Strong Increase of Durumwheatiron and Zinccontent by Field-Inoculation With

Strong increase of durumwheatiron and zinccontent by field-inoculation with arbuscularmycorrhizalfungiatdifferentsoilnitrogenavailabilities

Laura Ercoli1*, Arthur Schüßler2, Iduna Arduini3, Elisa Pellegrino1

1Institute of Life Sciences, Scuola Superiore Sant’Anna, Piazza Martiri della Libertà 33, 56127 Pisa, Italy; 2Symplanta GmbH& Co. KG, Milanweg 46, 26127 Oldenburg, Germany; 3Department of Agriculture, Food and Environment, University of Pisa, via del Borghetto 80, 56124 Pisa, Italy

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Table S1Dates of the three split application of the nitrogen (N) fertilizer and of the root samplings of the old (Cappelli) and modern (Svevo) durum wheat variety
Growth stage / Zadoks's code / Variety and dates / Operation
Old / Modern
- / - / November 4th, 2013 / November 4th, 2013 / Pre-seeding N fertilization (40 kg N ha-1)
- / - / November 4th, 2013 / November 4th, 2013 / Seeding
2 leaves unfolded / GS12 / January 10th, 2014 / January 10th, 2014 / 1st root sampling
5 leaves unfolded / GS15 / January 25th, 2014 / January 25th, 2014 / 1st topdressing N fertilization (40 kg N ha-1)
Pseudo stem erection / GS30 / March 14th, 2014 / March 14th, 2014 / 2nd topdressing N fertilization (40 kg N ha-1)
1st node detectable / GS31 / April 10th, 2014 / April 6th, 2014 / 2nd root sampling
Flag leaf ligule/collar just visible / GS39 / April 24th, 2014 / April 16th, 2014 / -
Physiological maturity / GS90 / July 7th, 2014 / July 1st, 2014 / 3rd root sampling

