3.1. Conventional and Organic N Trials
Grain yield and sedimentation value of conventionally produced cultivar Bussard were significantly influenced by the main effects treatment and year, but the interaction was not significant. Only N treatment affected crude protein content significantly, while free Asn showed significant differences concerning treatment and treatment-year interaction.
Yields were higher in 2007 than in 2008 with an average yield across all N treatments of 6.5 t ha−1
in 2007 and 5.2 t ha−1
in 2008. Yields increased with the applied amount of N and were highest with 6.8 and 7.0 t ha−1
across both years in treatments fertilized with 140 and 180 kg N ha−1
, respectively. Nevertheless, the maximum grain yield was reached at an N supply of 140 kg N ha−1
: A further increase in N supply did not lead to a further significant increase in grain yield. The treatments with an emphasized late application rate of N showed slightly reduced grain yields compared to their respective counterparts (Table 4
). Regarding the influence of total N fertilization independent of their distribution, increases in total N fertilization generally increase grain yield, unless fertilization exceeds a certain maximum [33
]. In both trial years, the grain yield results confirmed this assumption. The baking quality increased with increasing N input. The treatment 180 kg N ha−1
with an emphasized late application rate led to the highest crude protein content of 15.2%, which was 5% higher than in the unfertilized control. Similar to the crude protein content, the protein quality assessed by the sedimentation test also increased with increasing N supply and was highest in treatment 180-late with a mean value of 50 mL over both years. The sedimentation values can partially be influenced by the amount and a late N fertilization [34
]. This was also confirmed in this study, as sedimentation values increased with increasing N contents in the grain. The highest sedimentation values were reached by the highest N supply independent of the cropping system. Free Asn in the flour of cultivar Bussard was less influenced by N supply in 2007 under conventional farming, as the treatments with intensive N application did not show significantly higher Asn values compared to the unfertilized control (about 11.7 mg 100 g−1
flour-DM). However, in 2008, free Asn contents of cultivar Bussard were significantly different when comparing N60-late, N60, and N100-late with N140-late, N180, and N180-late (Table 4
). The highest free Asn contents of 17.8 mg 100 g−1
flour-DM were found when N amounts of 180 kg ha−1
were applied in 2008 without a late application rate. Determined values were about 43% higher than the free Asn value of the unfertilized control. Furthermore, the temporal distribution of N fertilization had no significant effect on free Asn levels. Results from Woolfolk et al. [36
] stated, that a late foliar application after flowering increased the total N content. They concluded that a late N supply, before or shortly after flowering may significantly enhance grain N content and finally the crude protein amount in winter wheat. Winkler and Schön [37
] found an increase of free Asn with increasing grain N concentration in barley. According to those studies, late N fertilization treatments may have led to an increased level of both crude protein and free Asn. However, only a significant increase of crude protein by late fertilization was found; therefore, it is assumed that synthesis of free Asn is genetically determined, and differences between cultivars will occur.
Under organic conditions, grain yield was only significantly influenced by treatment but not by year or treatment-year interaction. Hence, grain yield of cultivar Bussard is displayed combining years 2007 and 2008. Sedimentation value and crude protein content were both significantly influenced by the treatment and year but not by treatment-year interaction. In contrast to the conventional trial, under organic farming conditions free Asn content was only affected by year, but not by N treatment.
The highest grain yields (5.1 t ha−1) were achieved when slurry was applied with amounts of 100 kg N ha−1. The achieved grain yields were about 20% higher than the unfertilized control. The application of horn meal solely; however, did not increase grain yield significantly. This suggests that the mineralization of horn meal was slow, leading to late N availability. The high sedimentation value and crude protein content also indicated a late N availability.
Sedimentation value and crude protein content were lower in 2007 than in 2008 (34 compared to 44 units and 10.6% compared to 12.0%). Sedimentation values of the treatments S50, S50-H50 and H60 did not significantly differ from the control treatment while the treatments S100, S100-H20, H120, and H180 increased the sedimentation value significantly compared to the unfertilized control. The highest sedimentation value of 47 units was found when 180 kg horn meal ha−1
was applied. Compared to the unfertilized control, crude protein content increased significantly by about 1 to 2.9% if cv. Bussard was fertilized with N except for the application of 50 kg N ha−1
given as slurry. An amount of 180 kg N ha−1
horn meal led to the highest crude protein content of 13%, which was about 23% higher than the unfertilized control. For flour of organic origin, a lower baking quality is accepted. To reach a good baking quality, Brunner [38
] recommends a sedimentation value of 34 units and a crude protein content of 11.6%. Regarding our results, all treatments exceeded the suggested sedimentation value, while only treatments S100-H20 and H180 achieved the values for crude protein. However, a clear year effect was observed in this study as previously found by other authors for durum wheat [39
]. Specifically, in this study, significantly lower sedimentation values and crude protein values in 2007 compared to 2008 may be attributed to a higher N leaching-caused by the higher rainfall amount from May to July in 2007 (about 370 vs. 260 mm).
