European Journal of Agronomy 127 (2021) 126293

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European Journal of Agronomy

journal homepage: www.elsevier.com/locate/eja

Beneficial microbial species and metabolites alleviate soybean oxidative

damage and increase grain yield during short dry spells
Luiz Gustavo Moretti a, b, Carlos Alexandre Costa Crusciol a, *, João William Bossolani a, b,
Juliano Carlos Calonego a, Adônis Moreira c, Ariani Garcia a, Letusa Momesso a, b, d,
Eiko Eurya Kuramae b, d, *, Mariangela Hungria c
a
College of Agricultural Sciences, São Paulo State University (UNESP), Department of Crop Science, 18610-034, Botucatu, São Paulo, Brazil
b
Netherlands Institute of Ecology, NIOO-KNAW, Department of Microbial Ecology, 6708 PB, Wageningen, the Netherlands
c
Embrapa Soybean, Carlos João Strass Highway, Post Office Box 231, Londrina, PR, 86001-970, Brazil
d
Utrecht University, Institute of Environmental Biology, Ecology and Biodiversity, Padualaan 8, 3584 CH, Utrecht, the Netherlands

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

Keywords: Short dry spells are an important grain yield constraint in tropical regions. Plant growth-promoting bacteria
Antioxidant metabolism (PGPB) and their metabolites can mitigate the impact of drought stress by promoting changes in plant meta­
Glycine max bolism, physiology, and biochemistry. However, the effects of PGPB on soybean [Glycine max (L.) Merril] under
Photosynthesis
drought stress in tropical regions have not been established. The experiments were carried out under tropical
Plant growth-promoting bacteria
Water deficit
field conditions with short dry spells. Therefore, in this study we used a three-factorial trial to evaluate the effects
of bacterial consortium consisting of N2-fixing Bradyrhizobium japonicum (strain SEMIA 5079) and Bradyrhi­
zobium diazoefficiens (strain SEMIA 5080), the biocontrol agent Bacillus subtilis (strain QST 713), and the plant
growth-promoting Azospirillum brasilense (strains Ab-V5 and Ab-V6) with or without application of microbial
secondary metabolites (MSM, rhizobial metabolites enriched in lipo-chitooligosaccharides (LCOs)) during two
growing seasons. Photosynthetic pigments, gas exchange parameters, antioxidant enzyme activity and proline
concentrations in leaves, nodulation, plant growth development and grain yield were evaluated. The bacterial
consortium comprising Bradyrhizobium spp., A. brasilense strains and MSM application increased the contents of
chlorophyll a (14.5 %), chlorophyll b (30.8 %), total chlorophyll (17.2 %), and total carotenoids (27.3 %)
compared with Bradyrhizobium spp. treatment alone. This consortium also increased the net photosynthetic rate
(17.7 %), stomatal conductance (56.5 %), internal CO2 concentration in the substomatal chamber (8.3 %), and
transpiration (44 %) compared with plants that received the standard inoculation (Bradyrhizobium spp. only),
while reducing the leaf contents of hydrogen peroxide (− 18.8 %) and proline (− 29.4 %), lipid peroxidation
(− 15.9 %), and the activities of superoxide dismutase (− 18.2 %), catalase (− 21.2 %), and ascorbate peroxidase
(− 19.1 %). Taken together, the results indicate that a beneficial bacterial consortium comprising Bradyrhizobium
spp. and A. brasilense strains combined with MSM application can alleviate oxidative damage during dry spells.
Furthermore, this consortium improved soybean nodulation, plant growth development, and grain yield by up to
12.2 %.

1. Introduction agriculture worldwide (FAO, 2019). To guarantee global food security,

mechanisms to mitigate the effects of abiotic stresses caused by climate
Adverse environmental factors are increasingly impacting change have been investigated in several crop species (Fukami et al.,

Abbreviations: BNF, biological nitrogen fixation; ROS, reactive oxygen species; MD, malondialdehyde; SOD, superoxide dismutase; CAT, catalase; APX, ascorbate
peroxidase; PGPB, plant growth-promoting bacteria; MSM, microbial secondary metabolites; LCOs, lipo-chitooligosaccharides; IAA, indole-3-acetic acid; ACC
deaminase, 1-aminocyclopropane-1-carboxylate deaminase; A, net photosynthetic rate; gs, stomatal conductance; Ci, internal CO2 concentration in the substomatal
chamber; E, transpiration; FW, fresh weight; PH, plant height; NN, nodule number; NDW, nodule dry weight; RDW, root dry weight; SDW, shoot dry weight; GY,
grain yield.
* Corresponding authors.
E-mail addresses: [email protected] (C.A.C. Crusciol), [email protected] (E.E. Kuramae), [email protected] (M. Hungria).

https://doi.org/10.1016/j.eja.2021.126293
Received 6 October 2020; Received in revised form 13 April 2021; Accepted 13 April 2021
Available online 21 April 2021
1161-0301/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
L.G. Moretti et al. European Journal of Agronomy 127 (2021) 126293

