Design and timeline of the pot experiment
A cross-factorial pot experiment was performed in which spring wheat (Triticum aestivum L., cv. Fiorina) was cultivated in two soils: one with sufficient (HIGH Zn soil) and one with non-sufficient plant available Zn (LOW Zn soil). The design of the pot experiment consisted of three factors with two levels each; (i) green manure addition (no green manure (NO GM), Trifolium alexandrinum (also designated as clover) (+ GM)); (ii) soil x-ray irradiation (non-irradiated (NO IRR), x-ray irradiated (X-RAY IRR)); and (iii) ZSB inoculation ((NO ZSB), inoculated (+ ZSB)). Destructive soil and plant sampling was done at two time points, i.e. wheat flowering (i.e. 60 days after wheat planting for HIGH Zn and 66 days for LOW Zn) and wheat maturity (i.e. 123 days after wheat planting). For each time point, four experimental replicates were prepared. This resulted in 128 pots (4*2(GM)*2(IRR)*2(ZSB)*2(SOIL)*2(Sampling points)), all arranged in a completely randomized design on the glasshouse bench with rearrangement of the pots every second week.
Clover was grown and incorporated in half of the HIGH Zn and the LOW Zn soils before wheat was planted to all the treatments. There were therefore two successive plant cultures on the same soils in the + GM treatments, whereas only wheat was grown in the NO GM treatments. After green manure growth and incorporation (but before wheat planting), half of the experiment soils (with and without green manure) were x-ray irradiated and re-inoculated with the native soil bacteria. ZSB were inoculated to the wheat seedlings and to the soil at wheat planting and only to the soil at wheat flowering.
Origins, characteristics and initial fertilization of the two soils
The soil with sufficient Zn plant availability (HIGH Zn soil) was sampled from the long-term field trial “Zurich Organic Fertilizer Experiment” (ZOFE) conducted at the research institute for sustainability sciences (ISS) (Agroscope, Zurich Reckenholz, Switzerland) (Oberholzer et al. 2014). This old field experiment, established in 1949, aims at comparing the effects of different organic and inorganic fertilization regimes on crop yield and soil chemical and biological properties. The HIGH Zn soil was sampled from the farmyard manure treatment (FYM) mainly because it had the highest diethylene triamine penta-acetic acid (DTPA) extractable Zn (4.22 mg kg− 1) compared to other treatments of the ZOFE trial. The soil with non-sufficient plant Zn availability (LOW Zn soil) was imported from Turkey, Central Anatolia. The LOW Zn soil had DTPA extractable Zn concentrations below 0.5 mg kg− 1 (0.23 mg kg− 1), and can therefore be considered as low in plant available Zn (Cakmak et al. 1996).
Initial soils characteristics are described in Table 1. The HIGH Zn soil was a carbonate free and acidic (pH 5.5) while the LOW Zn soil contained carbonate and was slightly alkaline (pH 7.3). The LOW Zn soil had a higher clay content and cation exchange capacity compared to the HIGH Zn soil. Total Cd, N and Zn contents were higher in the HIGH Zn soil as compared to the LOW Zn. Likewise, DTPA extractable trace elements, Olsen P, microbial C and N biomasses and organic C were higher in the HIGH Zn than in the LOW Zn soil.
Table 1
Physicochemical and biological properties of the two soils used in this study. Soil analyses were performed before green manure sowing as an initial soil characterization.