If organically produced bakery goods are demanded, lower yields and lower baking qualities must be accepted [41
]. Bread bakery processing has to be adjusted to the lower protein contents of such flour [42
] to achieve acceptable products.
Free Asn contents were higher in 2007 (11%) than in 2008 (7.5%) and tended to increase with increasing amounts of N from 8.5 to 10 mg 100 g−1, however, no statistically significant difference could be found.
When comparing the free Asn contents of the three winter wheat cultivars dependent on N supply (unfertilized control vs. 180 kg N ha−1
), year, N treatment, cultivar, and the interaction year-nitrogen was significant under conventional farming, while N treatment, cultivar, and the interaction year-cultivar were significant under organic farming (Table 5
The three conventionally cropped winter wheat cultivars differed in their capacity to store free Asn in the flour, with Capo showing the lowest value of 6.8 mg 100 g−1
, followed by Bussard with 10.3 mg 100 g−1
, across years and N treatments (Table 6
). Cultivar Naturastar reached the highest level of free Asn (17.42 mg 100 g−1
). Also, the application of organic N increased free Asn contents in flour when averaged across years and cultivars. Cultivars differed in the same ascending order under organic conditions as under conventional conditions, with Capo having a free Asn content of 6.5 mg 100 g−1
, Bussard 7.2 mg 100 g−1
, and Naturastar 11.3 mg 100 g−1
flour-DM averaged across years and N treatments. Though Bussard and Naturastar had a slight trend to produce less free Asn in 2008 compared to 2007 if organically grown and under N supply, Capo had a slightly higher free Asn content of about 2.5 mg 100 g−1
in 2007 compared to 2008 if N was applied. In contrast, if cultivars grow under conventional farming conditions all three cultivars had in 2008 a higher level of free Asn. Thus, besides the year the cropping system seems to effect free Asn formation.
Finally, across years, N treatments and cropping systems cultivar Capo was found to exhibit the lowest free Asn level by up to 22% lower amounts when compared to Bussard and 42% when compared to Naturastar. When comparing the same N treatments, significant differences between cultivars were also found by Weber et al. [21
]. Stockmann et al. [43
] found a reduction potential of free Asn of around 60% for wheat cultivars grown under organic cropping terms. Postles et al. [18
] analyzed a significant increase in free Asn by up to 29% if tested rye cultivars were supplied with 200 kg N ha−1
compared to 1 kg N ha−1
. Nevertheless, they reported, that independent of N supply differences between cultivars in free Asn was not affected by N nutrition. Thus, combining cropping practices like N fertilization and choosing cultivars including a low potential to form free Asn will more effectively reduce free Asn than applying single measurements.
When pooling the means of free Asn values from the three field trials across both experimental years and correlating them with the N supply, a clear trend of increasing free Asn levels with an intensified N supply was obvious (Figure 3
). Contrary to a linear effect of increasing N amounts on crude protein content, the effect on free Asn followed a more quadratic function with moderate free Asn levels up to N amounts of 140 kg N ha−1
. Amounts of 180 kg N ha−1
or higher increased the probability of high free Asn contents considerably, while N supply below that amount led to free Asn values that did not differ considerably from the unfertilized controls. Similar findings were described by Weber et al. [21
] investigating one E-wheat cultivar (Enorm). They achieved an increase in free Asn by raising the level of N at different steps. Depending on the year, they found a significantly higher amount of free Asn at a level of 140 kg N ha−1
. According to the German Bundessortenamt, high baking quality can be expected from wheat lots (conventionally cropped) with crude protein contents of 13.3% or higher. According to the regression line, this critical crude protein content was met already with N amounts of 160 kg N ha−1
in the experimental years. In order not to exceed N supply, farmers are encouraged to carefully choose the amount of N as baking quality will not be affected negatively.
The overall correlation between crude protein and free Asn was relatively weak (Figure 4
), due to the fact that mean values of different cultivars and different trials were pooled. However, it was clear that considerably increased free Asn contents were found primarily if crude protein contents were 14% or higher. Also, a high scattering of free Asn, especially within untreated control without N supply and 180 kg N ha−1
, was present. Thus, it has to be considered that environment (=location and year) can affect free Asn levels considerably, as also shown by Curtis et al. [17
] for wheat and by Curtis et al. [8
] for rye. There is now clear evidence that free Asn accumulates in most, if not all, plant organs during periods of low rates of protein synthesis and a plentiful supply of reduced nitrogen [44
]. However, up to now information on how and why soil type, temperature, and precipitation affect grain Asn accumulation is missing. Corol et al. [45
] stated that especially during grain development low rainfall and high temperatures increased free Asn amount in grain. This has to be taken into account when interpreting our data, as the climate conditions during 2007 and 2008 could have had an impact.
In addition, a poor relation of crude protein and free Asn was found for both N trials, whereas the conventional S trial showed a good correlation for both traits (R2
0.71). This means that a higher amount of crude protein may lead to higher levels of free Asn. Corol et al. [45
] correlated free Asn with different quality traits of wheat wholemeal, and the closest relation was found for free Asn and protein content (r
= 0.507). Marschner [46
] reported an increase of amides if N fertilization was increased. Similar results concerning soluble N were reported by Gianibelli and Sarandon [47
]. Acknowledging that S fertilization had no effect on the level of free Asn, the increase in both crude protein and free Asn was mainly due to the high N treatment of 200 kg N ha−1
. Therefore, this high N supply could have led to an accumulation of soluble N, mainly as free Asn.
