Six weeks after the RW installation, the plants reached the heading stage (EC 59), and new root development was detectable over the whole surface of the observation plane (Fig. 1). In all treatments, comparative samplings were performed for undisturbed control (CP) plants and plants growing along the RW.
Shoot and root growth
In all treatments, no significant differences for dry shoot biomass were recorded between RW plants and the undisturbed CP plants (Fig. 2A). Among the different treatments, shoot biomass in MP-Int (was significantly reduced by 17 % (CP) and 28% (RW) compared with the CT-Ext treatment with reduced input of N fertilizers, fungicides and energy for soil tillage. This effect coincided with intense visible symptoms of powdery mildew on leaves particularly in the MP-Int treatment in both RW and CP plants (Suppl. Fig. S1).
A similar pattern was also observed for the root dry mass (RDM) and total root length (RL) of the excavated root systems, with no significant differences between RW and CP plants. In the MP-Int treatment, RDM decreased by 17% (CP) and 22% (RW) relative to CT-Ext. The RL showed an even more pronounced decline by 55% (CP) and 58% (RW) in the MP-Int treatment in comparison to CT-Ext (Fig. 2B-C). Additionally, intensive fertilization significantly reduced the root hair length (RHL) compared to extensive fertilization in both tillage practices (Fig. 2D), while the RW installation did not significantly affect RHL.
Plant nutritional status
The mineral nutritional status (Table 1) at flag leaf maturity was generally low or reached critical levels (Campbell, 2000) for the macronutrients nitrogen (N) and potassium (K) and for copper (Cu) as well as for zinc (Zn) as plant micronutrients . Compared to plants in the undisturbed CP, the foliar concentrations of K, magnesium (Mg), calcium (Ca), manganese (Mn), and Cu tended to be higher for all treatments in RW plants, although the respective differences were not significant in all cases.
Only in the CT-Ext treatment of RW plants, a reduction in N fertilization led to a notable 17% decrease in foliar N concentration compared to the CT-Int treatment. Similar trends were recorded also for MP-Ext vs. MP-Int (RW) and CT-Ext vs. CT-Int (CP). In RW, CT-Ext plants also showed significantly lower leaf concentrations of K, Mg, and sulfur (S) compared to CT-Int plants.
Cultivator tillage practice significantly reduced Mg (CT-Ext) and S concentrations (CT-Int and CT-Ext) in RW plants. In CP plants, CT significantly decreased Ca concentrations in CT-Int plants and Cu concentrations in CT-Ext plants. However, the nutrient concentrations did not decline below the deficiency thresholds (Campbell 2000).
Table 1 Impact of long-term farming practices and root window (RW) installation on A) the nutrient status of winter wheat (cv. Lemmy) in comparison to the undisturbed plants in the central plots (CP). MP = mouldboard plow tillage, CT = cultivator tillage, Ext = extensive N-fertilization without fungicides and growth regulators, Int = intensive N-fertilization including fungicides and growth regulators, CP = undisturbed central plots. Different letters indicate significant differences between farming practices according to three-way ANOVA, Tukey test: p≤0.05. B) P-values from ANOVA show the main effects and interactions of tillage practice, N-fertilization, and RW installation.