Parametera
|
HIGH Zn
|
LOW Zn
|
pH (CaCl2)
|
5.47
|
7.34
|
Clay (g kg− 1)
|
139
|
233
|
Textureb
|
sandy loam
|
sandy clay loam
|
Cation exchange capacity (cmol(+) kg− 1)
|
5.66
|
34.76
|
Maximum water holding capacity (H2O (ml) per kg soil DW− 1)
|
300
|
494
|
Total inorganic C (g kg− 1)c
|
<LOD
|
14.90
|
Total organic C (g kg− 1)
|
10.93
|
5.10
|
Total Cd (µg kg− 1)
|
352.7
|
67.8
|
Total N (g kg− 1)
|
1.04
|
0.38
|
Total Zn (mg kg− 1)
|
76.6
|
26.6
|
DGT extractable Cd (µg l− 1)c
|
0.427
|
<LOD
|
DGT extractable Zn (µg l− 1)c
|
15.6
|
<LOD
|
DTPA extractable Cd (µg kg− 1)
|
125.7
|
10.0
|
DTPA extractable Zn (mg kg− 1)d
|
4.22
|
0.23
|
Olsen P (mg kg− 1)
|
36.27
|
2.12
|
Basal soil respiration (mg CO2-C kg− 1 h− 1)
|
0.231
|
0.274
|
Microbial C (mg C kg− 1)
|
83.3
|
26.1
|
Microbial N (mg N kg− 1)
|
15.7
|
3.09
|
aApart from total soil N, DGT extractable Zn and Cd, basal soil respiration and microbial C and N biomasses, the rest of the soil parameters given in this table was obtained from Grüter et al. (2017b). |
b IUSS Working Group WRB (2014). |
cBelow limit of detection (< LOD). |
dFor LOW Zn, the DTPA extractable Zn was below 0.5 mg kg− 1, commonly considered as the Zn deficiency threshold (Cakmak et al. 1996). |
All essential macro- and micronutrients (except Zn) were added to the two soils at green manure sowing and N was added again at wheat planting, wheat tillering and wheat flowering resulting in total in 100 mg N added per kg− 1 soil (Table 2).
Table 2
Nutrients added before green manure sowing and N application throughout wheat growth.
Time of application
|
Element
|
Compound
|
Fertilization (mg kg soil-1)
|
Before green manure sowing
|
|
N
|
Mg(NO3)2. KNO3. Cu(NO3)2
|
25
|
|
P
|
KH2PO4
|
60
|
|
K
|
KH2PO4. K2SO4. KNO3
|
150
|
|
S
|
K2SO4. MnSO4
|
25
|
|
Fe
|
FeNaEDTA
|
10
|
|
B
|
H3BO3
|
0.691
|
|
Mn
|
MnSO4
|
0.703
|
|
Mg
|
Mg(NO3)2
|
17.2
|
|
Cu
|
Cu(NO3)2
|
0.081
|
|
Mo
|
Na2MoO4
|
0.061
|
At wheat planting, tillering and flowering (applied three times)
|
|
N
|
NH4NO3
|
25 at each time point
|
Green manure growth and incorporation
The green manure used in this study was the Fabaceae Trifolium alexandrinum L. cv. Elite II. Green manure seeds were purchased from Fenaco (UFA Samen, Winthertur, Switzerland). For each soil, four boxes (36 cm W *35 cm D *15 cm H) were filled with 14 kg of soil (dry weight equivalent). The initial fertilization was then applied to the eight boxes (Table 2), and soils were mixed carefully to ensure homogenous distribution of the nutrients. Half of the boxes, labelled as + GM, were used for growing T. alexandrinum while the other half, labelled as NO GM, were used as control treatment with no green manure amendment. Clover was sown directly on the soil surface at a rate of 0.08 g per kg− 1 soil (i.e. equivalent seeding density of 90 kg ha− 1). Soil moisture in the boxes was maintained around 60% water holding capacity by adding water frequently. All boxes (with and without green manure) were transferred to the glasshouse with climatic conditions set to a photoperiod of 16h with a light intensity of 50 kLux, a relative humidity of 65–70% and day/night temperature of 22°C/16°C.