2018a; Riar et al., 2018; Cerezini et al., 2019). Soybean [Glycine max (L.) different combinations of A. brasilense, B. subtilis, and rhizobial metab­
Merrill] is an important crop globally because of its use in protein, oil, olites) can alleviate the negative effects of dry spells in the field on
and biofuel production (Moretti et al., 2020a). In addition, due to its soybean carbon and antoxidant metabolism by increasing the intrinsic
high capacity for biological nitrogen fixation (BNF), soybean plays a key tolerance of soybean plants to abiotic stresses, thereby also improving
role in agriculture sustainability (Hungria and Mendes, 2015; Ciampitti grain yield. To test this hypothesis in practice, we established soybean
and Salvagiotti, 2018; Moretti et al., 2018). under tropical field conditions during two growing seasons (i.e., without
Soybean grain yield is affected by different environmental condi­ water supplementation and in region prone to dry spells during the crop
tions, especially pluvial precipitation and temperature (Chen and Wia­ cycle), aiming to mimic the field-real conditions of soybean cultivation
trak, 2010). Variations in the climate characteristics of tropical regions in rainfed areas.
used for cropping (Alvares et al., 2013; Cunningham, 2020) result in
differences in the intensity, frequency, and timing of water supply (Hu 2. Materials and methods
and Wiatrak, 2012; Battisti and Sentelhas, 2014). Water deficit is a
major limiting factor of soybean yield and can be mitigated at least 2.1. Site description
partially by using cultivars with appropriate physiological traits and
crop management (Battisti and Sentelhas, 2017). Two field experiments under rainfed conditions were carried out in
Several mechanisms of tolerance and resistance (e.g., expression of the 2016–2017 and 2017–2018 growing seasons with soybean at the
defence-related genes and phytohormones production) can be adopted Lageado Experimental Farm of São Paulo State University (UNESP),
by plants to deal with biotic and abiotic stresses. Among them, the major located in the municipality of Botucatu in the southeastern region of São
mechanism is to reduce the accumulation of reactive oxygen species Paulo State, Brazil (48◦ 26′ W, 22◦ 51′ S, elevation of 786 m above sea
(ROS) in plant tissues (dos Reis et al., 2017; Santos et al., 2017). ROS are level). According to the Brazilian soil classification system (Santos et al.,
byproducts of several metabolic pathways in various organelles, 2018), the soil is classified as a Latosol, which corresponds to the clas­
including chloroplasts, mitochondria, and peroxisomes (Gill and Tuteja, sifications of Ferralsol (Jahn et al., 2006) and Oxisol (Soil Survey Staff,
2010). ROS include free radicals resulting from the metabolism of ox­ 2014). According to the Köppen-Geiger climatic classification system
ygen (O2), such as superoxide radicals (O2− s), hydroxyl radicals (OH-s), (Alvares et al., 2013), the region has a mesothermal climate (Cwa), that
hydrogen peroxide (H2O2) and singlet oxygen (1O2). In plants, oxidative is, a humid subtropical climate with dry winters and hot summers. The
stress may be relieved by antioxidant enzymes such as superoxide dis­ average rainfall is 1,360 mm year− 1, and the mean annual air temper­
mutase (SOD; EC 1.15.1.1), catalase (CAT; EC: 1.11.1.6), and ascorbate ature is 20.7 ◦ C (50-year average) (Unicamp, 2020). Climatological data
peroxidase (APX; EC: 1.11.1.11). SOD, which dismutates O2 radicals for the study period were collected from meteorological stations located
(1O2) in the first excited electronic state to H2O2, is the first line of de­ near (approximately 400 m) the experimental location. The character­
fense against ROS (Gill and Tuteja, 2010); H2O2 can then be detoxified ization of short dry spells (approximately 15 days) in southeastern Brazil
to H2O by CAT and APX through the conversion of H2O2 to H2O and O2 during the monsoon season (from October to April) were comprehen­
(Alam and Ghosh, 2018). sively established by Cunningham (2020) based on an analysis of a
Stomatal and osmotic adjustments can occur through several historical series of 37 austral summer seasons (from 1979 to 2016).
mechanisms and channels. Inorganic ions such as sodium (Na+), po­ Soil texture (Donagema et al., 2017) and chemical (van Raij et al.,
tassium (K+), and chloride (Cl− ) drive most of the osmotic potential in 2001) properties at a depth of 0–0.20 m were determined prior to the
many plant species, while sugars and amino acids, especially proline, are establishment of the experiment and are presented in Table 1. The
the main osmoregulators in vascular plants (Alexieva et al., 2001; Ueda quantification of the autochthonous bacterial population capable of
et al., 2001). Oxidative damage caused by excess ROS affects cellular
components, resulting in membrane disruption via lipid peroxidation
Table 1
(Santos et al., 2017). However, the accumulation of solutes such as
Physicochemical and biological attributes (0–0.20-m depth) before sowing.
proline can protect cells against increased ROS levels (Juge et al., 2012;
Mauad et al., 2016). Increased proline biosynthesis in plant tissues Soil properties Unit Value
contributes to enzymatic stabilization and/or osmoregulation in Physical
response to abiotic stress (e.g., salinity, drought, heavy metals, or Clay g kg− 1 602
Silt g kg− 1 281
extreme temperature) (Torello and Rice, 1986; Abbasi et al., 2013;
Sand g kg− 1 117
Mauad et al., 2016). Bulk density g cm− 3 1.19
Plant growth-promoting bacteria (PGPB) and their metabolites, e.g.,
lipo-chitooligosaccharides (LCOs) and lipopolysaccharides (LPS), may Chemical
play a strategic role in alleviating the deleterious effects of ROS through pH (CaCl2) – 5.10
the production of various phytohormones, such as ethylene (Glick, Soil organic matter g kg− 1 26.2
Phosphorus–available (P resin) mg kg− 1 59.0
2012), cytokinins (Tien et al., 1979), gibberellins (Bottini et al., 1989), Calcium (Ca2+ resin) mmolc kg− 1
25.0
abscisic acid (Cohen et al., 2008), salicylic acid (Sahoo et al., 2014), Magnesium (Mg2+ resin) mmolc kg− 1
15.0
Exchangeable
indole-3-acetic acid (IAA), and jasmonic acid (Spaepen and Vanderley­ Potassium (K+ resin) mmolc kg− 1
3.90
den, 2015), among others (Fukami et al., 2018a), leading to the acti­ Aluminum (Al3+ KCl) mmolc kg− 1
2.00
1
Potential acidity (H + Al) mmolc kg− 42.0
vation of physiological and biochemical mechanisms that confer plant
S-Sulfate (S–SO2−
4 Ca(H2PO4)2) mg kg− 1 4.90
tolerance to abiotic and biotic stresses (Fukami et al., 2018b, c). Boron (B Hot water) mg kg− 1 0.40
Phytohormones produced by PGPB regulate processes and signaling Copper (Cu DTPA-TEAa) mg kg− 1 8.80
networks involved in plant responses that may ultimately lead to Iron (Fe DTPA-TEA) mg kg− 1 22.0
increased grain yields (Moretti et al., 2020a, b), including antioxidant Manganese (Mn DTPA-TEA) mg kg− 1 26.2
Zinc (Zn DTPA-TEA) mg kg− 1 2.10
metabolism (Fukami et al., 2017), exopolysaccharide production
Base saturation (BS) % 51.0
(Sandhya et al., 2010), osmotic adjustment (Cerezini et al., 2014), heavy Cation exchange capacity (CEC pH 7.0) mmolc kg− 1
86.0
metal mitigation (Bashan and de-Bashan, 2010), plant pathogen
biocontrol (Nicholson, 2002), and induced systemic tolerance (Pozo Biological
et al., 2008). However, the effects of beneficial microbes and microbial Most probable number CFUb g− 1
9.32 × 104
metabolites on soybean under water stress are poorly understood. a
DTPA-TEA, diethylenetriaminepentaacetic acid-triethanolamine.
We hypothesized that beneficial microbes (Bradyrhizobium spp. with b
CFU, colony forming units.