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Table S2 Total root length at tillering (GS12) and arbuscularmycorrhizal fungal (AMF)root colonization at tillering, stem elongation (GS31) and physiological maturity (GS90) of durum wheat. Variety (Var) x nitrogen fertilization (N fert) x AMF inoculation (AMF inoc) interaction
Var∗ / N fert / AMF inoc / Root dry weight at GS12 (mg plant-1)† / Total root length at GS12 (cm) / AMF root colonization at GS12 (%) / AMF root colonization at GS31 (%) / AMF root colonization at GS90 (%)
Old / 0-0-0 / -M / 21.1‡ / 36.2 / 4.0 / 20.7 / 32.1
+M / 28.9 / 59.6 / 10.3 / 33.6 / 28.4
0-40-40 / -M / 23.3 / 34.2 / 3.4 / 20.8 / 31.5
+M / 21.1 / 57.3 / 9.5 / 27.6 / 33.3
40-0-40 / -M / 40.4 / 33.7 / 3.5 / 25.2 / 31.0
+M / 48.3 / 56.1 / 9.6 / 31.6 / 27.8
40-40-0 / -M / 51.1 / 35.3 / 3.7 / 15.9 / 28.0
+M / 43.2 / 58.8 / 8.8 / 27.4 / 29.7
40-40-40 / -M / 57.7 / 34.5 / 3.5 / 26.3 / 24.4
+M / 61.1 / 82.8 / 10.1 / 23.0 / 24.8
Modern / 0-0-0 / -M / 54.7 / 37.0 / 5.1 / 27.0 / 30.2
+M / 44.3 / 38.6 / 11.5 / 37.3 / 32.7
0-40-40 / -M / 48.1 / 35.8 / 4.7 / 17.0 / 35.7
+M / 55.0 / 36.2 / 9.0 / 29.0 / 31.1
40-0-40 / -M / 47.3 / 35.6 / 5.8 / 19.7 / 29.5
+M / 58.0 / 36.1 / 7.6 / 28.7 / 28.2
40-40-0 / -M / 87.7 / 36.6 / 6.4 / 25.7 / 28.8
+M / 95.6 / 36.2 / 9.2 / 32.6 / 27.7
40-40-40 / -M / 79.9 / 37.8 / 4.8 / 27.6 / 25.9
+M / 84.4 / 37.6 / 9.0 / 28.3 / 27.3
∗Durum wheat varieties: Cappelli and Svevo are the old and the modern variety, respectively; N fert: 0-0-0, 0-40-40, 40-0-40, 40-40-0, 40-40-40 kg N ha-1 at pre-seeding, 5th leaf unfolded stage and pseudo stem erection, respectively; -M: mock inoculated; +M: inoculated with RhizophagusirregularisDAOM197198
†Significant at P = 0.002 see Table 1 and Figure 2
‡Values are means of three observations
Table S3 Relative abundance of molecular operational taxonomic units (MOTU) within the arbuscularmycorrhizalfungi(AMF) community (%) in the roots of old and modern durum wheat varieties inoculated with AMF (M) and not inoculated (NM)
Code / MOTU identifier / Old variety* / Modern variety
-M / +M / -M / +M
______%______
(1) / Ac1PI† / 10.2 / 5.7 / 8.3 / 3.9
(2) / Ac2PI / 11.7 / 5.1 / 9.2 / 3.8
(3) / Di1PI / 7.1 / 2.7 / 5.6 / 2.1
(4) / Scu1PI / 7.9 / 4.4 / 9.8 / 4.8
(5) / Scu2PI / 9.3 / 4.3 / 12.1 / 4.4
(6) / Fun1PI / 19.8 b‡ / 6.1 a / 16.8 b / 5.6 a
(7) / Fun2PI / 18.4 b / 8.8 a / 17.7 b / 2.9 a
(8) / Rh1PI / 2.8 a / 50.2 b / 5.2 a / 65.1 c
(9) / Rh2PI / 3.8 / 4.7 / 5.1 / 4.3
(10) / Glo1PI / 9.0 / 8.0 / 10.2 / 3.1
∗Durum wheat varieties: Cappelli and Svevo are the old and the modern variety, respectively; -M: mock inoculated; +M: inoculated with Rhizophagusirregularis DAOM197198
† MOTU names denote the most similar AM fungal phylotype, asrelated to sequences from the clone libraries, at genus level: Ac1PI and Ac2PI, Acaulospora; Di1PI, Diversispora; Scu1PI and Scu2PI, Scutellospora; Fu1PI and Fu2PI, Funneliformis; Rh1PI and Rh2PI, Rhizophagus; Glo1PI, uncultured Glomeromycota
‡Values in the same row followed by the same letter are statistically different at P ≤ 0.05

