Article

Cite This: J. Agric. Food Chem. 2019, 67, 1814−1822 pubs.acs.org/JAFC

Application of Trichoderma Strains and Metabolites Enhances

Soybean Productivity and Nutrient Content
Roberta Marra,*,†,‡ Nadia Lombardi,†,§ Giada d’Errico,†,‡ Jacopo Troisi,⊥,▽ Giovanni Scala,▽,○
Francesco Vinale,§ Sheridan L. Woo,‡,§,# Giuliano Bonanomi,†,‡ and Matteo Lorito†,‡,§
†
Department of Agricultural Sciences, University of Naples Federico II, 80055 Portici, Naples, Italy
‡
Task Force on Microbiome Studies, University of Naples Federico II, 80131 Naples, Italy
§
Institute for Sustainable Plant Protection, National Research Council, 80055 Portici, Naples, Italy
⊥
Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana″, University of Salerno, 84081 Baronissi, Salerno, Italy
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▽
Theoreo Srl, 84090 Montecorvino Pugliano, Salerno, Italy
○
Hosmotic Srl, 80069 Vico Equense, Naples, Italy
#
Department of Pharmacy, University of Naples Federico II, 80131 Naples, Italy
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ABSTRACT: Trichoderma fungi are effectively marketed worldwide as biocontrol agents and plant biostimulants on numerous
crops due to their demonstrated effects in direct antagonism against fungal pathogens and plant growth promotion. Here, we
examined the effects of single and combined applications of Trichoderma strains and their bioactive metabolites (BAMs)
harzianic acid (HA), 6-pentyl-α-pyrone (6PP), and hydrophobin1 (HYTLO1) on the growth, yield, and nutrient uptake of
soybean plants. Significant promotion of plant growth (up to 39%), as well as an increase in mineral content, was achieved with
BAMs, used alone or combined with T. harzianum. Interestingly, the treatments also increased the level of fatty acids (oleic,
linolenic, 11-eicosenoic, and stearic). This work demonstrates the usefulness of natural compound and microbe combinations to
enhance oilseed productivity, and reports for the first time the ability of Trichoderma and/or its BAMs to increase the lipid
content in harvested seeds.
KEYWORDS: Glycine max, plant biostimulants, bioactive metabolites, plant-microbe interaction, Trichoderma, oilseed crop

■ INTRODUCTION
Soybean, Glycine max (L.) Merr., is one of the most important
food crops with 313 million tons produced in 2016.1 This
legume is a rich source of high quality proteins (∼40% of the
dry weight) and oil (∼20% of the dry weight) and contains a
considerable amount of carbohydrates, amino acids, and
minerals that contribute to its nutritional value.2 Soybeans
and their derivatives have substantial economic importance in
a wide range of industrial applications, including animal feed
and, more recently, biodiesel production.3,4 They are the most
traded agricultural commodity, accounting for over 10% of the
total value of the global exchange.1 Numerous factors can
affect soybean productivity, including soil crusting, frost, hail,
insects, and diseases.5
The need to provide food for the increasing human
population challenges scientists and entrepreneurs to find
more effective tools to enhance sustainability while reducing
the negative impact to agriculture.6 These include using natural
compounds and microbes, generally referred to as plant
biostimulants, whose global market is predicted to increase
annually at a Compound Annual Growth Rate of 10.2% from
2017 to 2025.7 Biostimulants may in fact help plants to
overcome or tolerate stresses, therefore enhancing yield.8,9
Among the most studied cases of microbes able to
simultaneously exert beneficial effects on plant growth and
attack directly fungal pathogens are the filamentous fungi of
the genus Trichoderma.10−13 The interaction established in the
rhizosphere by Trichoderma has been related to the production
of bioactive metabolites (BAMs) able to induce systemic
resistance in the plant and enhance development.14−17 This
heterogeneous group of compounds includes secondary
metabolites, proteins, and peptides, with many of them
displaying multiple activities, i.e., antibiotic, siderophore,
plant growth regulator, and defense inducer.16
The use of BAMs, alone or in combination with the living
microorganism, may overcome some of the typical constraints
of biocontrol agent application, such as inoculum viability and
adaptation to environmental conditions, often resulting in a
loss of activity.18 In this work, we have determined the effect of
single and combined treatments of Trichoderma strain and
BAM-based formulations on the growth, productivity, and
nutrient content of soybean (G. max) plants grown in
greenhouse or field conditions. Different BAMs, fungal strains,
and application protocols were tested.
■ MATERIALS AND METHODS
Microbial Strains and Culture Conditions. In this work, three
different Trichoderma species (T. harzianum strains M10, T22, or
TH1, T. asperellum KV906, and T. virens strain GV41) were used.
Fungal cultures were obtained from the collection available at the
Received: November 23, 2018
Revised: January 14, 2019
Accepted: January 18, 2019
Published: January 18, 2019