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L.G. Moretti et al. European Journal of Agronomy 127 (2021) 126293

nodulating soybean in the soil was estimated by the most probable obtaining by growing individually Rhizobium tropici strain CIAT 899 (=
number (MPN) using soybean plants as trapping hosts, according to the CNPSo 103, = SEMIA 4077) and B. diazoefficiens strain USDA 110 (=
methodology described by O’Hara et al. (2016). CNPSo 56) in modified-YM medium (Hungria et al., 2016) enriched with
Liming to increase the base saturation (BS) of the topsoil (0–0.20 m inducer of nodulation genes for each species, resulting in metabolited
depth) to 70 % was conducted approximately 60 days prior to the enriched in lipo-chitooligosaccharides (LCOs) (Marks et al., 2013,
beginning of the experiment using dolomitic limestone [CaMg(CO3)2] 2015). Both strains are also are deposited at the “Diazotrophic and Plant
(280 g kg− 1 of calcium oxide—CaO, 180 g kg-1 of magnesium oxi­ Growth Promoting Bacteria Culture Collection of Embrapa Soja”. After
de—MgO, and 81 % of calcium carbonate equivalents—%ECaCO3) ac­ centrifugation, the supernatants of each bacterium were liophilized,
cording to the methodology described by Quaggio and van Raij (1997). resulting in the MSM product. Prior to soybean sowing, the lyophilized
MSM were resuspended in a mixture of acetonitrile and water (20 %;
2.2. Experimental design and treatments w/v) (Marks et al., 2015) and mixed in equal parts of metabolites of
R. tropici and B. of diazoefficiens.
A randomized complete block (RCB) design involving a 4 (inoculant
treatments) × 2 (metabolite applications) × 2 (growing seasons) facto­ 2.3.3. Inoculants
rial scheme was used with four replicates. The standard inoculation (SI) was carried out with liquid inoculants
The inoculation treatments factor was represented by (i) standard containing B. japonicum strain SEMIA 5079 and B. diazoefficiens strain
inoculation (SI) with Bradyrhizobium japonicum (strain SEMIA 5079) + SEMIA 5080 with a concentration of 7 × 109 cells mL− 1 and applied to
Bradyrhizobium diazoefficiens (strain SEMIA 5080) via seeds at sowing; provide 1.2 × 106 cells seed− 1. SI is a common practice in soybean
(ii) SI + foliar spraying with Bacillus subtilis (Bs) (strain QST 713) at the cultivation in tropical regions (Hungria et al., 2006, 2020).
V3 growth stage (Fehr and Caviness, 1977); (iii) SI + foliar spraying with The MSM concentration was adjusted to 1.0 mL L− 1, which corre­
A. brasilense (Ab) (strains Ab-V5 and Ab-V6) at the V4 growth stage; (iv) sponds to approximately 10-8 M, and applied at rate of 4 mL kg− 1 of
SI + foliar spraying with Bs at V3 + foliar spraying with Ab at V4. The seeds.
metabolite applications factor was represented by the presence or Foliar inoculation (by foliar spraying) of B. subtilis strain QST 713
absence of application of microbial secondary metabolites of (1 × 109 cells mL− 1) was performed at the V3 soybean phenological
B. diazoefficiens USDA 110 + Rhizobium tropici CIAT 899. stage (Fehr and Caviness, 1977) by applying 3 L of the inoculant in 200 L
It is worth mentioning that annual inoculation of soybean is a of water to 1 ha. Foliar spraying of A. brasilense strains Ab-V5 (= CNPSo
practice regularly adopted by the farmers, even in areas that have been 2083) and Ab-V6 (= CNPSo 2084) at a concentration of 2 × 108
inoculated for several years, such that nowadays about 80 % of the area cells mL− 1 was carried out at the V4 soybean phenological stage (Fehr
cropped with soybean in the contry is annually inoculated. Therefore, and Caviness, 1977) by applying 300 mL of inoculant in 150 L water to
standard inoculation was considered as the control treatment in our 1 ha.
study. The field experiments were conducted in the growing seasons of Foliar spraying with A. brasilense strains and B. subtilis was carried
2016–2017 and 2017–2018. out according to the technical recommendations of the manufacturers
and was always performed in the late afternoon (5:00 pm).
2.3. Microbial strains, metabolites and inoculants
2.4. Crop management practices history
2.3.1. Microbial strains
Bradyrhizobium japonicum strain SEMIA 5079 (= CPAC 15, = CNPSo Prior to soybean (cultivar TMG 1264 RR; Tropical Breeding & Ge­
07) and B. diazoefficiens strain SEMIA 5080 (= CPAC 7, = CNPSo 06) are netics, Cambé, PR, Brazil) sowing (Semeato, model Personale Drill 13,
used in commercial inoculants in Brazil since 1992 (Hungria et al., 2006; Passo Fundo, RS, Brazil) the experimental area was cultivated with black
Hungria and Mendes, 2015). The Brazilian strain selection program for oats (Avena strigosa Schreb.) to provide straw for the no-tillage system.
natural variants of B. japonicum and B. diazoefficiens adapted to envi­ Each plot consisted of 10 rows spaced 0.45 m apart, leading to a total
ronmental conditions was detailed previously by Hungria and Vargas plot area of 45 m2. The plots were separated by 0.5-m-wide rows and
(2000), and the strains used in this study are representative of the ma­ 1.5-m-wide terraces to avoid cross-contamination (bacteria and fertil­
jority of inoculants applied to soybean-cropped areas in the country. izer) from rainfall surface runoff. The soybean plants were fertilized
Inoculation with these strains is practiced even in areas that have with 300 kg ha− 1 of 00–20–20 (60 kg ha− 1 of P2O5 and 60 kg ha− 1 of
received inoculants for several years, as the annual inoculation allows K2O) in both growing seasons. Seeds were treated with fungicides
average grain yield increases of 8%. More information about the strains (carboxin + thiram at 100 g + 100 g a.i. 100 kg− 1 seeds) prior to
and their performance is available elsewhere (Hungria et al., 2020, inoculation and sowing. Seed inoculation was performed 1 h before
2006; Hungria and Mendes, 2015). sowing. Foliar inoculations were carried out with a tractor-mounted
Azospirillum brasilense strains Ab-V5 (=CNPSo 2083) and Ab-V6 sprayer. At phenological stage V4 (Fehr and Caviness, 1977), all treat­
(=CNPSo 2084) were selected, validated under field conditions and ments received a foliar application of 20 g ha-1 of molybdenum (Mo) (as
launched for the use in microbial inoculants for grasses in Brazil in 2009 Na2MoO4.2H2O) and 2 g ha-1 of cobalt (Co) (as CoCl2. 6H2O), which are
(Hungria et al., 2020), and for co-inoculation of soybean in 2013. required to maximize BNF with soybean (Embrapa, 2020). The man­
Nowadays estimates are that 25 % of the area cropped to soybean in agement of weeds, insects, and diseases were carried carried according
Brazil is co-inoculated with Bradyrhizobium and these two A. brasilense to the recommendations of Embrapa (2020) when necessary so these
strains. More information about the A. brasilense strains is available were not limiting factors.
elsewhere (Hungria et al., 2010; Santos et al., 2021).
B. japonicum, B. diazoefficiens and A. brasilense strains are deposited 2.5. Sampling, measurements and data analyses
at the “Diazotrophic and Plant Growth Promoting Bacteria Culture
Collection of Embrapa Soja” (WFCC Collection # 1213, WDCM Collec­ 2.5.1. Soil water content
tion # 1054), in Londrina, State of Parana, Brazil. Bacillus subtilis strain Prior to the establishment of the experiment, the soil water-holding
QST 713 was developed by Bayer CropScience (European Union, Reg. capacity was determined according to the tension table and the Richards
(EC) no. 839/2008). extractant chamber methods (Cassel and Nielsen, 1986), which allowed
the determination of the soil water potential (ψw). The climatological
2.3.2. Microbial metabolites water balance was monitored and calculated via electronic spreadsheets
The microbial secondary metabolites (MSM) were extracted according to the methods proposed by Rolim et al. (1998) following the