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Table S4Growth, yield and yield components of durum wheat. Variety (Var) x nitrogen fertilization (N fert) x arbuscularmycorrhizal fungal inoculation (AMF inoc) interaction
Var∗ / N fert∗ / AMF inoc∗ / Shoot dry weight at GS31 (g m-2)† / Grain yield (g m-2) / Straw (g m-2) / HI / Spikes (number m-2) / Kernels (number spike-1) / Mean kernel weight / Fertile spikelets (number spike-1) / Spike fertility index
Old / 0-0-0 / -M / 914.1‡ / 253.3 / 514.1 / 28.8 / 248.7 / 19.2 / 52.3 / 14.2 / 48.4
+M / 849.7 / 266.5 / 525.7 / 29.5 / 284.9 / 17.4 / 53.2 / 13.6 / 43.3
0-40-40 / -M / 765.1 / 473.1 / 887.3 / 31.3 / 252.0 / 38.8 / 52.5 / 16.1 / 56.7
+M / 446.4 / 471.9 / 1051.9 / 27.4 / 289.1 / 30.7 / 54.3 / 18.2 / 46.6
40-0-40 / -M / 1987.3 / 510.5 / 1013.4 / 31.4 / 213.2 / 49.4 / 49.6 / 18.5 / 46.8
+M / 2059.4 / 510.2 / 1072.6 / 29.8 / 307.3 / 37.6 / 44.7 / 18.2 / 50.5
40-40-0 / -M / 927.3 / 483.6 / 1089.5 / 27.8 / 221.9 / 48.5 / 48.4 / 20.6 / 49.5
+M / 1060.7 / 526.0 / 1125.8 / 29.2 / 210.2 / 48.1 / 52.3 / 20.1 / 41.7
40-40-40 / -M / 1500.5 / 469.4 / 1549.5 / 23.0 / 220.1 / 48.7 / 45.7 / 20.9 / 47.9
+M / 1849.3 / 479.6 / 1002.3 / 30.3 / 209.0 / 48.1 / 48.4 / 19.7 / 51.5
Modern / 0-0-0 / -M / 812.8 / 318.3 / 460.6 / 36.6 / 302.6 / 24.4 / 43.9 / 13.7 / 68.1
+M / 941.3 / 282.2 / 298.9 / 41.6 / 321.8 / 20.8 / 43.7 / 12.2 / 66.0
0-40-40 / -M / 1898.8 / 397.5 / 503.3 / 37.8 / 330.9 / 28.8 / 43.1 / 13.6 / 63.1
+M / 2070.9 / 467.2 / 595.2 / 37.9 / 336.3 / 33.7 / 41.6 / 14.3 / 69.0
40-0-40 / -M / 918.3 / 520.9 / 510.8 / 43.2 / 352.9 / 37.1 / 40.4 / 16.0 / 73.4
+M / 1065.2 / 447.6 / 499.8 / 41.1 / 304.7 / 34.9 / 42.9 / 16.0 / 68.7
40-40-0 / -M / 1342.0 / 466.1 / 545.8 / 43.3 / 258.0 / 45.8 / 41.5 / 15.3 / 69.9
+M / 1899.9 / 431.3 / 487.6 / 41.4 / 288.9 / 37.1 / 40.7 / 16.1 / 79.5
40-40-40 / -M / 1023.8 / 531.0 / 526.4 / 42.2 / 356.8 / 32.8 / 45.0 / 15.7 / 63.2
+M / 1208.3 / 614.1 / 686.5 / 40.0 / 411.1 / 38.2 / 39.5 / 17.0 / 70.0
∗Durum wheat varieties: Cappelli and Svevo are the old and the modern variety, respectively; N fert: 0-0-0, 0-40-40, 40-0-40, 40-40-0, 40-40-40 kg N ha-1 at pre-seeding, 5th leaf unfolded stage and pseudo stem erection, respectively; -M: mock inoculated; +M: inoculated with Rhizophagusirregularis DAOM197198
†See Table 2 for statistical significance
‡Values are means of three observations

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Table S5Grain concentration of nutrients of durum wheat. Variety (Var) x nitrogen fertilization (N fert) x arbuscularmycorrhizal fungal inoculation (AMF inoc) interaction
Var∗ / N fert / AMF inoc / N concentration (g kg-1)† / P concentration (g kg-1) / Fe concentration (mg kg-1) / Zn concentration (mg kg-1)
Old / 0-0-0 / -M / 14.8‡ / 6.2 / 25.7 / 11.0
+M / 20.1 / 6.7 / 34.7 / 20.7
0-40-40 / -M / 15.7 / 5.4 / 23.0 / 13.0
+M / 19.1 / 9.5 / 39.0 / 24.3
40-0-40 / -M / 10.0 / 6.6 / 23.7 / 12.0
+M / 13.3 / 4.0 / 31.3 / 20.7
40-40-0 / -M / 13.0 / 4.9 / 22.0 / 12.0
+M / 13.8 / 4.6 / 37.3 / 25.3
40-40-40 / -M / 12.7 / 5.2 / 22.7 / 13.0
+M / 20.7 / 3.5 / 31.3 / 25.0
Modern / 0-0-0 / -M / 17.4 / 5.5 / 23.3 / 13.0
+M / 18.1 / 7.6 / 37.3 / 26.3
0-40-40 / -M / 21.5 / 7.5 / 21.7 / 13.3
+M / 14.7 / 5.7 / 30.3 / 24.7
40-0-40 / -M / 13.2 / 6.3 / 24.3 / 14.0
+M / 12.8 / 5.8 / 37.7 / 27.7
40-40-0 / -M / 22.2 / 5.9 / 19.3 / 15.0
+M / 22.0 / 5.2 / 32.7 / 23.3
40-40-40 / -M / 21.2 / 4.9 / 23.0 / 11.7
+M / 21.2 / 5.9 / 37.7 / 24.0
∗Durum wheat varieties: Cappelli and Svevo are the old and the modern variety, respectively; N fert: 0-0-0, 0-40-40, 40-0-40, 40-40-0, 40-40-40 kg N ha-1 at pre-seeding, 5th leaf unfolded stage and pseudo stem erection, respectively; -M: mock inoculated; +M: inoculated with Rhizophagusirregularis DAOM197198
†See Table 2 for statistical significance
‡Values are means of three observations