Two weeks after green manure sowing, the LOW Zn soil was inoculated with Rhizobium leguminosarum (strain reference: DSMZ No. 30139) to ensure effective nodulation of the clover. This rhizobium strain was obtained from the German Collection of Microorganisms and Cell Cultures (https://www.dsmz.de/). For this purpose, R. leguminosarum was incubated and multiplied for 4 days on a rotary shaker at 28°C in Yeast Extract Mannitol (YEM) broth prepared with yeast extract 1 g l− 1, mannitol 10 g l− 1, K2HPO4 0.5 g l− 1, MgSO4 0.2 g l− 1 and Nacl 0.1 g l− 1. To inoculate LOW Zn, 20 ml of this R. leguminosarum culture was dispensed per kg− 1 dry soil. R. leguminosarum was also inoculated in LOW Zn where no green manure was grown. The HIGH Zn soil was not inoculated with this rhizobium as preliminary tests highlighted infectious potential of this soil. Nodules were found on clover roots in both soils after 6 weeks of growth.
At this time, the green manure shoots were cut 1 cm above soil surface, pooled separately for each soil to a single green manure sample, dried at 60°C, weighed for biomass estimation and milled homogeneously. From each of the two green manure samples, ca. 3 g of dry shoot matter were taken for total C, N, Zn and Cd total element analyses (cf. “Plant analyses”). Soils were left for 3 weeks under the same conditions in the glasshouse to allow for green manure root decomposition. Only then, milled green manure shoots were transferred to their respective + GM box and soils were mixed again homogeneously. After an additional week of equilibration in the glasshouse, soils were transferred to 4°C until further use.
Soil x-ray irradiation and re-inoculation with native bacteria
The goal of soil x-ray irradiation and re-inoculation was to obtain soils containing only bacterial communities. In this context, half the volume of NO GM and + GM soils (i.e. containing green manure residues) were transported to Synergyhealth (Däniken, Switzerland, http://www.synergyhealthplc.com/us) where they were x-ray irradiated with a dose range of 25 to 75 kGy. Such levels of irradiation have been reported to kill soil bacteria, fungi and invertebrates (McNamara et al. 2003). Soil sterility was checked after irradiation by inoculating Nutrient Agar (NA) with 10-fold serial dilutions according to Trevors (1996) (data not shown). In order to re-introduce native soil bacterial communities in x-ray irradiated soils, 6.25 mL of filtered soil suspensions (25% (w:v) in 0.3 g L− 1 NaCl) were added per kg− 1 dry soil (the same volume of sterile NaCl 0.3 g L− 1 was added to each NO-IRR treatment). This was done following the procedure described by Aghili et al. (2014), except that we used two additional filtration steps, one through Whatman No. 42 filter paper (i.e. 2.5 µm pore size) in order to exclude fungi mycelia and spores, and a final filtration step through 0.22 µm pore size membranes to concentrate 10-fold the obtained soil filtrates so as to reduce their volume. The 3-step filtration procedure used to prepare each soil filtrate (referred later as “bacterial wash”) is described in details in the supplementary material (Sect. 1). Each bacterial wash was prepared from the corresponding NO IRR soil.
ZSB inoculation
Half of the pots were inoculated with a mix consortia of ZSB including three strains of Pseudomonas (P. chlororaphis, P. moraviensis, P. syringae; accession numbers from 16S rRNA gene sequences blasted against the NCBI nr database: FJ652611.1, HG000013.1, AY574913.1), two strains of Plantibacter flavus (both HE716918.1) and one strain of Streptomyces narbonensis (KU324447). Two of the inoculated strains (S. narbonensis and P. moraviensis) were isolated from the HIGH Zn soil itself, and the four others from a soil with very similar chemical properties in Costerousse et al. (2018). In the same study, the six inoculated ZSB were selected out of 115 Zn solubilizing isolates based on their abilities to produce organic acids, release siderophores and dissolve scarcely soluble Zn salts (Zn oxide and Zn carbonate) in plate and broth assays. To multiply ZSB for wheat seedlings and soil inoculation, ZSB were grown separately overnight in Nutrient Broth (NB) (peptone 5 g l− 1 (BD Difco, Allschwil, Switzerland), meat extract 3 g l− 1 (Sigma-Aldrich, Buchs, Switzerland)) with shaking at 28°C. To quantify viable bacterial cells in NB, relation between optical density (OD) at 600 nm with the number of Colony Forming Units (CFU) per mL was used.