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L.G. Moretti et al. European Journal of Agronomy 127 (2021) 126293

procedure of Thornthwaite and Mather (1995) to determine the real atmospheric CO2, 1100 μmol m− 2 s− 1 of photosynthetically active ra­
evapotranspiration (ETr). The climatological water balance of the two diation (PAR) supplied by LED lamps, 25–27 ◦ C leaf chamber temper­
experimental growing seasons is shown in Fig. 1. ature, and 60–70 % relative humidity. The minimum equilibration time
In both growing seasons, at the R5 growth stage (beginning of seed for each set of measurements was 3 min. The measurements were per­
development) (Fehr and Caviness, 1977), which corresponds to seeds formed between 10:00 and 12:00 am. The following parameters were
with a length of approximately 3.0 mm in the pod at one of the four determined: net photosynthesis rate (A; μmol CO2 m− 2 s− 1), stomatal
uppermost nodes on the main stem, photosynthetic pigments, gas ex­ conductance (gS; mol H2O m− 2 s− 1), internal CO2 concentration in the
change, oxidative stress, antioxidant metabolism, and proline content substomatal cavity (Ci; μmol mol− 1), and transpiration (E; mmol H2O
were measured in the third fully expanded leaves of plants under m− 2 s− 1).
moderate drought from short dry spells.
2.5.4. Oxidative stress and antioxidant metabolism
2.5.2. Photosynthetic pigments To evaluate the contents of H2O2 and malondialdehyde (MDA) as
Chlorophyll a (chl a), chlorophyll b (chl b), total chlorophyll (total well as the activities of SOD (EC:1.15.1.1), CAT (EC:1.11.1.6) and APX
chl) and total carotenoid (total car) contents were determined in the (EC:1.11.1.11), sampling was performed on the same leaflets taken at
third fully expanded leaves from five discs cut between the edge and the the R5 growth stage to assess the gas exchange parameters. The sampled
central rib of the soybean leaves using a paper punch (0.5 cm in diam­ tissue was then flash-frozen in liquid N and stored at − 80 ◦ C.
eter) at the R5 growth stage. The discs were incubated for 24 h in capped Lipid peroxidation was evaluated according to the method of Heath
glass vials containing 2 mL of N, N-dimethylformamide (DMF) and and Packer (1968). To calculate MDA content, a molar extraction co­
wrapped in aluminum foil according to the methodology proposed by efficient of 155 mM− 1 cm− 1 was used, and the results were expressed in
Lichtenthaler (1987). Pigment concentrations were quantified using the nanomoles of MDA per gram of fresh weight (FW). The H2O2 content
spectrophotometric method at wavelengths of 664, 647, and 480 nm for was determined according to the method of Alexieva et al. (2001). The
chlorophyll a, b and carotenoids, respectively. The pigment concentra­ content was calculated based on a calibration curve and expressed in
tions were determined as proposed by Wellburn (1994). micromoles of H2O2 per gram of FW. Extractions for enzyme analysis
were performed according to the methodology described by Silva et al.
2.5.3. Gas exchange (2020). SOD activity was evaluated as described by Giannopolitis and
Gas exchange was evaluated via nondestructive analysis with a Ries (1977), and the results were expressed in units (U) of SOD per
portable gas exchange device (LI-6400 infrared gas analyzer (IRGA), LI- milligram of protein. CAT activity was evaluated as described by Aze­
COR Biosciences Inc., Lincoln, NE, USA). Samples were taken from the vedo et al. (1998), and the results were expressed in micromoles per
central leaflet of the third fully expanded leaves and intact trifoliate leaf minute per milligram of protein. APX activity was evaluated according
from the plant apex of the main stem of 10 plants per plot. The pa­ to Gratão et al. (2008), and the results were expressed in nanomoles per
rameters of the instrument were as follows: 380–400 mol mol− 1 minute per milligram of protein.

