral student Kristina Grozić has been supported in
part by the “Young researchers’ career development project – training of
doctoral students” under the Croatian Science Foundation project DOK-
2018-09-1841.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2021.129961.
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