© 2019 American Chemical Society 1814 DOI: 10.1021/acs.jafc.8b06503

J. Agric. Food Chem. 2019, 67, 1814−1822
Journal of Agricultural and Food Chemistry
Department of Agricultural Sciences of the University of Naples
Federico II and cultivated bimonthly on Potato Dextrose Agar (HI-
MEDIA, Pvt. Ltd., Mumbai, India) at 25 °C. Spore suspensions were
adjusted with water to the desired concentrations. The strains used in
this work were selected according to their biocontrol and/or plant
growth promotion activities as determined in previous experiments
carried out in our laboratory.
Fungal Metabolites. The Trichoderma BAMs harzianic acid (HA,
produced by T. harzianum strain M10) and hydrophobin1 (HYTLO1,
produced by T. longibrachiatum strain MK1) were extracted from
fungal culture filtrates as previously described.17,19 The Trichoderma
metabolite 6-pentyl-α-pyrone (6PP) was purchased from Sigma-
Aldrich (St. Louis, MO, USA). These metabolites showed abilities to
promote root development, induce plant resistance, and control
fungal pathogens.15−17
BAM solutions were prepared by diluting the compound with
distilled water to the final concentration used for the treatments. For
HA and 6PP, 0.1% ethyl acetate was added to facilitate resuspension
and successively evaporated under cabinet flow.
Plant material. The plant growth promotion (PGP) activity of
different combinations of Trichoderma strains and BAMs was tested
on soybean (Glycine max (L.) Merr., variety Sultana) in vitro and
under greenhouse or field conditions, using different modes of
applications. Experimental conditions are reported in details in the
following paragraphs.
PGP Activity of Trichoderma Strains in the Greenhouse. The
growth promotion activity of different Trichoderma strains (GV41,
T22, KV906, M10, TH1) on soybean plants was tested under
greenhouse conditions. Ten day-old plant roots were immersed for 30
min in a 30 mL fungal spore suspension (1 × 107 spore mL−1) and
then transferred into 14 cm diameter pots containing sterile soil (10
biological replicates per treatment). Water-treated plants were used as
controls. Treatments were repeated after 10 days by irrigating the
plants with the same solutions used for the root dip (30 mL per
plant). The pots were setup in a completely randomized design, and
the plants were grown under greenhouse conditions (day/night
temperature 28 °C/18 °C; humidity 50−60%; day length 14 h) and
watered when necessary. Stem and root lengths (SL, RL), plant fresh
and dry weights (FW, DW), and number of branches (NB) were
evaluated 45 days after sowing.
Application of Trichoderma Strains or BAMs to Soybean
Seeds. Soybean seeds were surface-sterilized with 1% (v/v)
hypochlorite solution, washed extensively with sterilized water, and
then dipped in a 50 mL Trichoderma KV906 spore suspension (1 ×
106 spore mL−1) or BAMs solution (HA and 6PP were both applied
at 1 × 10−6 M; HYTLO1 was used at 1 × 10−8 M). Water-treated
seeds were used as controls. Seeds were incubated for 2 h at 25 °C
under agitation (200 rpm), then transferred to Petri plates (140 mm
diameter) containing filter paper soaked with sterile water and
allowed to germinate for 3 days in a dark room at 25 °C. The
seedlings were transferred into 14 cm diameter pots containing sterile
soil, with 4 seeds per pots and 3 replicates per treatment. The plants
were grown under greenhouse conditions as described above. In vitro
seed germination and root length were measured, respectively, 2 and 3
days after sowing. Plant growth (SL, RL, FW) under greenhouse
conditions was measured 15 days after sowing.
Trichoderma Strain and BAMs Applied Singly or in
Combination in Greenhouse. Fungal spore suspensions (T.
asperellum KV906) and metabolite (HA, 6PP or HYTLO1) solutions
were tested singly and in combination (strain + single BAM) on 10
day-old soybean plants. Treatments (30 mL solution per plant) were
first applied by root dip, followed after 2 weeks by soil irrigation (30
mL solution per plant). The KV906 spore suspension was 1 × 107
spore mL−1, while BAMs solutions were applied at 1 × 10−6 M for HA
and 6PP, and at 1 × 10−8 M for HYTLO1. The combined
applications were prepared immediately before use. Plants were grown
in greenhouse as described above and biometric parameters (SL, RL,
FW, DW, NB) were evaluated 30 days after seeding.
Plant Treatments in Field Conditions. Experimental Site. The
field experiment was conducted during the 2016 planting season in a
1815
Article
field trial located at 40.81° N latitude, 14.34° E longitude, and 47 m
above mean sea level, at the Department of Agricultural Sciences of
the University of Naples Federico II, Portici (Naples, Italy). The plot
size was approximately 15 m × 7 m. The soil of the site was sandy
(78% sand, 12% silt, 10% clay) with pH 7.1 in water and nonsaline
[EC (1:2.5 soil/ water) 0.46 dS/m] and contained 17.3 g kg−1 of
organic carbon, available nitrogen (N; 1.77 g kg−1), and available
phosphorus (P2O5; 107.16 mg kg−1), and showed a carbon-to-
nitrogen (C/N) ratio of 9.77. The climate of the region is typically
Mediterranean. Since this field was never used previously to cultivate
soybean, a commercial bioformulation (Umostart, Sipcam, Italy),
based on selected nitrogen fixing rhizobia (Bradyrhizobium japonicum,
Rhizobium melioti, and Rhizobium leguminosarum bv. viciae) was
applied according to the manufacturer’s instructions as a nutritional
substrate to promote nodulation and root development.
Experimental Design and Treatments. The experiment consisted
of nine treatments, including water control, two Trichoderma strains
(KV906 and T22), three BAM solutions (HA, 6PP, and HYTLO1)
used singly, and three strain-BAM combinations (KV906 + HA,
KV906 + 6PP, and KV906 + HYTLO1). The field trial was arranged
in a completely randomized block design.
The treatments were performed once at the time of sowing as a
seed coating, and three more times to the soil every 30 days. Coating
was carried out in plastic bags (6 mL of coating solution for about 100
seeds). Seeds were mixed with the suspension used for coating. For
seed treatment the spore suspensions were 1 × 108 sp mL−1, while the
BAMs solutions contained 1 × 10−5 M of HA or 6PP and 1 × 10−6 M
of HYTLO1. A 10-fold dilution was used for soil irrigation with fungal
spores and/or metabolites. For the combined application, the
metabolites were added to the spore suspension at the time of
inoculation after adjusting for the desired concentration.
Crop Management. Soybean seeds were manually sown on 28/
04/2016 in rows, 80 cm apart at a depth of 4−5 cm and keeping 15
cm plant intervals, while 50 cm distance was used to avoid
contaminations among treatments. Each treatment consisted of at
least 25 plants with 3 replicates. Irrigation was applied two or three
times per week according to the crop needs but was interrupted 2
weeks before harvesting. The crop was harvested at maturity after 4
months.
Plant Sampling and Analyses. Soybean seed and plant samples
were collected from each plot, placed in paper bags, and oven-dried at
65 °C for 48 h. The growth and yield parameters measured included:
stem length (SL) and weight (SW), root weight (RW), number of
pods/plant (NPP), pods total weight/plant (PTW), number of seeds/
plant (NSP), seeds total weight/plant (STW), weight of 100 seeds
(W100S), number of seeds/pod/plant (NSPP), and single pod
weight/plant (PWP).
Chemical analyses consisted in the determination of protein, lipid,
and mineral content in harvested seeds. Crude proteins were
measured using the Kjieldahl method.20 Briefly, 2 g of oven-dried
seed sample were subjected to digestion at 450 °C (VELP, UK model
DKL) with 30 mL of 96% H2SO4 in the presence of K2SO4 (7 g) and
CuSO4 (0.7 g). Digested samples were alkalinized with 45% NaOH
and then subjected to steam-distillation (VELP, UK model UDK149).
The condensed distillate was gathered in an Erlenmeyer flask
containing 25 mL of 0.25 N H2SO4. The sulfuric acid not neutralized
by the ammonia present in the distillate was titrated with 0.25 N
NaOH until pH = 7.0 was reached. The ammonia rate was converted
into protein using 6.25 as conversion factor.
Raw lipid determination was carried out according to the AOAC
International method (920.85).21 Ten grams of dried sample were
weighed in a Soxhlet extraction thimble. Three grams of anhydrous
Na2SO4 were added, and absorbent cotton was used as a seal. Lipids
were extracted with hexane by using a Soxhtraction device (VWR
International Pbi). The hexane was removed with vacuum-packed
distillation followed by incubation in a stove at 105 °C for 1 h. The
extracted lipid weight was compared to the initial 10 g of the sample.
Esterification of fatty acids was carried out as previously reported
by melting solid lipid in an oven at 50 °C in order to determine its
composition.22 A drop of lipid was transferred into a 1.5 mL vial with
DOI: 10.1021/acs.jafc.8b06503
J. Agric. Food Chem. 2019, 67, 1814−1822
Journal of Agricultural and Food Chemistry Article

