1. Introduction

2. Materials and Methods

3. Results

4. Discussion

4.1. Comparison of seasonal impact on yield and quality

Grain yield in 2020 showed no significant difference between variety and Nrate but FCR infection impacted GY in both varieties reducing it with the higher dose of inoculum having more impact in most situations. In the 2021 season higher yields were obtained (overall mean 2020, 2.0 t/ha; 2021, 4.8 t/ha) with much more responsiveness to applied N in both varieties but no significant effect from FCR inoculation. Yield increases were similar between varieties in 2021. Grain yield is responsive to applied N fertiliser in durum wheat as N fertilization is largely considered to be the main factor affecting yield and protein content [15] where the N is converted into protein and biomass. This was reflected in the GP responses being blunted in 2020 (range 1.04%) compared to more responsive at all N rates in 2021 (range 2.4%), especially between 40-160N. These increases had expected effects on technological quality traits. Nitrogen fertilization contributes to increases in protein content when fertilizer rates satisfy the requirements of both yield and protein synthesis [16,17]. Indeed, in 2021 traits significantly affected by Nrate were GY, GP, WG, TW, TGW, HVK, SKHI, SY, Semo only, L*, b*, MPT, RBD, MPH and GI compared to 2020 where only GP, WG, TGW, b* and Semo only were significantly affected by Nrate. In 2021 both varieties responded similarly to applied N with DBA Lillaroi starting GP lower than Jandaroi. Response was larger at 80N and above compared to lower rates. If the soil N at sowing is high then the plant relies less on the applied N to fill the grain with protein. GP content obtained in the mature grain depends on other factors such as water and N availability, length of grain filling, weather conditions prevailing during grain filling (like higher rainfall, lower temperatures prolonging flowering and grain filling period), partitioning of N and dry matter to the grain and source-sink relations with leaves and stems [18]. An inverse relationship between grain yield and grain N concentration has been reported in durum wheat [19,20,21,22,23]. Against this trend was a strong positive correlation between GY and GP in 2021 (r = 0.84, p<0.05) but not in 2020. This relationship can be affected by soil fertility, water availability etc. [18]. The combination of above average rainfall and high rates of N application satisfying the requirements for both yield and protein synthesis could explain this positive correlation in 2021 [16]. Based on the GP responses, both varieties appear to have similar abilities to transfer the applied N to more grain protein. Grains were smaller in 2020 than in 2021 (mean TGW 2020, 41.8 g compared to 2021, 46.4 g) and this could also result in the higher protein in that season (mean 0N GP 13.8% in 2020 vs. 12.7% at 160N in 2021).

Genetic differences in grain weight were evident between the two varieties and DBA Lillaroi was selected during its breeding to achieve high TGW and milling potential and the data supports this as do other studies [24,25]. The overall TGW achieved are typical for durum wheat grown in the NNSW region under non-drought situations as was the case in the two seasons of this study. Jandaroi TGW did not respond to Nrate while DBA Lillaroi tended to decrease but was not consistent in 2020 and hardly changed in 2021. FCR had no impact on TW in both varieties in 2021 because there was very little FCR expression despite the infection from the inoculation but reduced TW for Jandaroi in 2020 significantly from 78.7 kg/hl to 77.6 kg/hl. This is not a large change and of no commercial significance as these levels are above the trading discount of 76 kg/hl. There was some evidence of weather damage in the visual appearance of the kernels especially in 2021 where much lower HVK was found. Rainfall at or near harvest (1st December 2021) of 191 mm in November (Table 1) could have been responsible for the low falling numbers from 185-290 sec in 2021 that was not found in the 2020 season with only 3 mm of rain near harvest, 24th November, 2020 (mean FN 571 sec). While this is below the minimum GTA trading value of 300 sec required for acceptable grain [3], evidence from the literature suggests only if weather damage is severe in durum wheat (FN < 100 sec or thereabouts), is there any influence on pasta colour and cooking quality [27].