2.5.5. Proline content

Proline content was determined according to Torello and Rice (1986)
in the same leaflets collected at the R5 growth stage used for the other
analyses. The absorbance at wavelengths of 647 and 664 nm was
determined via a spectrophotometer, and the results were expressed per
gram of FW, as described by Mauad et al. (2016).

2.5.6. Crop yield

Additionally, at the R5 stage, five plants per plot were harvested and
separated into shoots and roots according to the methodology described
by Hungria et al. (2006). In the laboratory, shoots were separated from
roots and the latter were carefully washed and placed in a forced-air
dryer at 65 ◦ C until constant weight was obtained (approximately
72 h). Nodules were removed from roots and dried again. From this
material, the nodule number (NN), nodule dry weight (NDW), root dry
weight (RDW), and shoot dry weight (SDW) were determined.
At physiological maturity, plants were harvested from a 15-m2 area
from the central part of each plot and estimated the final population of
plants, plant height, position of insertion of the first pod, numbers of
branches and pods per plant, and number of grains per pod. Grain yield
(kg ha− 1) and thousand-grain weight (W1000) were converted to values
on a dry weight basis by correcting for 13 % moisture. The moisture was
determined with an automatic measuring device (Gehaka G650i, Brazil).

2.6. Statistical analyses

All data were initially analyzed via the Shapiro-Wilk test (Shapiro
and Wilk, 1965) for normality and the Levene test for homoscedasticity
(Levene, 1960), both at p < 0.05; the UNIVARIATE procedure of SAS
version 9.4 was used for the analysis (SAS Institute, 2015). The data
Fig. 1. Climatological water balance at the experimental site during the 2016- were also tested for sphericity by the Bartlett test (Bartlett, 1937) via the
2017 and 2017-2018 growing seasons. ETc, crop evapotranspiration; ETr, real FACTOR procedure of SAS version 9.4 (SAS Institute, 2015). The results
evapotranspiration; R, rainfall. The arrows indicate the key time points in indicated that all data were distributed normally (W ≥ 0.90) and
the study. exhibited no sphericity. The data were then analyzed with the linear

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L.G. Moretti et al. European Journal of Agronomy 127 (2021) 126293

mixed-effect model by the PROC MIXED procedure of SAS and with the 3.2. Gas exchange
Satterthwaite approximation to determine the denominator degrees of
freedom for the fixed-effects tests. The blocks and spatial interactions The effects of the different treatments on gas exchange parameters
between neighbouring blocks were considered as random effects, were similar to those on photosynthetic pigments, highlighting the
whereas bacterial consortium, microbial metabolites, growing season, benefits of foliar spraying of A. brasilense and MSM application
and their interactions were considered as fixed effects. The results were (Table S2). Interaction effects of bacterial consortium and MSM inocu­
reported as the least square means and were separated by the probability lation on the net photosynthesis rate (A) (p ≤ 0.01), stomatal conduc­
of differences option (PDIFF). The means were compared using the least tance (gs) (p ≤ 0.01), internal CO2 concentration in the substomatal
significant difference (LSD) test. The main factors and interactive effects cavity (Ci) (p ≤ 0.01) and transpiration (E) (p ≤ 0.01) were detected
were considered statistically significant at p ≤ 0.05. The heatmap was (Table S2). Additionally, A, gs, Ci, and E increased significantly by up to
built calculating the Pearson correlation coefficients (p ≤ 0.05) and only 17.7 %, 56.5 %, 8.3 %, and 44.0 %, respectively, in all plants receiving SI
the significant correlations are shown. plus foliar spraying of A. brasilense strains and MSM application (Fig. 3A-
D) compared to plants receiving SI alone.
3. Results
3.3. Oxidative stress, antioxidant metabolism and proline content
3.1. Photosynthetic pigments
Interaction effects of bacterial consortium and MSM inoculation on
The photosynthetic pigments of soybean plants did not differ be­ H2O2 content (p ≤ 0.01), lipid peroxidation (assayed via the MDA
tween those that received SI plus inoculation with B. subtilis and those content) (p ≤ 0.01), SOD (p ≤ 0.01), CAT (p ≤ 0.01), and APX (p ≤
that received SI (Bradyrhizobium spp. only) (Table S1). However, plants 0.01) activities, and proline content (p ≤ 0.01) in soybean leaf tissue
inoculated with A. brasilense strains exhibited higher concentrations of were detected (Table S3). In addition, H2O2 content, lipid peroxidation
these pigments compared to plants that were not inoculated with rates, and proline content were highest in plants receiving SI only
A. brasilense, and application of MSM further increased these levels. A (Fig. 4A-B-F). By contrast, plants receiving SI plus foliar spraying with
significant interaction between bacterial consortium and MSM appli­ A. brasilense strains and MSM application exhibited 18.8 % lower H2O2
cation was observed (p ≤ 0.01; Table S1) for both chlorophyll and content, 29.4 % lower proline content, and 15.9 % lower lipid peroxi­
carotenoid contents in soybean leaves at the R5 reproductive stage in dation in leaf cell membranes. Moreover, this treatment resulted in
both growing seasons (Fig. 2). Plants treated with SI plus A. brasilense significant decreases in SOD (− 18.2 %), CAT (− 21.2 %), and APX
and MSM exhibited increases in chl a, chl b, total chl, and total car (− 19.1 %) activities compared to SI treatment alone (Fig. 4C-E).
contents of up to 14.5 %, 30.8 %, 17.2 %, and 27.3 %, respectively,
compared with the plants receiving SI only. The results for the two
growing seasons were similar.

Fig. 2. Chlorophyll a (chl a), chlorophyll b (chl

b), total chlorophyll (total chl), and total ca­
rotenoids (total car) in soybean leaves at the R5
reproductive stage as a function of bacterial
consortium and microbial metabolites under
field conditions. Different lowercase letters
indicate significant differences between treat­
ments, and different uppercase letters indicate
significant differences between the presence
and absence of application of microbial sec­
ondary metabolites by Fisher’s least significant
(LSD) test at p ≤ 0.05. The error bars express
the standard error of the mean of the two
growing seasons (n = 8).