Table 1. Effects of the Applications of Different Trichoderma Strains (GV41, T22, KV906, M10, TH1) on the Growth of
Soybean Plants under Greenhouse Conditionsa
stem length root length fresh weight No. of branch-
(cm plant−1) (cm plant−1) (g plant−1) dry weight (g plant−1) es plant−1 No. of pods plant−1
treatment b
mean ± SE % mean ± SE % mean ± SE % mean ± SE % mean ± SE % mean ± SE %
CTRL 68.1 ± 1.8 a - 10.2 ± 0.5 a - 14.3 ± 1.1 ab - 2.48 ± 0.9 a - 7.1 ± 0.1 ab - 5.1 ± 0.3 a -
GV41 93.5 ± 5.7 bc +37 11.5 ± 0.4 ab - 15.9 ± 0.7 b - 2.47 ± 0.6 a - 7.3 ± 0.1 bc - 4.2 ± 0.2 bc -
T22 60.3 ± 3.1 a - 11.5 ± 0.5 ab - 12.8 ± 0.8 a - 1.97 ± 0.8 a - 6.7 ± 0.1 a - 4.3 ± 0.2 bc -
KV906 85.3 ± 3.2 bc +25 12.4 ± 0.7 b +21 16.5 ± 0.8 b - 2.74 ± 1.1 b +10 7.7 ± 0.1 c +8 4.8 ± 0.3 ab -
M10 95.8 ± 4.6 c +41 10.9 ± 0.4 ab - 15.8 ± 0.6 b - 2.46 ± 1.0 a - 7.7 ± 0.2 c +8 4.0 ± 0.2 c -
TH1 83.0 ± 3.6 b +22 12.2 ± 0.6 b +19 14.9 ± 1.1 ab - 2.43 ± 0.9 a - 7.5 ± 0.2 bc - 3.8 ± 0.3 c -
a
Treatments were applied at the time of transplant (root dip) and to the soil after 10 days. Results were collected 45 days after sowing. Data
represent the mean value of 10 biological replicates ± standard error (SE). Different letters in a column indicate statistically significant differences
for P < 0.05. Significant increments compared to control (CTRL) are reported as %. bT. harzianum strains M10, T22, TH1; T. asperellum strain
KV906; T. virens strain GV41.

Table 2. Effects of Single Applications of Trichoderma Strain KV906 or Metabolites (HA, 6PP, HYTLO1) to Soybean Seeds in
Vitro and in Vivoa
in vitro in vivo
−1 −1
germination (%) root length (cm plant ) root length (cm plant ) stem length (cm plant−1) fresh weight (g plant−1)
treatment mean ± SE % mean ± SE % mean ± SE % mean ± SE % mean ± SE %
CTRL 76.2 ± 1.5 a - 4.5 ± 0.1 a - 15.6 ± 0.4 a - 33.9 ± 1.3 a - 2.1 ± 0.1a -
HA 76.9 ± 1.5 a - 6.0 ± 0.1 b +33 16.7 ± 0.9 a - 38.6 ± 1.0 b +14 2.2 ± 0.1a -
6PP 72.1 ± 2.5 a - 5.0 ± 0.2 a - 15.2 ± 0.6 a - 40.5 ± 0.1 b +19 2.1 ± 0.1a -
HYTLO1 71.4 ± 0.9 ab - 4.7 ± 0.3 a - 17.6 ± 0.7 a - 39.4 ± 0.7 b +16 2.4 ± 0.1a -
KV906 66.7 ± 0.4 b - 4.6 ± 0.3 a - 14.8 ± 0.2 a - 40.7 ± 0.4 b +20 2.3 ± 0.1 a -
a
Germination rate and root length in vitro were determined on Petri plates, respectively, 2 and 3 days after treatments. Germinated seeds were then
transferred to sterile soil and plants were grown for 15 days in greenhouse. Data represent the mean value of three biological replicates ± standard
error. Each replicate consisted of four plants. Different letters in a column indicate statistically significant differences for P < 0.05. Significant
increments compared to control (CTRL) are reported as %.