Fig. S1Average ten-day maximum and minimum temperature and cumulated rainfall during the wheat growing cycle (from 4th of November 2013 to 1st of July 2014). Daily weather data were obtained from a meteorological station located within 100 m from the experimental plots

Fig. S2Neighbor-Joining (NJ) tree of arbuscularmycorrhizal fungal (AMF) public sequences based on the partial nuclear small subunit ribosomal RNA gene (≈ 800 bp; AML1/AML2 fragment). The tree is rooted with a sequence of Corallochytriumlimacisporum(L42528). The NJ tree was created using sequences from morphologically defined AMF samples, including most AMF species listed in the phylotaxonomic classification of Schüßlerand Walker (2010). Scale bar: evolutionary distance, computed using the Kimura 2-parameter method, shown as base substitutions per site. Bootstrapping is based on 1000 replicates and bootstrapping values ≥ 50 are shown. Analyses were conducted with MEGA7. The analysis involved 65 nucleotide sequences plus theoutgroup. In the figure, AMF orders and families are indicated. The alignment is available from an open-access database at The arrows indicate the position of the Molecular Operational Taxonomic Unit (MOTU) retrieved in this study

Fig. S3Phylogenetic placement of arbuscularmycorrhizal fungal (AMF) sequences derived from roots of old (Cappelli) and modern(Svevo) durum wheat inoculated with AMF and not inoculated. The Neighbor-Joining (NJ) analysis is based on the partial nuclear small subunit ribosomal RNA gene sequences (SSU ≈ 800 bp;AML1/AML2 fragment). The NJ truncated tree is rooted with a sequence of Corallochytriumlimacisporum(L42528) anddivided into six portions (from (a) to (f)).Red branches show the position of each portion of the tree. Clades of sequences (Molecular Operational Taxonomic Units, MOTUs) were affiliated to species from Acaulospora (Ac1PI; Ac2PI),Diversispora (Di1PI),Scutellospora (Scu1PI; Scu2PI),Funneliformis(Fu1PI; Fu2PI),Rhizophagus (Rh1PI; Rh2PI) anduncultured Glomeromycota (Glo1PI). The MOTU positionsare shown by the square bracket and the name. The evolutionary distances (scale bar), computed using the Kimura 2-parameter method,represent the number of base substitutions per site. Bootstrapping is based on 1000 replicates. Analyses were conducted with MEGA7.The analysis involved 406nucleotide sequencesplus the outgroup. Sequences obtained in the present study are shown in bold with their accession numbers prefixed bythe internal clone identifier referring to the treatment (a, NM old variety; b, M old variety; c, NM, modern variety; d, M, modern variety), the replicate plot (from 1 to 3), the number of clone and the MOTU name with the suffix of the location of the study (Pisa, PI). All new sequences have been submitted to the EMBL nucleotide sequence database (accession numbers: LN714838-LN715198)

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