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L.G. Moretti et al. European Journal of Agronomy 127 (2021) 126293

Fig. 3. Net photosynthetic rate (A), stomatal

conductance (gS), internal CO2 concentration in
the substomatal chamber (Ci), and transpiration
(E) of soybean leaves at the R5 reproductive
stage as a function of bacterial consortium and
microbial metabolites under field conditions.
Different lowercase letters indicate significant
differences between treatments, and different
uppercase letters indicate significant differ­
ences between the presence and absence of
application of microbial secondary metabolites
by Fisher’s least significant (LSD) test at p ≤
0.05. The error bars express the standard error
of the mean of the two growing seasons (n = 8).

3.4. Crop yield 4. Discussion

Positive interactions effects of bacterial consortium (Bradyrhizobium For successfully realizing attainable yield, soybean cultivation in
plus A. brasilense strains) and rhizobial metabolites application were South America requires 450–800 mm of rainfall (Embrapa, 2020). The
observed on soybean nodule number (NN, 19 %), nodule dry weight maximum crop requirement for water occurs between flowering and
(NDW, 21 %), root dry weight (RDW, 17.2 %) and shoot dry weight grain filling, with ETr of ~7–8 mm day− 1 (Embrapa, 2020) and a crop
(SDW, 20.6 %) when compared to exclusively SI (p ≤ 0.01, Table S4) coefficient (Kc) of ~1.5 (Farias et al., 2001). In the present study, total
(Fig. 5A-D). At physiological maturity, significant interactions effects of rainfall was 789 and 705 mm in the 2016–2017 and 2017–2018 growing
bacterial consortium and MSM application on soybean plant height seasons, respectively. However, there was an uneven distribution of rain
(PH), number of pods, thousand-grain weight (W1000) and grain yield during the crop cycle. Short dry spells with negative hydric balance
(GY) were detected (p ≤ 0.01, Table S5). Plant height, pods, W1000 and lasting 17 and 14 days were observed in the first and second growing
GY increased considerably when plants received SI plus foliar applica­ seasons, respectively, both prior to the R5.1 reproductive stage (Fig. 2)
tion of A. brasilense strains and MSM application, by up to 7.6 % (PH), and during the critical stage of early grain filling. According to Flexas
25.3 % (pods), 5.0 % (W1000) and 12.2 % (GY), respectively, compared et al. (2004), the stress level can be quantified via stomatal conductance,
to SI alone (Fig. 6A-D). No benefits of foliar application of B. subtilis were where values ≥ 0.2 mol H2O m− 2 s− 1 represent well-watered plants,
observed. Overall, significant effects were not observed in the final values of 0.1–0.2 mol H2O m− 2 s− 1 represent moderate drought-stressed
population of plants (mean = 288,886 plant ha− 1), position of insertion plants, and values ≤ 0.1 mol H2O m− 2 s− 1 represent severely
of the first pod (mean =12 cm), number of branches per plant drought-stressed plants. According to this classification, in both growing
(mean = 3), nor in the number of grains per pod (mean = 2.2) (data not seasons the plants treated with SI only were under moderate drought
shown). stress at the R5 stage.
Short dry periods during the summer cropping season are common in
3.5. Correlation analysis between plant measurements tropical regions (Silva et al., 2019) and, combined with the high tem­
perature and evapotranspiration typical of this period of the year,
Changes in pigments concentrations correlated positively with all greatly impact crop productivity, particularly during critical periods of
photosynthetic parameters from soybean gas exchange measurements, plant development such as flowering and grain filling (Basu et al., 2016).
and both correlated with soybean plant responses, including root In addition, water restriction during the reproductive stage (R1–R5;
nodulation and dry weight, as well as production of shoot, pods and beginning of flowering until grain formation) leads to several physio­
grains (Fig. 7). On the other hand, oxidative stress and antioxidant logical, morphological, and biochemical changes resulting in loss of leaf
metabolism correlated negatively with all other parameters studied and turgor, premature flower dropping, pod abortion and ultimately lower
positively with themselves. grain yields (Nelson et al., 2005).
Chlorophyll is the main photosynthetic pigment in the

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L.G. Moretti et al. European Journal of Agronomy 127 (2021) 126293

Fig. 4. Hydrogen peroxide (H2O2) content,

malondialdehyde (MDA) content, superoxide
dismutase (SOD), catalase (CAT), and ascorbate
peroxidase (APX) activities, and proline content
in soybean leaves at the R5 reproductive stage
as a function of bacterial consortium and mi­
crobial metabolites under field conditions.
Different lowercase letters indicate significant
differences between treatments, and different
uppercase letters indicate significant differ­
ences between the presence and absence of
application of microbial secondary metabolites
by Fisher’s least significant (LSD) test at p ≤
0.05. The error bars express the standard error
of the mean of the two growing seasons (n = 8).

Calvin–Benson–Bassham (CBB) cycle (Croft et al., 2017; Busch, 2020). effects of LCOs on photosynthesis and plant growth acceleration by
Reduced chlorophyll content decreases the light energy uptake and light stimulating mitotic activity in the meristematic tissue of leaves (Khan
utilization capacity of plants, resulting in a decrease in photosynthesis et al., 2020).
(Ansari et al., 2019). Carotenoids are responsible for plastid adaptation Our results also demonstrated that, compared with plants inoculated
to stress conditions such as light and energy dissipation and avoiding exclusively with Bradyrhizobium spp., the combination of Bradyrhi­
excessive ROS production (Rodriguez-Concepcion et al., 2018). One of zobium spp. inoculation with A. brasilense and MSM treatment improved
the main effects of drought in higher plants is reduction and/or inhibi­ the efficiency of gas exchange, including stomatal conductance and
tion of photosynthesis (Reddy et al., 2004), as reflected by decreased transpiration. Regulating stomatal conductance under conditions of low
synthesis of chlorophyll and carotenoids. As schematized in Fig. 8, our hydric availability is key to improving water-use efficiency (Gupta et al.,
field experiments in this tropical region clearly demonstrated that the 2020). Our results suggest that A. brasilense plus MSM positively influ­
bacterial consortium represented by seed treatment with Bradyrhizobium enced stomatal opening and closing, which directly affected the process
spp. and rhizobial metabolites (MSM) and foliar application of of gas exchange and contributed to increased biomass accumulation. A
A. brasilense at the V4 stage increased photosynthetic pigments, namely, previous study conducted on the same site also reported important roles
chl a, chl b, total chl, and total car. However, the increased growth of of MSM in Bradyrhizobium-soybean symbiosis (Moretti et al., 2020b).
plants inoculated with MSM could be at least partially related to indirect This previous study suggested that a more developed root system