a glass Pasteur pipet heated to 50 °C. One milliliter of hexane was
heated at 50 °C and added to the drop of lipid, and then the mixture
was slowly chilled to 25 °C. Next, 100 μL of 2 N KOH−CH3OH
solution was added. The vial was vortexed for 5 min and then left
under static conditions for 5 min in order to enable a complete
stratification of the hexanic portion, which contains the methyl ester
fraction of the fatty acids. Chromatographic separation was carried
out according to ref 23 with a GC Shimadzu (mod. GC2010P),
equipped with a Phenomenex ZB-WAX column 30 m, internal
diameter 0.25 mm, 0.25 μm thickness. GC conditions were as follows.
Carrier gas: He. Linear velocity: 40 cm s−1. Injector temperature: 220
°C. FID temperature: 250 °C. Oven program: 170 °C for 8 min, 2
°C/min to 185 °C for 10 min, 1 °C/min to 190 °C for 12 min, 10
°C/min to 240 °C for 5 min.
Seed mineral content was evaluated on ashes obtained according to
the International AOAC official method (900.02).21 About 10 g of
oven-dried seed samples were weighed in a capsule previously
calibrated at 550 °C for 4 h and chilled in a silica gel dryer.
Subsequently, the samples were burned on a little flame and then
incubated overnight in a muffle furnace (Heraeus model K1251F).
Then, ashes were chilled in a silica gel dryer and weighed soon after
reaching room temperature. Ash was dissolved in 2% ultrapure nitric
acid. The determination of the elements of interest was performed
using quadrupole inductively coupled plasma mass spectrometry
(ICP-QMS, model 820-MS, Bruker Daltonics, Billerica, MA). The
operational parameters were as follows. Plasma flow: 18 L min−1.
Auxiliary flow: 1.8 L min−1. Sheath Gas: 0.14 L min−1. Nebulizer flow:
Number of scan replicates: 10. Number of replicates for sample: 5.
High purity gases (99.9999% He, produced by SALDOGAS Srl, Italy;
99.9999% H2 produced by the DBS H2 generator PGH2-300) were
used to minimize the potential problems caused by unidentified
reactive contaminant species in the cell. High radio frequency power
(1400 W) helped maintain plasma stability. All chemicals were of the
highest commercially available purity grade. Before use, all glassware
and plastic containers were cleaned using 10% ultrapure grade HNO3
for at least 24 h and then rinsed copiously with ultrapure water before
use. Calibration solutions were prepared from multielemental
standard stock solutions of 20.00 mg L−1. Calibration curves were
obtained using nine calibration solutions. Reagent blanks containing
ultrapure water were additionally analyzed in order to control reagents
purity and laboratory equipment. Determinations were performed
using a mix solution of internal standards (6Li, 45Sc, 72Ge, 89Y,
103Rh, 159Tb, 165Ho, 209 Bi) at 10 μg L−1 online aspired with a T
union with the sample and standard solution.
Statistical Analysis. All obtained data were analyzed by one-way
ANOVA using SPSS 15.0 software, and significant differences among
treatments were separated using S−N−K (Student− Newman−
Keuls) and Fisher’s Least Significant Difference (LSD) post hoc tests
at the 0.05 level of significance.
Multivariate statistics (principal component analysis, PCA) was
used to address the relationship between experimental treatments and
soybean seed quality in terms of mineral (i.e., Al, Ca, Cu, Fe, K, Li,
Mg, Mn, Na, and Zn) and lipid content (i.e., arachidic, behenic,
eicosenoic lignoceric, linoleic, linolenic, oleic, margaroleic, margaric,
myristic, palmitic, palmitoleic, and stearic acids).

■
0.98 L min−1. RF power: 1.40 kW. Pump rate: 4 rpm. Stabilization
delay: 20 s. First Extraction Lens: −40 V. Second Extraction Lens: RESULTS
−166 V. Third Extraction Lens: −234 V. Corner Lens: −208 V.
Mirror Lens left: 29 V. Mirror Lens right: 26 V. Mirror Lens bottom: Effects of Trichoderma Strains or BAMs on Plant
30 V. CRI parameters were as follows. Skimmer Gas (H2) at 50 mL Growth in Vitro and in Greenhouse. Different Trichoderma
min−1. Sample Gas (He) at 10 mL min−1. dwell time, 10000 μs. strains belonging to the harzianum (M10, T22, TH1),
1816 DOI: 10.1021/acs.jafc.8b06503
J. Agric. Food Chem. 2019, 67, 1814−1822
Journal of Agricultural and Food Chemistry Article

Table 3. Effects of Trichoderma Strain KV906 and/or Metabolites (HA, 6PP, HYTLO1) Applications on the Growth of
Soybean Plants under Greenhouse Conditionsa
stem length (cm plant−1) root length (cm plant−1) fresh weight (g plant−1) dry weight (g plant−1) No. of branches plant−1
treatment mean ± SE % mean ± SE % mean ± SE % mean ± SE % mean ± SE %
CTRL 71.0 ± 3.8 a - 23.8 ± 0.7 abc - 3.7 ± 0.4 a - 1.9 ± 0.5 a - 4.0 ± 0.1 a -
HA 101.5 ± 5.4 bc +43 20.3 ± 1.6 a - 5.7 ± 0.5 bc +53 2.4 ± 0.4 ab - 5.3 ± 0.2 bc +31
6PP 118.8 ± 1.5 c +67 24.8 ± 1.9 bc - 6.2 ± 0.2 c +68 3.7 ± 0.6 c +47 6.0 ± 0.1 c +50
HYTLO1 84.5 ± 7.2 ab - 23.8 ± 1.5 abc - 4.2 ± 0.3 ad - 1.8 ± 0.1 a - 4.8 ± 0.5 ab -
KV906 80.3 ± 1.8 a - 26.3 ± 1.5 c - 4.2 ± 0.2 ad - 1.7 ± 0.2 a - 4.0 ± 0.12 a -
HA + KV906 104.0 ± 2.8 c +46 31.0 ± 0.6 d +31 4.8 ± 0.3 bd +29 2.8 ± 0.5 b +22 5.5 ± 0.2 bc +37
6PP + KV906 109.8 ± 7.5 c +55 20.5 ± 1.0 a - 4.7 ± 0.4 abd - 4.1 ± 0.6 c +56 5.3 ± 0.5 bc +31
HYTLO1 + KV906 109.3 ± 1.9 c +54 22.0 ± 0.5 ab - 5.0 ± 0.3 bd +34 1.9 ± 0.3 a - 6.0 ± 0.1 c +50
a
Treatments were applied at the time of transplant (root dip) and to the soil after 10 days (watering). Results were collected 30 days after sowing.
Data represent the mean value of four biological replicates ± standard error (SE). Different letters in a column indicate statistically significant
differences for P < 0.05. Significant increments compared to control (CTRL) are reported as %.