A)
|
Macronutrients [g kg-1 shoot DM]
|
|
Micronutrients [mg kg-1 shoot DM]
|
|
Ctotal
|
Ntotal
|
P
|
K
|
Mg
|
Ca
|
S
|
|
Cu
|
Fe
|
Mn
|
Zn
|
Deficiency Threshold#
|
|
|
30.0
|
|
1.5
|
|
20.0
|
|
1.0
|
|
1.5
|
|
1.0
|
|
|
3.0
|
|
25.0
|
|
15.0
|
|
15.0
|
|
MP-Int-RW
|
449.9
|
abc
|
37.13
|
a
|
2.14
|
ab
|
18.40
|
a
|
1.82
|
a
|
6.56
|
a
|
4.57
|
a
|
|
4.58
|
a
|
51.40
|
b
|
52.61
|
a
|
14.93
|
ab
|
MP-Ext-RW
|
450.4
|
abc
|
35.88
|
ab
|
2.54
|
a
|
17.25
|
ab
|
1.63
|
ab
|
5.69
|
bc
|
3.44
|
bcd
|
|
4.79
|
a
|
66.64
|
ab
|
52.90
|
a
|
15.82
|
ab
|
CT-Int-RW
|
453.8
|
a
|
37.88
|
a
|
2.39
|
ab
|
19.13
|
a
|
1.73
|
ab
|
5.91
|
ab
|
3.88
|
b
|
|
4.04
|
ab
|
67.64
|
ab
|
57.14
|
a
|
15.40
|
ab
|
CT-Ext-RW
|
448.8
|
bc
|
31.38
|
b
|
2.13
|
ab
|
15.65
|
bc
|
1.40
|
c
|
5.33
|
bcd
|
2.92
|
e
|
|
4.15
|
ab
|
100.87
|
a
|
55.31
|
a
|
16.58
|
ab
|
MP-Int-CP
|
450.0
|
abc
|
36.01
|
ab
|
2.04
|
b
|
12.03
|
d
|
1.55
|
bc
|
5.88
|
ab
|
3.79
|
bc
|
|
3.99
|
ab
|
56.14
|
ab
|
39.92
|
b
|
18.31
|
a
|
MP-Ext-CP
|
451.7
|
ab
|
37.89
|
a
|
2.39
|
ab
|
13.63
|
cd
|
1.38
|
c
|
5.05
|
cd
|
3.38
|
cd
|
|
4.69
|
a
|
89.81
|
ab
|
39.33
|
b
|
19.79
|
a
|
CT-Int-CP
|
451.1
|
ab
|
38.89
|
a
|
2.44
|
ab
|
14.30
|
cd
|
1.38
|
c
|
4.72
|
d
|
3.54
|
bcd
|
|
3.63
|
bc
|
61.46
|
ab
|
41.70
|
b
|
15.72
|
ab
|
CT-Ext-CP
|
446.2
|
c
|
35.14
|
ab
|
2.43
|
ab
|
12.15
|
d
|
1.39
|
c
|
4.93
|
d
|
3.19
|
de
|
|
3.08
|
c
|
52.29
|
ab
|
39.26
|
b
|
12.31
|
b
|
#Deficiency thresholds of macro- and micro-nutrients after Campbell (2000)
B)
|
Ctotal
|
Ntotal
|
P
|
K
|
Mg
|
Ca
|
S
|
Cu
|
Fe
|
Mn
|
Zn
|
Tillage practice (TP)
|
0.4255
|
|
0.3338
|
|
0.2941
|
|
0.9670
|
|
0.0004
|
|
>0.0001
|
|
>0.0001
|
|
>0.0001
|
|
0.1572
|
|
0.2507
|
|
0.0059
|
|
Fertilization intensity (Fertl-int)
|
0.0084
|
|
0.0006
|
|
0.0767
|
|
0.0044
|
|
>0.0001
|
|
>0.0001
|
|
>0.0001
|
|
0.3896
|
|
0.0761
|
|
0.4408
|
|
0.9661
|
|
Root Window (RW)
|
0.1659
|
|
0.1202
|
|
0.7022
|
|
>0.0001
|
|
>0.0001
|
|
>0.0001
|
|
0.0250
|
|
0.0005
|
|
0.6177
|
|
>0.0001
|
|
0.2583
|
|
TP x Fertl-int
|
0.0001
|
|
>0.0001
|
|
0.0006
|
|
0.0012
|
|
0.8154
|
|
0.0036
|
|
0.6146
|
|
0.0170
|
|
0.2313
|
|
0.6411
|
|
0.1291
|
|
TP x RW
|
0.0197
|
|
0.6217
|
|
0.0298
|
|
0.3262
|
|
0.1986
|
|
0.5052
|
|
0.0075
|
|
0.1487
|
|
0.0112
|
|
0.6412
|
|
0.0008
|
|
Fertl-int x RW
|
0.6173
|
|
0.0104
|
|
0.4685
|
|
0.0211
|
|
0.0054
|
|
0.0545
|
|
>0.0001
|
|
0.7471
|
|
0.1801
|
|
0.6179
|
|
0.1864
|
|
TP x Fertl-int x RW
|
0.7029
|
|
0.4800
|
|
0.2428
|
|
0.3989
|
|
0.0094
|
|
0.0855
|
|
0.5710
|
|
0.0392
|
|
0.2918
|
|
0.8409
|
|
0.0898
|
|
Expression of stress-related genes in the leaf tissue of winter wheat
As indicators for the physiological stress level of the investigated wheat plants, 28 target genes involved in nutrient transport/metabolism and (a)biotic stress responses as well as regulatory and signal transduction genes were selected for expression analyses in the tissue of flag leaves (Behr et al. 2024). The clustering of the farming practices (Treatment) showed a clear separation along the tillage practices with an overall higher expression level for the MP treatments compared to a lower expression level for the CT treatments (Fig. 3A). The fertilization intensity (Int vs. Ext) had only a minor impact on the gene expression clustering pattern. CP and RW plants showed highly similar responses of gene expression patterns to different farming practices. Clustering was confirmed also by PERMANOVA analysis (Table 2).