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L.G. Moretti et al. European Journal of Agronomy 127 (2021) 126293

Fig. 5. Nodule number, nodule dry weight,

root dry weight, shoot dry weight in soybean at
the R5 reproductive stage as a function of bac­
terial consortium and microbial metabolites
under field conditions. Different lowercase let­
ters indicate significant differences between
treatments, and different uppercase letters
indicate significant differences between the
presence and absence of application of micro­
bial secondary metabolites by Fisher’s least
significant (LSD) test at p ≤ 0.05. The error
bars express the standard error of the mean of
the two growing seasons (n = 8).

increases soil exploration and, in turn, the distribution and activity of of these genes was always greatest when foliar spraying was combined
soybean roots, which might indirectly ensure plant metabolic activities with the application of rhizobial metabolites. This previous study cor­
and soybean establishment and favor tolerance to moderate water roborates our results and suggests that the persistence of oxidative stress
restriction. is reduced when live cells (only Azospirillum spp.) are applied in com­
The gradual loss of photosynthetic capability due to drought condi­ bination with rhizobial metabolites.
tions results in accumulation of electrons in photosystems I and II, Proline is an important stress marker molecule associated with
leading to increased ROS levels and lipid peroxidation (Croft et al., osmoregulation that prevents dehydration under negative soil water
2017). ROS can severely damage the photosynthetic apparatus, resulting potential (ψw) (Goswami et al., 2016). In our study, proline content was
in lipid peroxidation (Loix et al., 2018). To mitigate the effects of significantly lower in soybean leaves of plants treated with the con­
oxidative stress and reduce cell damage, a variety of enzymes in anti­ sortium comprising Bradyrhizobium spp., MSM and A. brasilense strains
oxidant metabolism are activated to inactivate ROS (Farooq et al., compared with plants treated with SI alone, suggesting an important
2019). In the present study, the H2O2 concentration and lipid peroxi­ impact of the former consortium on avoiding oxidative damage and
dation rate in soybean leaves were greater in plants inoculated just with reducing the accumulation of proline in tissues under water stress.
Bradyrhizobium spp. and with B. subtilis by foliar spraying at the V3 stage. Similar, Fukami et al. (2018b) reported that inoculation of maize with
By contrast, the lower activities of SOD, CAT and APX in soybean leaves Azospirillum reduced proline synthesis, and the decrease in proline
of plants treated with Bradyrhizobium spp. + MSM + A. brasilense indi­ content was correlated with the maintenance of plant metabolism under
cate an increased ability to mitigate ROS compared with inoculation abiotic stress. Similar results were reported in wheat (Triticum aestivum
with Bradyrhizobium alone. Indeed, SOD, CAT and APX activities have L.) with a consortium of seeds inoculated with Planomicrobium chinense
been used as important indicators of PGPB-induced plant resistance to and Bacillus cereus followed by foliar spray of salycilic acid one month
abiotic stresses, intracellular ROS removal, reduced membrane peroxi­ later, where leaf proline content, lipid peroxidation and antioxidant
dation, stabilization of membrane permeability and improved plant enzyme activity were decreased, and drought effects were decreased
photosynthesis (Sandhya et al., 2010; Fukami et al., 2018b, c). Inter­ (Khan and Bano, 2019).
estingly, in chickpea (Cicer arietinum L.) Khan et al. (2019) reported that Drought stress significantly affected the concentration of chlorophyll
proline content, lipid peroxidation and activities of antioxidant enzymes and carotenoids (Fig. 2), photosynthetic capacity and carbon assimila­
(CAT, APOX, POD, and SOD) increased in response to drought stress but tion of plants (Fig. 3), influencing root and shoot the dry weight (Fig. 5),
decreased due to a consortium of Bacillus spp. with the plant growth seriously reflecting 1000-grain weight (Fig. 6). The photoassimilates
regulators salicylic acid and putrescine, indicating physiological ad­ produced in the source leaves, mainly sucrose, are the main components
justments to reduce drought impacts, and our results support this of C metabolism and amino acid biosynthesis for the development of the
assumption. Fukami et al. (2017) reported that foliar spraying of maize grains (Du et al., 2020).Water stress during the grains filling phase
(Zea mays L.) with A. brasilense resulted in regulation of oxidative stress inhibited the synthesis and accumulation of carbohydrates, probably by
genes in leaves (APX1, APX2, SOD2, SOD4); however, the up-regulation blocking cell division (Awasthi et al., 2014; Sehgal et al., 2017), which

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L.G. Moretti et al. European Journal of Agronomy 127 (2021) 126293

Fig. 6. Plant height, pods, soybean 1000-grain

weight (W1000) and grain yield (GY) as a
function of bacterial consortium and microbial
metabolites under field conditions. Different
lowercase letters indicate significant differences
between treatments, and different uppercase
letters indicate significant differences between
the presence and absence of application of mi­
crobial secondary metabolites by Fisher’s least
significant (LSD) test at p ≤ 0.05. The error
bars express the standard error of the mean of
the two growing seasons (n = 8).

Fig. 7. Heatmap of the Pearson correlation coefficients matrix

between soybean physiological, biochemical and agronomic
parameters. Only significant correlations at p ≤ 0.05 are
shown. Chlorophyll a (Chl a), chlorophyll b (chl b), total
chlorophyll (Total chl), total carotenoids (Total car), net
photosynthesis rate (A), stomatal conductance (gs), internal
CO2 concentration (Ci), transpiration (E), hydrogen peroxide
(H2O2), malondialdehyde (MDA), superoxide dismutase (SOD),
catalase (CAT), ascorbate peroxidase (APX), proline, nodule
number (NN), nodule dry weight (NDW), root dry weight
(RDW), shoot dry weight (SDW), plant height (PH), pods,
thousand-grain weight (W1000) and grain yield (GY).