asperellum (KV906), or virens (GV41) species were tested for
their ability to promote soybean growth in greenhouse (Table
1). The spore suspensions were applied to the roots of 10 day-
old seedlings, and the treatment was repeated after 2 weeks by
soil irrigation. In several cases, stem (SL) and root lengths
(RL), plant dry weight (DW), and number of floral branches
(NB) were significantly enhanced (Table 1). In particular,
strains M10, GV41, KV906, and TH1 increased SL,
respectively, by 41, 37, 25, and 21% compared to control
(CTRL). KV906 produced the highest difference compared to
CTRL of RL (+21%), DW (+10%), and NB (+8%). Therefore,
this fungus was used for the experiments described below.
Application to soybean seeds without subsequent drenching
of Trichoderma KV906 spores or the BAMs HA, 6PP, and
HYTLO1 did not increase germination rate compared to
control (CTRL; Table 2). However, the metabolite HA
significantly enhanced RL (+33%) in vitro and SL (+14%) in
vivo. Interestingly, under greenhouse conditions, SL was
increased by about 19, 16, and 20% vs water-treated seeds
when, respectively, 6PP, HYTLO1, or KV906 were used
(Table 2).
Single and combined applications of metabolite solutions
(HA, 6PP, and HYTLO1) and/or spore suspension of T.
asperellum strain KV906 were tested under greenhouse
conditions (Table 3). We observed that several agronomic
traits were enhanced by Trichoderma applications, including SL
(up to 67%), RL (up to 31%), FW (up to 68%), DW (up to
56%), and NB (up to 50%). The metabolites HA and 6PP,
alone or in combination with KV906, were found to increase
significantly (P < 0.05) the values for the majority of the
parameters considered. Interestingly, some combinations
showed synergistic plant growth promotion (PGP) activity
compared to single treatments. For example, the application of
HYTLO1 and KV906 increased SL, FW, and NB compared to
control more than by using the strain or metabolite alone
(Table 3).
Effects of Trichoderma Strains and/or BAMs on
Growth and Productivity in Field Conditions. The effects
on soybean plants treated with Trichoderma strains (T22,
KV906) and/or metabolites (HA, 6PP, HYTLO1) in field
conditions were also investigated. SL and RW were not
affected by either single or combined applications of strains or
BAMs as compared to the control, and no significant
differences were observed in the number of seeds per pod
(Table 4). In contrast, stem weight (SW), number of pods per
plant (NPP), pod total weight (PTW), number of seeds per
1817
plant (NSP), seeds total weight (STW), and weight of 100
seeds (W100) were significantly enhanced (P < 0.05) by
different treatments vs the control (Table 4). Overall, the
highest promotion of soybean growth and yield were obtained
by treating the plants with 6PP or with the strain KV906 plus
HA or 6PP (Table 4).
At the end of the experiment, 6PP-treated plants showed an
enhanced development (+26% SL, +26% NPP, +32% PTW)
and yield-related traits, including NSP (+29%), STW (+39%),
and W100 (+8%). Similar results were obtained with the
combination of 6PP and KV906, but not with the Trichoderma
KV906 alone (Table 4). The hydrophobin HYTLO1, applied
alone or combined with the fungal strain did not increase
significantly (P < 0.05) the soybean growth or productivity
compared to water-treated plants. Moreover, no differences in
nodules formation were observed (data not shown).
Effects of Trichoderma Strains or BAMs on Nutrient
Uptake. Soybean grown in field plots treated with the
Trichoderma spore suspensions and/or the BAMs HA, 6PP, or
HYTLO1 showed enhanced nutrient concentration, including
Na, Zn, Al, and Li, compared to controls (Table 5). The
highest increase was observed for Na (up to 58%), Al (up to
89%), and Li (up to 150%), while no significant differences
were found in Fe, Mg, Ca, Mn, or Ni content. Treated seeds
showed also a decrease in K, Cu, and Se concentration
compared to controls (Table 5).
A satisfactory ordination of the seed quality (based on
mineral content) as resulting from treatment application in
field plots was provided by principal component analysis
(PCA), with the first two eigenvalues accounting for 63.4%
(45.3 and 18.1% for PC1 and PC2, respectively) of the total
variance (Figure 1). In Figure 1A,B are reported the loading
vectors and scores of grain quality in term of mineral content
in the bi-dimensional space. The first PCA component (PC1)
was positively correlated to Mg content, while Li, Ca, K, Zn,
and Cu had an opposite effect. Differently, the amount of Na
and Al was positively related to the PC2, while a negative
correlation was observed for Fe and Mg. The control contained
more Fe and Mn, while some treatments (e.g., HYTLO1 +
KV906) showed different chemical traits characterized by a
high Ca and Li content. Interestingly, 6PP used alone
increased the mineral concentration mainly of Na (32.73 mg
kg−1), Al (0.22 mg 100 g−1), and Li (37.41 μg 100 g−1), while
the combinations of BAMs and strain KV906 determined a
reduction of several macro- and microelements.
DOI: 10.1021/acs.jafc.8b06503
J. Agric. Food Chem. 2019, 67, 1814−1822
 Table 4. Effects of Trichoderma Strains (T22, KV906) and/or Metabolites (HA, 6PP, HYTLO1) on the Growth and Productivity of Soybean Plants under Field Conditionsa
stem length stem weight root weight No. of pods pod total weight No. of seeds seeds total weight weight of 100 seeds No. of seeds single pod weight
treatment (cm plant−1) (g plant−1) (g plant−1) plant−1 (g plant−1) plant−1 (g plant−1) (g plant−1) pod−1 (g plant−1)
CTRL 59.8 ± 2.6 a 69.7 ± 5.4 a 14.7 ± 0.4 ab 86.2 ± 6.5 ab 51.4 ± 4.7 ab 190.5 ± 15.8 ab 32.8 ± 4.1 a 17.1 ± 0.6 ab 2.2 ± 0.1 a 0.59 ± 0.01 ab
T22 56.5 ± 0.2 ab 73.8 ± 13.7 ab 14.2 ± 1.0 ab 87.0 ± 9.1 ab 54.5 ± 8.3 ab 198.7 ± 21.0 ab 34.8 ± 5.5 a 17.4 ± 0.8 ac 2.3 ± 0.1 a 0.61 ± 0.03 ab
HA 56.0 ± 2.8 ab 73.4 ± 5.1 a 9.7 ± 0.3 ab 95.2 ± 7.8 bc 56.0 ± 4.0 b 216.0 ± 15.7 bcd 36.0 ± 2.6 a 16.6 ± 0.1 b 2.3 ± 0.1 a 0.59 ± 0.01 ab
6PP 50.4 ± 1.9 cd 87.8 ± 3.2 c 14.2 ± 0.5 ab 108.8 ± 4.9 d 67.8 ± 2.7 c 246.0 ± 11.3 e 45.7 ± 1.8 b 18.5 ± 0.3 d 2.3 ± 0.1 a 0.62 ± 0.02 bc
HYTLO1 48.2 ± 3.2 c 59.7 ± 3.7 d 8.0 ± 0.2 a 78.1 ± 3.6 a 47.6 ± 3.0 a 178.6 ± 7.7 a 31.8 ± 2.5 a 17.8 ± 0.7 acd 2.3 ± 0.1 a 0.61 ± 0.02 ab
KV906 51.2 ± 0.6 cd 68.9 ± 4.3 ad 11.1 ± 0.6 ab 87.8 ± 5.6 ab 54.0 ± 3.3 ab 201.1 ± 10.2 ab 35.8 ± 2.5 a 17.8 ± 0.4 acd 2.3 ± 0.1 a 0.62 ± 0.02 bc
HA+KV906 58.5 ± 1.9 a 88.4 ± 10.0 c 18.5 ± 0.9 b 107.0 ± 8.0 cd 67.7 ± 8.2 c 242.0 ± 27.2 de 43.7 ± 6.7 b 17.9 ± 0.8 cd 2.3 ± 0.1 a 0.62 ± 0.03 bc
6PP + KV906 58.3 ± 7.0 ab 84.8 ± 2.7 bc 13.3 ± 0.6 ab 100.4 ± 4.2 cd 65.9 ± 1.2 cd 232.1 ± 11.0 cde 41.2 ± 2.5 bc 17.3 ± 0.4 ac 2.3 ± 0.1 a 0.66 ± 0.05 c
HYTLO1 + 54.0 ± 2.3 bd 71.8 ± 4.5 a 14.8 ± 0.9 ab 93.9 ± 6.4 bc 57.0 ± 4.4 bd 207.8 ± 10.8 bc 36.6 ± 2.6 ac 17.5 ± 0.9 ac 2.2 ± 0.1 a 0.60 ± 0.01 ab
KV906
a
Treatments were applied to the seeds (coating) and monthly to the soil (watering). Results were collected after 4 months. Data represent the mean value of three biological replicates ± standard error
(SE). Each replicate consisted of 25 plants. Different letters in a column indicate statistically significant differences for P < 0.05.
Journal of Agricultural and Food Chemistry

