Strong increase of durum wheat iron and zinc content by field-inoculation with arbuscular
mycorrhizal fungi at different soil nitrogen availabilities
Laura Ercoli1*, Arthur Schüßler2, Iduna Arduini3, Elisa Pellegrino1
1
Institute 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
1
Table S1 Dates 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
th
th
2 leaves unfolded
GS12
January 10 , 2014
January 10 , 2014
1st root sampling
th
th
5 leaves unfolded
GS15
January 25 , 2014
January 25 , 2014
1st topdressing N fertilization (40 kg N ha-1)
th
th
Pseudo stem erection
GS30
March 14 , 2014
March 14 , 2014
2nd topdressing N fertilization (40 kg N ha-1)
1st node detectable
GS31
April 10th, 2014
April 6th, 2014
2nd root sampling
th
th
Flag leaf ligule/collar just visible
GS39
April 24 , 2014
April 16 , 2014
Physiological maturity
GS90
July 7th, 2014
July 1st, 2014
3rd root sampling
2
Table S2 Total root length at tillering (GS12) and arbuscular mycorrhizal 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
N fert
AMF Root dry weight
Total root
AMF root
AMF root
AMF root
Var∗
inoc
at GS12
length at
colonization at
colonization at
colonization at
(mg plant-1)†
GS12 (cm)
GS12 (%)
GS31 (%)
GS90 (%)
Old
0-0-0
0-40-40
40-0-40
40-40-0
40-40-40
Modern 0-0-0
0-40-40
40-0-40
40-40-0
40-40-40
-M
21.1‡
36.2
4.0
20.7
32.1
+M
28.9
59.6
10.3
33.6
28.4
-M
23.3
34.2
3.4
20.8
31.5
+M
21.1
57.3
9.5
27.6
33.3
-M
40.4
33.7
3.5
25.2
31.0
+M
48.3
56.1
9.6
31.6
27.8
-M
51.1
35.3
3.7
15.9
28.0
+M
43.2
58.8
8.8
27.4
29.7
-M
57.7
34.5
3.5
26.3
24.4
+M
61.1
82.8
10.1
23.0
24.8
-M
54.7
37.0
5.1
27.0
30.2
+M
44.3
38.6
11.5
37.3
32.7
-M
48.1
35.8
4.7
17.0
35.7
+M
55.0
36.2
9.0
29.0
31.1
-M
47.3
35.6
5.8
19.7
29.5
+M
58.0
36.1
7.6
28.7
28.2
-M
87.7
36.6
6.4
25.7
28.8
+M
95.6
36.2
9.2
32.6
27.7
-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, 40-040, 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 Rhizophagus irregularis DAOM197198
†
Significant at P = 0.002 see Table 1 and Figure 2
‡
Values are means of three observations
3
Table S3 Relative abundance of molecular operational taxonomic units (MOTU) within
the arbuscular mycorrhizal fungi (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*
-M
Modern variety
+M
_____________________________
(1)
Ac1PI
(2)
†
-M
%
+M
_____________________________________
10.2
5.7
8.3
3.9
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 Rhizophagus irregularis
DAOM197198
†
MOTU names denote the most similar AM fungal phylotype, as related 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
4
Table S4 Growth, yield and yield components of durum wheat. Variety (Var) x nitrogen fertilization (N fert) x arbuscular mycorrhizal fungal inoculation (AMF inoc) interaction
Var∗
N fert∗
AMF
inoc∗
Old
0-0-0
-M
+M
-M
+M
-M
+M
-M
+M
-M
+M
-M
+M
-M
+M
-M
+M
-M
+M
-M
+M
0-40-40
40-0-40
40-40-0
40-40-40
Modern 0-0-0
0-40-40
40-0-40
40-40-0
40-40-40
Shoot dry
weight at GS31
(g m-2)†
Grain yield
(g m-2)
Straw
(g m-2)
HI
914.1‡
849.7
765.1
446.4
1987.3
2059.4
927.3
1060.7
1500.5
1849.3
812.8
941.3
1898.8
2070.9
918.3
1065.2
1342.0
1899.9
1023.8
1208.3
253.3
266.5
473.1
471.9
510.5
510.2
483.6
526.0
469.4
479.6
318.3
282.2
397.5
467.2
520.9
447.6
466.1
431.3
531.0
614.1
514.1
525.7
887.3
1051.9
1013.4
1072.6
1089.5
1125.8
1549.5
1002.3
460.6
298.9
503.3
595.2
510.8
499.8