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L.G. Moretti et al. European Journal of Agronomy 127 (2021) 126293

Fig. 8. Schematic graphic representing the treatments and corresponding results in the field experiment.

reduced energy demand and promoted the early completion of the grain 2019; Khan and Bano, 2019), abiotic stresses may be mitigated. There­
filling process, resulting in considerable losses in productivity (Fig. 6). In fore, the consortium identified in our study represents an effective
this study, plants treated only with SI presented significant decrease in strategy to increase soybean grain yield by improving plant osmotic
nodulation, inhibition of root and shoot growth development, photo­ adjustment, maintenance of physiological processes and biological ni­
synthetic activity, and decrease in thousand-grain weight and yield. trogen fixation during the short dry spells that occur frequently in
Taken together (Fig. 7), the results indicate that a beneficial bacterial tropical regions. This study illustrates the feasibility of identifying
consortium comprising Bradyrhizobium spp. and A. brasilense strains beneficial bacterial consortia that can promote plant growth and elicit
combined with MSM application help plants in the accumulation of plant tolerance to abiotic stress, which represents an important
osmolytes, antioxidants metabolism and upregulate stress-responsive biotechnological strategy to achieve sustainable food production and
gene for a better acquisition of drought tolerance, ensuring nodula­ security.
tion, root and shoot development, improving the photosynthetic activity
of the plant and the production of photoassimilates and, finally, the Funding information
filling of the grains.
Benefits in plant growth, yield and tolerance to abiotic and biotic This study was funded by the São Paulo Research Foundation
stresses with the use of consortia of plant growth-promoting bacteria (FAPESP) (Grant #2016/23699-8 and Grant #2018/14892-4).
and microbial metabolites or other synthetic plant-growth regulators
have been increasingly reported (e.g., Fukami et al., 2017; Khan et al., CRediT authorship contribution statement
2019; Khan and Bano, 2019; Marks et al., 2015, 2013; Moretti et al.,
2020b, 2020a). Recently, Liu et al. (2020) proposed an interesting Luiz Gustavo Moretti: Conceptualization, Methodology, Data
approach based on observations of microbiomes of plants submitted to curation, Software, Formal analysis, Investigation, Writing - original
stresses, the concept of a "DefenseBiome", that would include the design, draft, Writing - review & editing. Carlos Alexandre Costa Crusciol:
construction and application of beneficial microbial synthetic commu­ Supervision, Project administration, Funding acquisition. João William
nities; however, as shown in the studies mentioned above as well as in Bossolani: Visualization, Data curation, Writing - review & editing.
ours, this could also be reached by searching the right combinations of Juliano Carlos Calonego: Methodology, Data curation, Software.
microbial cells and metabolites. Adônis Moreira: Methodology, Data curation, Software. Ariani Garcia:
In our study, the beneficial effects of the consortium of Bradyrhi­ Visualization, Data curation, Writing - review & editing. Letusa
zobium spp., MSM and A. brasilense strains on alleviating soybean water Momesso: Visualization, Data curation, Writing - review & editing.
stress under field conditions resulted in improvements in the 1000-grain Eiko Eurya Kuramae: Supervision, Project administration, Funding
weight and in grain yield by up to 574 kg ha− 1 compared with inocu­ acquisition. Mariangela Hungria: Supervision, Project administration,
lation with Bradyrhizobium spp. alone. Most importantly, as in other Funding acquisition.
studies using different consortia of bacteria with microbial metabolites
or synthetic plant regulators (Fukami et al., 2017; Khan et al., 2020,

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L.G. Moretti et al. European Journal of Agronomy 127 (2021) 126293

Declaration of Competing Interest plants. Plant Growth Regul. 54, 97–103. https://doi.org/10.1007/s10725-007-9232-
9.
Croft, H., Chen, J.M., Luo, X., Bartlett, P., Chen, B., Staebler, R.M., 2017. Leaf chlorophyll
The authors report no declarations of interest. content as a proxy for leaf photosynthetic capacity. Glob. Chang. Biol. 23,
3513–3524. https://doi.org/10.1111/gcb.13599.
Cunningham, C., 2020. Characterization of dry spells in Southeastern Brazil during the
Acknowledgments monsoon season. Int. J. Climatol. 40, 4609–4621. https://doi.org/10.1002/
joc.6478.
The authors acknowledge the National Council for Scientific and Donagema, G.K., Viana, J.H.M., Almeida, B.G., Ruiz, H.A., Klein, V.A., Dechen, S.C.F.,
Fernandes, R.B.A., 2017. Granulometric analysis. In: Teixeira, P.C., Donagema, G.K.,
Technological Development (CNPq) for an award for excellence in
Fontana, A., Teixeira, W.G. (Eds.), Soil Analysis Methods Manual. Embrapa Solos,
research to the second, fourth, fifth, and ninth authors as well as the Soil Brasília, DF, pp. 95–116.
Biotechnology Laboratory (Embrapa Soybean) and Netherlands Institute dos Reis, A.R., de Queiroz Barcelos, J.P., de Souza Osório, C.R.W., Santos, E.F., Lisboa, L.
A.M., Santini, J.M.K., dos Santos, M.J.D., Furlani Junior, E., Campos, M., de
of Ecology (Royal Netherlands Society of Arts & Sciences). Publication
Figueiredo, P.A.M., Lavres, J., Gratão, P.L., 2017. A glimpse into the physiological,
number 7188 of the Netherlands Institute of Ecology (NIOO-KNAW). biochemical and nutritional status of soybean plants under Ni-stress conditions.
Environ. Exp. Bot. 144, 76–87. https://doi.org/10.1016/j.envexpbot.2017.10.006.
Du, Y., Zhao, Q., Chen, L., Yao, X., Zhang, H., Wu, J., Xie, F., 2020. Effect of drought
Appendix A. Supplementary data stress during soybean r2–r6 growth stages on sucrose metabolism in leaf and seed.
Int. J. Mol. Sci. 21, 1–19. https://doi.org/10.3390/ijms21020618.
Supplementary material related to this article can be found, in the Embrapa, 2020. Tecnologias de Produção de Soja, Sistemas de Produção 17. Embrapa
Soja, Londrina, PR, Brazil.
online version, at doi:https://doi.org/10.1016/j.eja.2021.126293.
FAO, 2019. The state of food security and nutrition in the world 2019. State Food Secur.
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