Sampling and Measurements

The effects of the treatments were evaluated on plants in terms of changes in leaf structure, photosynthesis, and plant growth and productivity, also unraveling whether changes in photosynthesis are linked with modifications in leaf functional anatomical traits and photochemistry.

Sampling for anatomical analyses was done at 57 DAS on the 3th fully expanded trifoliate leaf from the top of the plant. More specifically, 3 middle leaflets from 3 plants per treatment were cut and immediately submerged in the chemical fixative FAA (40% formaldehyde – glacial acetic acid – 50% ethanol, 5:5:90 by volume). After 2 weeks of fixation, leaflets were halved under a dissection microscope (SZX16, Olympus, Germany) to obtain two twin groups of subsamples: one devoted to the quantification of stomata traits, the other to the analysis of lamina cross sections.

For the analysis of stomata, three strips of lamina adaxial epidermis were peeled off from each subsample and mounted on microscope slides with distilled water. Epidermal peels were analyzed under a transmitted light microscope (BX60, Olympus) and digital images were collected by means of a digital camera (CAMEDIA C4040; Olympus), avoiding main veins. Digital images were analyzed through the software program Analysis 3.2 software (Olympus) to quantify stomata frequency and size. More specifically, stomata frequency was expressed as number of stomata per surface unit (mm²), counted in two regions per peel. Stomata size was quantified by measuring the length of at least 15 guard-cells (pole to pole) and the width of the same cells in the median position.

To obtain cross sections of the leaf lamina, the subsamples were cut under the SZX16 dissection microscope to obtain subsamples of 5 mm × 5 mm from the median part of the leaflet, avoiding the main vein. These subsamples were dehydrated in an ethanol series (up to 90%) and embedded in the acrylic resin JB4 (Polysciences, Warrington, PA, USA). Thin cross sections (5 μm) were cut by means of a rotary microtome, stained with 0.025% Toluidine blue in 0.1 M citrate buffer at pH 4 (Reale et al., 2012) and mounted with Canadian Balsam. The sections were analyzed under a BX60 light microscope and digital images were collected at different magnifications. By means of the Olympus Analysis 3.2 software, the mesophyll was characterized by measuring the thickness of palisade and spongy parenchyma tissues and the quantity of intercellular spaces in the spongy tissues. The thickness of palisade and spongy tissues were measured in five positions along the lamina, avoiding veins. The incidence of intercellular spaces was measured as the percentage of tissue occupied by intercellular spaces over a given surface, in six regions along the leaf lamina.

The light fast kinetics curve was performed at a single leaf level on non-inoculated plants, using a portable Infra Red Gas Analyzer WALZ HCM 1000 (Walz, Effeltrich, Germany) (Supplementary Figure S1). The curve was determined on the middle leaflet of the 2nd and 3th fully expanded trifoliate leaves from the top of the plant (two leaves per plant, three plants). Increasing PPFDs (0, 50, 100, 250, 500, 1000, 1500, and 2000 μmol m^-2 s^-1) were obtained by using a built-in halogen lamp, and the conditions inside the leaf chamber were kept constant: temperature 25°C, RH 70%, ambient CO₂ concentration.

During plant cultivation, NP was measured on the same leaf types chosen for the light response measurements (two leaves per plant, three plants per treatment), at ambient light intensity (420 μmol m^-2 s^-1) and the same leaf chamber conditions. Measurements were carried out in the different phenological phases: vegetative growth (30 DAS), flowering (44 DAS), pod setting (57 DAS). NP was not detectable during the pod filling, because of the difficulty to position the leaves in the leaf chamber in presence of symptoms of senescence (wilting and curling).

On the same leaves, chlorophyll a fluorescence were determined using a portable FluorPen FP100 max fluorometer (Figure ​Figure22), equipped with a light sensor (Photon System Instruments, Brno, Czech Republic), at room temperature (26°C). The ground fluorescence F_o was induced on 30′ dark adapted leaves, by a blue LED internal light of about 1–2 μmol photons m^-2 s^-1. The maximal fluorescence level in the dark F_m was induced by 1 s saturating light pulse of 5.000 μmol photons m^-2 s^-1. The maximum quantum efficiency of PSII photochemistry was calculated as (F_m-F_o)/F_m. The measurements in the light were conducted at PPFD of 420 μmol (photons) m^-2 s^-1 at the canopy level. The PSII quantum yield (QY) was determined by means of an open leaf-clip suitable for measurements under ambient light, according to Genty et al. (1989). QY was used to calculate the linear electron transport rate (ETR), according to Krall and Edwards (1992). Non-photochemical quenching (NPQ) was calculated as described by Bilger and Björkman (1990), according to the following formula: NPQ = (F_m/F_m′)-1. Measurements started at flowering (44 DAS), as significant differences in NP between the treatments were detected, and repeated during pod setting (57 DAS) and pod filling (84 DAS).

After fluorescence determinations, the leaf greenness was estimated using a colorimeter (Chlorophyll Meter Konica-Minolta SPAD 502), and expressed as SPAD units, in six plants per treatment (two leaves per plant, five measurements per leaf), at flowering (44 DAS). Measurements were made at the central point of the leaflet between the midrib and the leaf margin. In the same samples, chlorophyll a and b content was determined by extraction in acetone and spectrophotometer lecture (Jeffrey and Humphrey, 1975), using a Hach DR 4000 Spectrophotometer (Hach Company, Loveland, CO) on one 2-cm² leaf sample per leaf.

Growth analysis during the growing cycle was based on non-destructive measurements of plant height and leaf number, determined every 7 to 10 days, on six plants per treatment. Plant leaf area (LA) was estimated by the values of leaf length and width, using the formula of Wiersma and Bailey (1957), based on the specific soybean leaf types and shapes.

Soybean seeds were harvested when pods had turned brown (average water content 14%). At each harvest, yield data [fresh weight (FW) of pods and seeds] were determined per single plant. Plant productivity was measured as grams of seeds per plant^-1 (edible biomass).

At the end of the experiment, plants were collected to determine FW and dry matter (DM), and their partitioning in roots, stems and leaves (non-edible biomass). Measurements were carried out on six plants per treatment (two plants × double gully). DM was measured after oven-drying at 60°C until constant weight.

The concentration of the main cations (K⁺, Ca²⁺, Mg²⁺) and anions (NO₃^-) in the recirculating nutrient solution and in the leaf tissues was determined using an ion exchange chromatographer (ICS-3000, Dionex, Sunnyvale, CA, USA). Nutrient solution samples were collected at 7-day intervals, starting from the first inoculation of the recirculating solution. Leaf analysis was performed on water extract of DM of 5 healthy, fully expanded leaves per treatment randomly sampled, during the flowering phase.