In addition to environmental conditions and N treatments, the cropping system also had an impact on free Asn (different symbols in Figure 3
). Across N treatments and cultivars, organically treated samples (black triangles) showed up to 18% lower free Asn compared to conventional farming. While for single cultivars, a reduction of 23% was possible by choosing organically grown cultivars. This may favour the assumption that the level of free Asn is generally lower in organic farming systems due to a lower N supply. This is in agreement with studies of Stockmann et al. [22
], who realized a significant reduction potential of free Asn (up to 30%) if wheat cultivars were grown under organic farming conditions.
3.2. Conventional S trial
Grain yield, crude protein, and sedimentation values were significantly influenced by treatment and year, but not by the interaction of both. Free Asn content was significantly influenced by the treatment and year-treatment interaction, but not by year. Since S level of flour samples was only influenced significantly by treatment, it is given as mean of both years.
Independent of S and N treatment, grain yield in 2007 ranged from 4.2 to 7.5 t ha−1
and from 4.3 to 7.5 t ha−1
in 2008 (Table 7
). While N application led to a significant yield increase, S supply did not change grain yield significantly. Similar results were found by Pompa et al. [48
] and Rossini et al. [40
], where the effect of a foliar S supply was tested and no significant effect on grain yield was found.
Randall et al. [49
] and Luo et al. [35
] recommended that plants did not suffer from S deficiency if grain S concentration is higher than 0.12% and N/S ratios in grains are below 17:1. All grain S concentrations analyzed in this trial, including the control treatments, exceeded 0.12% (Table 7
) and the N/S ratio was below 17:1. Thus, it can be assumed that no S deficiency occurred. In addition, Dai et al. [50
] reported that the time of S availability is important as sufficient S supply, especially during grain filling, will invert high levels of free Asn. It can be assumed that in our trial soil S availability during grain filling was sufficient.
Crude protein contents ranged from 9.6% to 14.6% in 2007 while in 2008 it was significantly lower, ranging from 8.5% to 13.9% (Table 7
). N fertilization levels of 200 kg ha−1
resulted in significantly higher crude protein contents of around 14%. Comparing type and amount of S fertilization within the same N amount applied, only a significant impact on crude protein was found for K20-N2 and elS-N2. The S content of flour samples varied from 0.13% to 0.19% across years (Table 7
). All treatments except treatment KEp26-N1 produced significantly higher S contents than both control treatments. Consistent effects were found neither for the type nor for the amount of S supply.
The analysis of flour concerning free Asn showed means varying from 6.9 to 21.9 mg 100 g−1
in 2007 while in 2008 means ranged from 9.3 to 23.8 mg 100 g−1
). The S supply had no influence on free Asn et al.
Weber et al. [51
] investigated the effect of S fertilizer kieserite and an additional N supply of 180 kg ha−1
and found similar results. They concluded that an additional S application for lowering free Asn is not constructive if the S amount within the soil is sufficient. Nevertheless, if soils are poor in S, free Asn level can increase dramatically. This was revealed by Muttucumaru et al. [25
], who showed that free Asn content in wheat grain increased up to 30 times under S deficiency. Similar results were reported by Granvogl et al. [24
] from a greenhouse experiment with a summer wheat cultivar. They found a high increase of free Asn in flour of S poor wheat, which finally revealed the AA formation strongly. However, it might be a cereal species influenced output as Postles et al. [18
] reported that there was no effect of S increasing free Asn in grain samples of five rye cultivars. Thus, it seems that S fertilization is more linked to protein-rich cereal species above all wheat, where S is needed to form storage proteins, not accumulating free Asn. Köhler et al. [52
] and Shewry et al. [26
] postulated that storage protein composition changes if S availability is limited, due to a limited formation of S rich protein fractions. They concluded that this leads to an increase of protein fractions low in S and boosts the amount of N structures, e.g., aspartic acid and free Asn. Besides those results, the working group of Postles et al. [18
] also stated that S supply could minimize the effect of high N availability on free Asn formation. They found two cultivars which showed a reduced level of free Asn if high N was available and S was applied. Curties et al. [53
] reported that in case of S supply the level of free Asn was less influenced by year and the cultivar was more stable in producing free Asn amounts. In our study, similar effects of S on free Asn formation were not found. There was no clear reduction effect of S comparing N1 with 100 kg N ha−1
and N2 with 200 kg N ha−1
within the single S treatments. However, one needs to keep in mind that, in our study, N was not applied without S. Maybe a treatment of N1 (100 kg N ha−1
) and N2 (200 kg N ha−1
) without an S application could have revealed other results. Nevertheless, most studies concerning the effect of S on free Asn formation were carried out as pot trials, including soils poor in S as well as field trials where S was deficient [24