In the + ZSB treatments, the 6 strains were inoculated to the wheat seedlings (i.e. prior to planting) and twice to the soil (i.e. at wheat planting and flowering). This was done to promote survival and persistence of the inoculated strains in soil and at the interface of the wheat roots throughout the pot experiment. Indeed, previous soil inoculation studies with phosphorus solubilizing bacteria have shown that density of inoculated strains may decrease within one month after inoculation (Meyer et al. 2017a; Mosimann et al. 2017).
For seedlings inoculation, wheat seeds were germinated (cf. next section of the materials and methods, “Pot experiment”) and four days old seedlings were dipped for 2 h in a 2-liter bacterial suspension containing the equivalent of 2 x 107 CFU mL− 1 of each ZSB. For soil inoculation, NB aliquots containing 8 x 109 CFU from each ZSB were pooled and centrifuged at 3000g for 10 min. Using NaCl 0.3 g L− 1, bacterial cells were washed twice and cell concentration adjusted to 8 x 106 CFU mL− 1 of each ZSB. At wheat planting and flowering, 12.5 mL from this mixed ZSB suspension was added per kg of dry soil and hence, the equivalent of 105 CFU of each ZSB was added per g of soil at both time points (or 7.5 x 107 CFU per pot− 1).
Pot experiment
The pots were previously washed with hot water, sprayed with ethanol 70% and air dried. The following solutions were applied and mixed to the soils prior to potting (i.e. per kg dry weight); (i) 12.5 mL of initial N fertilization solution (containing 25 mg of N in the form of NH4NO3; Table 2), (ii) 6.25 mL of filtered soil suspension (bacterial wash) in X-RAY IRR treatments and (iii) 12.5 mL of mixed ZSB suspension in + ZSB treatments (the same volumes of sterile NaCl 0.3 g L− 1 was added to NO IRR and NO ZSB pots). After mixing the soils thoroughly, samples were taken for soil analyses at wheat planting and 750 g of soil (dry weight equivalent) were placed into each pot.
When setting up the pot experiment, all wheat seeds (Triticum aestivum L., cv. Fiorina) were surface sterilized with 10% H2O2 for 30 min and 0.3% previcur fungicide (propamocarb and fosetyl, Bayer) for 20 min. After rinsing with autoclaved nanopure water, the seeds were placed on autoclaved sand for 4 days during which germination and primary growth of the seedlings occurred. Half of the seedlings were inoculated with ZSB. The pots were transferred to a glasshouse bench and two wheat seedlings were planted per pot (thinned to only one plant per pot one week later). Climatic conditions were set to a photoperiod of 14h (light intensity of 12 kLux), a relative humidity of 40–45% and day/night temperature of 20°C/14°C. Soil moisture in the pots was raised on a balance to ca. 60% water holding capacity by watering every second day.
Soil analyses
Soil analyses were performed at three time points: before green manure sowing (i.e. for the initial soil characterization; Table 1), at wheat planting (i.e. on soil samples taken after N, bacterial wash, ZSB mixed suspension and/or NaCl solutions were added) and at wheat flowering. At this time point, half of the pots were sampled destructively for soil and wheat shoot analyses. The remaining half of the pots were maintained until wheat maturity where no soil but only wheat roots, shoots and grains were sampled and analyzed. No soil analyses were performed specifically at wheat planting on the + ZSB soil treatments as ZSB consortia had just been inoculated and had had no time to take action.