1818
Table 5. Effects of Trichoderma Strains (T22, KV906) and/or Metabolites (HA, 6PP, HYTLO1) on Soybean Seed Mineral Content at Harvesta
macroelements microelements
−1 −1 −1 −1 −1
Na (mg kg Ca (mg 100 g Mg (mg 100 g Fe (mg 100 g Zn (mg 100 g Cu (mg 100 g−1 Mn (mg 100 g−1 Al (mg 100 g−1 Li (μg 100 g−1
treatment DW) K (mg 100 g−1 DW) DW) DW) DW) DW) DW) DW) DW) DW)
CTRL 24.27 ± 2.70 cd 552.09 ± 5.90 bcd 243.06 ± 7.74 a a 170.94 ± 31.65 ab 6.76 ± 0.51 a 5.72 ± 0.18 ab 1.22 ± 0.03 d 1.52 ± 0.21 a 0.14 ± 0.02 ab 22.09 ± 1.19 a
T22 29.39 ± 3.35 de 542.06 ± 6.20 b 254.14 ± 30.65 a 150.86 ± 12.91 ab 5.02 ± 0.83 a 5.47 ± 0.17 a 1.04 ± 0.03 a 1.18 ± 0.13 a 0.12 ± 0.02 a 25.12 ± 3.30 c
HA 38.44 ± 1.92 f 518.25 ± 1.96 a 251.65 ± 17.45 a 173.18 ± 25.40 ab 5.86 ± 0.73 a 5.86 ± 0.20 ab 1.15 ± 0.04 bcd 1.52 ± 0.10 a 0.13 ± 0.01 ab 22.57 ± 1.84 a
6PP 32.73 ± 1.74 ef 558.93 ± 2.85 d 263.06 ± 24.74 a 149.91 ± 28.04 ab 6.50 ± 0.95 a 6.22 ± 0.23 bc 1.20 ± 0.03 cd 1.18 ± 0.05 a 0.22 ± 0.02 cd 37.41 ± 2.42 b
HYTLO1 15.62 ± 2.38 ab 521.44 ± 1.14 a 240.65 ± 14.18 a 183.01 ± 21.77 b 6.06 ± 0.78 a 5.65 ± 0.14 a 1.11 ± 0.03 abc 1.39 ± 0.21 a 0.27 ± 0.03 d 16.49 ± 1.92 a
KV906 16.67 ± 1.80 ab 548.36 ± 3.44 cd 258.93 ± 8.95 a 146.28 ± 17.70 ab 6.43 ± 0.36 a 6.20 ± 0.18 bc 1.22 ± 0.04 d 1.38 ± 0.10 a 0.15 ± 0.01 ab 35.88 ± 4.23 b
HA + KV906 13.17 ± 1.49 a 561.84 ± 1.55 bc 249.05 ± 30.92 a 144.26 ± 23.49 ab 6.04 ± 1.14 a 6.41 ± 0.20 c 1.20 ± 0.03 cd 1.29 ± 0.19 a 0.11 ± 0.01 a 40.20 ± 1.62 b
6PP + KV906 22.36 ± 1.32 bc 520.69 ± 0.98 a 236.08 ± 7.12 a 173.01 ± 2.85 ab 5.19 ± 0.07 a 5.59 ± 0.07 a 1.07 ± 0.06 ab 1.15 ± 0.12 a 0.20 ± 0.04 bc 23.69 ± 3.28 a
HYTLO1 + 25.98 ± 2.89 cd 557.69 ± 3.17 d 261.55 ± 38.88 a 115.68 ± 12.20 a 4.71 ± 0.56 a 6.23 ± 0.19 bc 1.23 ± 0.04 d 1.51 ± 0.23 a 0.24 ± 0.01 cd 55.33 ± 3.17 c
KV906
a
Data refer to seed dry weight (DW) and represent the mean value of three biological replicates ± standard error (SE). Different letters in a column indicate statistically significant differences for P <
0.05.
Article