A principal coordinate analysis (PCoA) of gene expression levels confirmed the separation of samples by tillage practice and a very narrow clustering pattern between CP and RW samples, especially for MP-Int and MP-Ext treatments (Fig. 3B). A PERMANOVA analysis showed that tillage practice had the strongest influence on the gene expression and explained 64.1 % of the variance. The location (CP vs. RW) did not show a significant influence (Table 2).
Table 2 PERMANOVA analysis based on Bray-Curtis distances (10,000 permutations) performed on gene expression profiles of 28 selected genes expressed in leaves of winter wheat grown in central plots or with root windows (location) under different management practices of LTE Bernburg. *p < 0.05, ** p< 0.01, ***p < 0.001.
Factor
|
Explained Variance (%)
|
P-Value
|
Tillage
|
64.1
|
<0.001 ***
|
N-Fertilization intensity
|
2.8
|
<0.05 *
|
Location (RW vs CP)
|
0.9
|
<0.328
|
Tillage:Fertilization
|
2.3
|
<0.086
|
Tillage:Location
|
2.6
|
<0.069
|
Fertilization:Location
|
4.8
|
<0.014 **
|
Residuals
|
22.5
|
|
Composition of microbial communities
NMDS analysis showed a separate clustering of RH and RA samples with distinct subclusters depending on the tillage practice. Microbial communities from CP and RW samples were highly similar (Fig. 4). This was confirmed by PERMANOVA analysis which revealed the habitat (RH vs. RA) and the tillage practice (MP vs. CT) as the main shaping factors, whereas the sampling location (CP vs. RW) did only marginally influence the microbial community composition (Table 3). This became also apparent at the level of the top 30 most abundant bacterial/archaeal (Fig. 5) and fungal genera (Fig. 6). In both cases, clustering was according to the habitat followed by tillage practice. The relative abundance of major genera was similar in CP and RW plants. Thus the heatmap of the dominant bacterial genera (Fig. 5) revealed several taxa with higher relative abundance in the rhizosphere of CP and RW-grown wheat plants compared to root-affected soil such as Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Devosia, Pseudoxanthomonas, Saccharimonadales, Massilia and Pedobacter. The heatmap of the 30 dominant fungal genera (Fig. 6) indicated a higher variability but showed that Zymoseptoria, Cystofilobasidium and Filobasidium were enriched in the rhizosphere of both CP and RW-grown plants.
Table 3 PERMANOVA analysis based on Bray-Curtis distances (10,000 permutations) of bacterial/archaeal and fungal communities of winter wheat (cv. Lemmy; EC 59) obtained from undisturbed central plots (CP) and root windows (RW). Missing factor combinations are not significant. *p < 0.05, **p < 0.01, *** p < 0.001.
Factor
|
Explained variance [%]
|
Bacterial/archaeal community
|
Fungal community
|
Location (RW vs CP)
|
1.3
|
1.6*
|
Habitat
|
23.5***
|
39.5***
|
Tillage practice
|
11.4***
|
12.3***
|
N-fertilization
|
2.2*
|
7.5***
|
Tillage practice x N-fertilization
|
1.8*
|
2.9**
|
Tillage practice x Habitat
|
3.9***
|
3.6***
|
Tillage practice x Location
|
1.3
|
1.8*
|
Habitat x Location
|
1.2
|
1.4*
|
Residuals
|
45.9
|
25.3
|
Additionally, we tested the effect of the different agricultural practices (tillage, N-fertilization intensity) on the microbial community composition separately in CP and RW samples. In both sampling locations, similar influences of tillage and N-fertilization intensity were observed. RW and CP analysis showed in agreement that tillage exhibited a much stronger effect on the microbial community than N-fertilization intensity, which was even more pronounced in RH in the case of bacterial/archaeal communities (Supplementary Tables S2 and S3).