Table 3 summarizes the various soil analyses performed at each time point and how the soil samples were prepared for analysis. Clay content, soil texture, cation exchange capacity, maximum water holding capacity, soil organic and inorganic C content, total element concentrations, DTPA extractable Zn and Cd and Olsen P data were obtained from Grüter et al. (2017b), where the analytical methods can also be found. The methods used for the remaining soil analyses (soil pH, total soil N, DGT extractable Zn and Cd, soil respiration, microbial C and N contents and soil mineral N concentrations) can be found in the supplementary material (Sect. 2).
Table 3
List of soil analyses performed at the different time points.
|
Time point
|
Soil preparation
|
Parameters
|
Before green manure sowing
|
Wheat planting
|
Wheat flowering
|
pH
|
x
|
x
|
x
|
b
|
Clay
|
x
|
|
|
b
|
Texture
|
x
|
|
|
b
|
Cation exchange capacity
|
x
|
|
|
a
|
Maximum water holding capacity
|
x
|
|
|
a
|
Soil organic and inorganic C content
|
x
|
|
|
b
|
Total element concentrations
|
x
|
|
|
b
|
Total soil N
|
x
|
x
|
x
|
b
|
DGT extractable Zn and Cd
|
x
|
x
|
x
|
a
|
DTPA extractable Zn and Cd
|
x
|
|
|
b
|
Olsen P
|
x
|
|
|
b
|
Soil respiration
|
x
|
|
x
|
a
|
Microbial C
|
x
|
|
x
|
a
|
Microbial N
|
x
|
|
|
a
|
Soil mineral N (NO3− and NH4+)
|
|
x
|
x
|
a
|
aparameters measured on moist soil. Fresh soil samples were always kept at 4 degrees between their collection and their analyses. |
banalyses performed on oven dried soil (40°C), sieved 2 mm and ground with a vibratory disc mill. For texture analysis, the soil was not ground. |
Plant analyses
Milled and oven dried green manure shoot samples (60°C) were taken before it was incorporated to the corresponding soil. At wheat flowering, half of the pots were removed from the glasshouse and the wheat shoots were cut 1 cm above the ground, dried for 5 days at 40°C and milled. At wheat maturity, the plants were harvested following the same procedure and the wheat shoots (i.e. including chaff) were separated from the wheat grains. At wheat maturity only, the roots were washed with tap water, and dried at 40°C for wheat root biomass determination. Number of ears, grains per ear and grains per pots were also reported at wheat maturity.
Plant Zn and Cd concentrations were measured by digesting 200 mg of dried plant material in 2 mL 11M HNO3 for 30 min at ca. 250°C and a pressure of 40 bar in the high pressure single reaction change microwave system (turboWave, MWS microwave systems, Leutkirch, Germany). Zn and Cd concentrations in the extracts were measured by IPC-MS (7500ce, Agilent Technologies, Santa Clara, CA USA). Total N concentrations in green manure and wheat shoots and grains were determined with an NCS analyser (FlashEA 1112 Series, Thermo Fisher Scientific Inc., Waltham, MA USA).
We used the root staining procedure described by Phillips and Hayman (1970) to look for mycorrhizal structures and compare root mycorrhizal colonization among the treatments at wheat flowering.
Statistical analyses
Statistical analyses were performed using the JMP® software, Version 12.2.0 (SAS Institute Inc., Cary, NC, USA). Graphics were constructed also with JMP® or using GraphPad Prism, Version 7.0a (GraphPad Software, La Jolla, CA). Two-way and three-way analyses of variance (ANOVA) were used to test the effects of the factors (green manure addition, soil x-ray irradiation, ZSB inoculation) factors and their interactions. Statistical analyses were performed separately for the two soils. Tukey’s HSD post hoc test was performed to further assess significances of the differences between the means. The “Fit Y by X” function of JMP® was used for Pearson linear regressions. When needed, data were log, square root or Box-Cox transformed to fit normal distribution of their residuals which was verified using the Shapiro-Wilk W test.