J. Agric. Food Chem. 2019, 67, 1814−1822

DOI: 10.1021/acs.jafc.8b06503
Journal of Agricultural and Food Chemistry
Figure 1. Principal component analysis (PCA) showing loading plot (left) and scores (right) of soybean seed mineral (A,B) and fatty acid content
(C,D) as a function of single or combined application of Trichoderma strains (T22, KV906) or metabolites (HA, 6PP, HYTLO1) in field plots, as
compared to control (CTRL).
Protein content was almost unaffected by treatments in the
field (Table 6). Instead, total lipids increased up to 17%
following the application of single active principles (strains or
BAMs) as compared to combinations or controls (Table 6).
Interestingly, the content of both saturated and unsaturated
fatty acids in soybean seeds was affected by Trichoderma spore
applications: all the treatments increased the contents of
stearic, oleic, linolenic, and 11-eicosanoic acids, while
decreasing linoleic acid. Among the BAMs, 6PP used alone
enhanced the content of stearic (+40%) and 11-eicosanoic
(+19%) acids, while HA, alone or in combination with strain
KV906, increased the level of 11-eicosanoic acid by 12 to 21%,
and linolenic acid up to 39% (Table 6).
The eigenvalues of the first two components of the PCA
based on fatty acid content accounted for 51.2% (27.5 and
23.7% for PC1 and PC2, respectively) of the total variance
(Figure 1C,D). The first PCA component was mainly related
to the content of the two most abundant acids, linoleic acid
(positively) and oleic acid (negatively). The PC2, instead, was
associated with fatty acids that are quantitatively less abundant
(Figure 1C). Noteworthy, on PC1 the control was clearly
separated from all other treatments with a Trichoderma strain
and/or a BAM. The control, the only one in the upper right
quadrant, had the highest linoleic and the lowest oleic acid
content. Other treatments (e.g., HA, HYTLO1, 6PP + KV906,
KV906, and HA + KV906) fell, instead, in the left quadrants
(Figure 1D), associated with an oleic acid content higher than
that in control.
1819
Article
■ DISCUSSION
Trichoderma spp. are the active ingredients of numerous
biopesticides and biofertilizers because of their ability to
contrast fungal pathogens and promote plant growth by
implementing various modes of action.13,18,24 They are fast-
growing fungi found worldwide that can establish in many
different types of soil environments, showing a notable ability
to thrive in diverse climatic conditions. Recently, some of the
benefits provided to the plants have been associated with the
secretion of several bioactive molecules.16,17 Some Trichoderma
BAMs, such as harzianic acid, 6-pentyl-α-pyrone, and hydro-
phobin1, have been also involved in biocontrol, due to their
ability to reduce disease symptoms and stimulate the
expression of defense-related genes in the plant.14−17
In this work, we investigated the growth promotion activity
of selected Trichoderma strains and metabolites, applied either
individually or in combination, on soybean plants. In a
preliminary screening performed on seedlings inoculated with
Trichoderma strains, the highest effect in terms of SL, RL, DW,
and NB was obtained using T. asperellum strain KV906.
Therefore, this strain was used to test the combination of
spores and BAMs. In greenhouse conditions a single seed
treatment with the BAM HA, 6PP, or HYTLO1, as well as with
KV906 spore suspension, increased stem length up to 20%
compared to control. However, we found no effects on seed
germination rate of the tested cultivar (almost 100%
germination after 3 days). This finding is in contrast with
studies reporting enhanced seed germination rate in soybean
treated with different Trichoderma species.25−27 The high
germination rate of the soybean variety used for our
DOI: 10.1021/acs.jafc.8b06503
J. Agric. Food Chem. 2019, 67, 1814−1822
Journal of Agricultural and Food Chemistry Article

experiments or the mode and timing of treatment applications

Data refer to seed dry weight (DW) and represent the mean value of three biological replicates ± standard error (SE). Different letters in a column indicate statistically significant differences for P <
11-eicosenoic acid C20:1 (n
(root dip at the transplant and soil irrigation after 10 d) may

− 9) (g 100 g−1fat)

b,c

0.31 ± 0.01 de
explain this discrepancy. Thus, we tested if combinations of the

cd
bc

0.29 ± 0.01 bc
d

b
e
a
0.01
0.01
0.01
0.01
0.01
0.01
0.01
living microbe and bioactive molecules in multiple applications
(to the seed and/or to the soil) could have a beneficial effect
Table 6. Effects of Trichoderma Strains (T22, KV906) and/or Metabolites (HA, 6PP, HYTLO1) on Soybean Seed Content of Proteins and Lipids at Harvesta

±
±
±
±
±
±
±
more pronounced than each component applied singly. Our
0.26
0.29
0.29
0.31
0.30
0.28
0.33
data indicated that under greenhouse conditions either a
combined application or spores or BAMs used singly increased
significantly stem length (from 43 to 67%), fresh weight (from
linolenic acid C18:3

29 to 68%), dry weight (from 22 to 56%), and number of floral

bcd
(g 100 g−1fat)

cd

6.10 ± 0.05 cd
bc branches (from 31 to 50%), as compared to control. In field
d
d

6.17 ± 0.16 d
b
e
0.03
0.07
0.11
0.06
0.07
0.17
0.08
conditions, 6PP singly or in combination with KV906
±
±
±
±
±
±
± enhanced significantly PGP traits in soybean, including stem
unsaturated

5.45
6.28
7.59
6.10
5.81
6.06
5.78
length, number of pods, plant weight, and yield parameters,
such as seed number, total seed weight, and average weight of
100 seeds, as compared to water control or T. harzianum T22,
linoleic acid C18:2

51.43 ± 0.01 abc

de
bc

ab

which is a reference strain used in commercial formulations.18

(g 100 g−1fat)

e
a

52.37 ± 0.32 c
f
0.52
0.31
0.32
0.28
0.55
0.25
0.42

Similar, but less pronounced, beneficial effects were observed

±
±
±
±
±
±
±

following HA + KV906 application, but not with the

56.80
54.02
52.09
55.20
50.49
51.05
54.85

hydrophobin HYTLO1.
It is well documented that Trichoderma spp. may improve
plant fitness and productivity also in the field, especially when
oleic acid C18:1 (n −

growth conditions are unfavorable, such as during salt stress.28

fatty acids

9) (g 100 g−1fat)

b
b

b
a

a
c
c

23.28 ± 0.34 c

23.30 ± 0.21 c
0.41
0.27
0.13
0.25
0.43
0.13
0.14

These fungi have been reported to stimulate soybean root

system and enhance nodulation by helping the metabolism of
±
±
±
±
±
±
±

nodulating bacteria.27,29,30 Rudresh and colleagues have

20.46
22.24
22.25
20.80
23.66
23.22
21.98

indicated that the application of a microbial consortium

consisting of selected strains of Rhizobium, Trichoderma, and
Bacillus enhance growth, nutrient uptake, and yield of chickpea
arachidic acid C20:0

b,c,d

(Cicer aritenium L.) possibly due to an increased availability of

(g 100 g−1fat)

cd

bc
d

b
e

0.50 ± 0.01 e

0.40 ± 0.01 a

nutrients and a release of growth promoting substances

0.01
0.01
0.01
0.01
0.01
0.01
0.01

produced by the microbes.31 However, in our work we did

±
±
±
±
±
±
±

not observe differences among treatments in nodule formation.