545.8
487.6
526.4
686.5
28.8
29.5
31.3
27.4
31.4
29.8
27.8
29.2
23.0
30.3
36.6
41.6
37.8
37.9
43.2
41.1
43.3
41.4
42.2
40.0
Spikes
Kernels
(number m-2) (number spike-1)
248.7
284.9
252.0
289.1
213.2
307.3
221.9
210.2
220.1
209.0
302.6
321.8
330.9
336.3
352.9
304.7
258.0
288.9
356.8
411.1
19.2
17.4
38.8
30.7
49.4
37.6
48.5
48.1
48.7
48.1
24.4
20.8
28.8
33.7
37.1
34.9
45.8
37.1
32.8
38.2
Mean kernel
weight
Fertile spikelets
(number spike-1)
Spike fertility
index
52.3
53.2
52.5
54.3
49.6
44.7
48.4
52.3
45.7
48.4
43.9
43.7
43.1
41.6
40.4
42.9
41.5
40.7
45.0
39.5
14.2
13.6
16.1
18.2
18.5
18.2
20.6
20.1
20.9
19.7
13.7
12.2
13.6
14.3
16.0
16.0
15.3
16.1
15.7
17.0
48.4
43.3
56.7
46.6
46.8
50.5
49.5
41.7
47.9
51.5
68.1
66.0
63.1
69.0
73.4
68.7
69.9
79.5
63.2
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 Rhizophagus irregularis DAOM197198
†
See Table 2 for statistical significance
‡
Values are means of three observations
5
Table S5 Grain concentration of nutrients of durum wheat. Variety (Var) x nitrogen fertilization (N
fert) x arbuscular mycorrhizal fungal inoculation (AMF inoc) interaction
N fert
AMF
N
P
Fe
Zn
Var∗
inoc
concentration
concentration
concentration
concentration
(g kg-1)†
(g kg-1)
(mg kg-1)
(mg kg-1)
Old
0-0-0
0-40-40
40-0-40
40-40-0
40-40-40
Modern
0-0-0
0-40-40
40-0-40
40-40-0
40-40-40
-M
14.8‡
6.2
25.7
11.0
+M
20.1
6.7
34.7
20.7
-M
15.7
5.4
23.0
13.0
+M
19.1
9.5
39.0
24.3
-M
10.0
6.6
23.7
12.0
+M
13.3
4.0
31.3
20.7
-M
13.0
4.9
22.0
12.0
+M
13.8
4.6
37.3
25.3
-M
12.7
5.2
22.7
13.0
+M
20.7
3.5
31.3
25.0
-M
17.4
5.5
23.3
13.0
+M
18.1
7.6
37.3
26.3
-M
21.5
7.5
21.7
13.3
+M
14.7
5.7
30.3
24.7
-M
13.2
6.3
24.3
14.0
+M
12.8
5.8
37.7
27.7
-M
22.2
5.9
19.3
15.0
+M
22.0
5.2
32.7
23.3
-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 Rhizophagus
irregularis DAOM197198
†
See Table 2 for statistical significance
‡
Values are means of three observations
6
Fig. S1 Average 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
7
Fig. S2 Neighbor-Joining (NJ) tree of arbuscular mycorrhizal 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 Corallochytrium limacisporum (L42528). The NJ tree was created
using sequences from morphologically defined AMF samples, including most AMF species listed
in the phylotaxonomic classification of Schüßler and 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 the outgroup. In the
figure, AMF orders and families are indicated. The alignment is available from an open-access
8
database at https://sites.google.com/site/restomedpeatland/microbiology. The arrows indicate the
position of the Molecular Operational Taxonomic Unit (MOTU) retrieved in this study
9
10
11
12
13
14
15
Fig. S3 Phylogenetic placement of arbuscular mycorrhizal 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 Corallochytrium limacisporum (L42528) and divided 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 (Ac1 PI;
Ac2PI), Diversispora (Di1 PI), Scutellospora (Scu1 PI; Scu2 PI), Funneliformis (Fu1 PI; Fu2 PI),
Rhizophagus (Rh1 PI; Rh2 PI) and uncultured Glomeromycota (Glo1 PI). The MOTU positions are
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 406
nucleotide sequences plus the outgroup. Sequences obtained in the present study are shown in bold
with their accession numbers prefixed by the 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)
16