0.46
0.44
0.45
0.50
0.42
0.49
0.44

Some Trichoderma strains have been found to enhance plant

nutrient uptake and nitrogen use efficiency,10,32 improve iron
assimilation through the release of siderophores,33,34 and
stearic acid C18:0

0.041 a
(g 100 g−1fat)

4.57 ± 0.07 d
0.01 b
0.05 b

0.06 b
0.01 b
0.10 e

0.03 c

4.18 ± 0.07 c

support the solubilization of soil phosphates and micro-

saturated

nutrients.35 In this work, a significant increase of macro- and

±
±
±
±
±
±
±

microelement content in seeds was caused by single or

3.46
3.88
3.92
4.84
3.91
3.76
4.12

combined applications of Trichoderma strains and/or BAMs.

This may be related to the PGP effect and the consequent yield
increase, and possibly is due to a more efficient micronutrient
palmitic acid C16:0

b,c

a,b
cd
(g 100 g−1fat)

13.14 ± 0.11 e
a

11.95 ± 0.21 c

assimilation by the root system supported by an improved

f
f
0.04
0.14
0.11
0.10
0.10
0.21
0.20

solubilization. The ability of Trichoderma spp. to increase the

±
±
±
±
±
±
±

bioavailability of insoluble or sparingly soluble minerals, such

12.00
11.84
12.40
11.28
14.35
14.15
11.49

as P, Fe, Zn, Cu, and Mn has been previously reported.36 In

particular, a beneficial effect on mineral nutrition was observed
in wheat and white lupin plants inoculated with T.
(g 100 g−1 DW)

cd

cd
cd
ab

bc

asperellum.33,34 It was associated with the production of

d

14.71 ± 0.08 d
a

12.21 ± 0.08 a
total lipids

0.02
0.05
0.10
1.07
0.14
0.07
0.06

siderophores able to counteract the reduced bioavailability of

micronutrients, particularly in low acidic soils.37,38 In addition,
±
±
±
±
±
±
±
12.52
12.35
14.44
14.67
14.50
14.37
13.50

Altomare and coauthors demonstrated the ability of T.

harzianum strain T22 to solubilize phosphates and other
compounds (Fe2O3, MnO2, Zn) by using a chelation or a
(g 100 g−1 DW)

de

31.76 ± 0.14 de
cd

bc
ab

bc

30.50 ± 0.64 bc
e
a

reduction mechanism and suggested a role of this process in

0.18
0.40
0.04
0.22
0.25
0.29
0.16
protein

biocontrol and PGP.35 However, our data indicated that the

±
±
±
±
±
±
±

concentration of several minerals, e.g., Ca, Mg, Fe, or Mn, does

31.21
29.18
30.50
29.86
32.56
30.48
31.73

not vary significantly in harvested grains collected from the

field. In any case, most of the studies carried out so far have
analyzed nutrient uptake in treated plants (above or below
HYTLO1 +
treatment

HYTLO1

ground parts) and not in seeds.29,33,34,36,39

KV906

KV906

KV906
KV906
CTRL

6PP +
HA +

In a recent work, Yadav and coauthors evaluated the effect of

0.05.
T22

6PP
HA

two rhizosphere-competent microbes (Pseudomonas f luorescens

a

1820 DOI: 10.1021/acs.jafc.8b06503

J. Agric. Food Chem. 2019, 67, 1814−1822
Journal of Agricultural and Food Chemistry
and T. asperellum), used either singly or in combination, on the
growth and nutritional quality of chickpea.40 Inoculated plants
showed the maximum increase in minerals, proteins,
carbohydrates, and other nutrients also in the edible parts. In
this work we measured total lipids as well as saturated and
unsaturated fatty acid concentration in the seeds produced by
treated plants. We observed that the lipid profile was affected
by Trichoderma or BAM application, with saturated (palmitic,
stearic, arachidic) and unsaturated (oleic, linoleic, linolenic,
11-eicosanoic) fatty acids over- or down-accumulated
compared to controls. The highest increase in fatty acid as
well as in total lipids concentration was obtained using the
BAMs HA and 6PP singly or in combination with the T.
asperellum strain KV906. Interestingly, treatments with
Trichoderma or BAMs produced seeds more rich in oleic
acid (C18:1) but reduced the content of linoleic acid (C18:2)
compared to controls. A higher content of monounsaturated
lipids in soybean seeds increases the value of the product in
terms of heart-healthy properties and stability in food
processing. To the best of our knowledge, this is the first
study reporting the ability of Trichoderma strains and/or
metabolites to affect lipid content and modify the fatty acid
profile of seeds harvested from any crop. This finding may
support a more efficient use of soybean in the food and
beverages (i.e., bean, meal, oil), as well as the automotive
industry (i.e., lubrificants), or for the manufacturing of
biobased plastics and biodiesel.
Our results highlight the usefulness of fungal bioactive
metabolites used either alone or in combination with beneficial
microbes to improve yield and product quality of important
leguminous plants such as soybean. The data presented here,
combined with those reported in the literature for other
important crops, can help to develop new generation of plant
biofertilizers and bioprotectants more effective than those
available on the market today. Moreover, the design of
microbial-based consortia providing multiple benefits to staple
crops could be synergistically combined with genetic
manipulation or plant breeding programs, thus increasing
yield quality of products and sustainability.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: robmarra@unina.it.
ORCID
Roberta Marra: 0000-0003-2110-7539
Jacopo Troisi: 0000-0003-2962-7379
Funding
This work was supported by the following projects: MIUR-
PON [grant number Linfa 03PE_00026_1; grant number
Marea 03PE_00106]; MIUR-GPS [grant number Sicura
DM29156]; Regione Puglia [grant number SIX 1410-12/06/
2015]; Grant contract no. KENYA-AID: 10306/CEFA/KEN
“Strengthening livelihoods of rural agro-pastoralists in Kitui
East sub county, Kitui County, Kenya.” The funders had no
role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors thank Paul Koppert and Harald Mikkelsen for the
useful discussion and ideas, and Koppert Biological Systems
1821
Article
Inc. for the financial and technical support of this work. The
authors gratefully thank Prof. Maria A. Rao (University of
Naples Federico II) for the soil analysis.
■
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Journal of Agricultural and Food Chemistry Article

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1822 DOI: 10.1021/acs.jafc.8b06503

J. Agric. Food Chem. 2019, 67, 1814−1822