The percentage of kernels that are vitreous is an important grade determinant for durum wheat because of its impact on semolina milling yield and end-product quality. Durum wheat with high HVK (>80) and test weight will generally give high semolina yield with a minimum number of white starchy particles [27]. According to the grading standards in Australia [3], durum is graded as, ADRI HVK >80%; ADR2 HVK 70-80%; ADR3 <70% which means that despite what the grain protein is, grain could be downgraded at grain receival centres from low HVK with consequent financial penalties for growers. Grain vitreousness was affected in 2021 but not in 2020 where HVK average values >95% (data not shown) probably because protein was >13% and unresponsive to applied N. In 2021, the increase in N fertilisation led to an increase in HVK which is mostly likely due to the increase in GP that has been found by others [28,29]. High grain protein content allows the formation of a compact grain structure due to the starch granules in the amyloplasts surrounded by the protein matrix and this creates reduced air spaces in the grain structure resulting in a vitreous or “glassy” appearance, i.e. high HVK score. In contrast, a less compact grain structure has many open spaces, with a lower density endosperm [28] and the protein matrix is discontinuous creating opaque appearance due to light diffraction and diffusion at the void spaces [28]. There is generally a strong correlation between GP and HVK as observed in the 2021 data (GP vs. HVK, r = 0.81, P<0.001, Fig 11B) in line with other reports [2,26]. This relationship differed between Jandaroi and DBA Lillaroi with the former variety showing a more gradual change and holding the HVK mostly above 75% even at 9-10% GP, whereas DBA Lillaroi slope was larger with HVK<80% as GP declined below 11.5% reaching values as low as 42%. Given that HVK>80 ensures top grade (ADR1) in Australia, Jandaroi for most field plots met this requirement whereas DBA Lillaroi did not especially at Nrates below 80 kg/ha. Even with no applied N, many Jandaroi samples achieved HVK>80% whereas for DBA Lillaroi, the applied N was found to be critical in achieving the highest grade and needed 80N minimum, with downgrading to the lowest grade at ≤20N. This would strongly impact the price a grower would receive. Jandaroi has more potential to achieve a higher GP than DBA Lillaroi in the NNSW environment although quite often lower in yield [25]. So despite the known relationship between HVK and GP, this was not observed for Jandaroi since even at very low GP 9-11%, the HVK was mostly >75%. It has been noted by others that this strong association between protein and HVK is inconsistent [2] and Sieber et al. [30] found large variation in grain protein in non-vitreous kernels. A possible explanation is related to the protein composition and how that influences grain structure. At the same Gli/Glu ratio range of 0.65-0.79, Jandaroi had mean HVK 89 ± 8.3% and GP 11.4 ± 1% while DBA Lillaroi had mean HVK 70 ± 14.6% with a similar GP 10.8 ± 1.1%, showing that despite very similar GP and Gli/Glu between the varieties, the grain structure in Jandaroi must be quite different to DBA Lillaroi to account for this large difference in HVK, so other factors are operating. The question then is why is Jandaroi able to hold its HVK so high at low protein? Samson et al. [28] found variety Néodur was able to synthesise protein more efficiently from applied N than other varieties examined (like the case for Jandaroi vs. DBA Lillaroi in this study) and showed the highest density kernels, consistent with high HVK. We found that as HVK increased the gliadin to glutenin ratio increased in both durum varieties (Figure 14). This result is consistent with other studies showing preferential accumulation of gliadin in vitreous kernels [2,26]. Samson et al. [28] suggested that gliadin proteins facilitate the formation of vitreous endosperm by providing better adhesion of the protein matrix to starch granules during kernel desiccation. Our data suggests this would at least have some dependence on the genotype but none of the above studies examined the relationship between HVK, GP and Glu/Gli in multiple genotypes. Further analysis of the glutenin proteins during grain maturation should confirm if Jandaroi does synthesise gliadins faster during grain development than DBA Lillaroi and this may assist the formation of a “tighter grain structure” which translates into higher mature HVK levels in this variety irrespective of grain protein content making Jandaroi a better variety when soil N levels could be low but more studies are needed to confirm this.

Grain weight, TW and vitreousness can contribute to milling yield and purity (ash content and bran specks in semolina) that can impact pasta appearance and colour [31]. For the trait Semo only in 2020 there was a tendency for this to decline with increased N but only significantly for DBA Lillaroi (Figure 6). However, in 2021 there was a much greater change (increase) in Semo only which was very clear for DBA Lillaroi, increasing significantly from 20N peaking at 80N while Jandaroi showed little response to applied N (Figure 6). This was less apparent for trait SY% with smaller increases with applied N, also noting no change in SY% for both varieties in 2020 (Figure 5). The increase in Semo only in DBA Lillaroi in 2021 could be due to a good response from applied N for HVK and less so for TGW that was not the case for Jandaroi. It seems that DBA Lillaroi has more potential to improve its milling potential than Jandaroi possibly by translating the N into more starch synthesis through higher starch synthase activity or improving the milling potential of this variety by changes in endosperm to bran ratio or the separability of the bran or other factors [32]. However, further testing across more environments is needed to confirm this response which is very interesting showing potential to manipulate milling potential with applied N that is genotype dependent. Interestingly, despite DBA Lillaroi at Nrates <80 kg/ha (2021 data only) having HVK<75, the Semo only and SY% were still superior to Jandaroi and even at 0N DBA Lillaroi has higher milling yield than Jandaroi receiving 160N (for SY% only). It was noted by Fu et al., [2] that low protein durum can have acceptable milling yield even with HVK<70%. Other factors come into play in particular genetic factors [25]. DBA Lillaroi genetic tendency to produce high TGW would contribute to higher semolina yield than Jandaroi and not so much, if at all, from HVK contributions. In particular the N response seen in TGW in DBA Lillaroi and not in Jandaroi would help in achieving a higher semolina yield. Fu et al. [2] found using composite durum wheat samples varying in GP from 14.5 to 9.9% that providing HVK>70%, the semolina yield did not change with GP content but semolina yield would decline significantly if low HVK coincided with low grain protein. This could explain why semolina yield in Jandaroi did not respond to N fertilisation (even at GP ~10.5% with 0N) because mean HVK>80%. Whereas for DBA Lillaroi at N<80 kg/ha where HVK was <75% and GP<11.5% that this combination resulted in the grain producing significantly less semolina on milling, as observed. In 2020 season both GP >13% and HVK >96% were high which could explain the lack of SY response to applied N in that season. High HVK confers superior milling performance and high wheat protein can mitigate the negative impact of low HVK on durum milling by reducing the size of the starchy areas in the piebald kernels [2].

Semolina colour is an important characteristic in durum because high semolina yellowness tends to translate to high pasta yellowness although during milling and pasta processing loss of colour can occur [33]. Higher protein semolina is known to be duller (lower L*) as protein affects brightness/redness of semolina [34] and the semolina L* decreased with increasing N rate (data not shown). High HVK grain can negate the effect of duller semolina due to high grain protein resulting in the final pasta still remaining a bright yellow colour [2]. DBA Lillaroi has a higher semolina b* than Jandaroi because this variety has a higher natural yellow pigment level in the endosperm, as a result of years of breeding and selection to enhance semolina b* levels [25]. While there was no significant changes in semolina b* in 2020, in 2021 there was a significant increase for DBA Lillaroi but not Jandaroi with increasing N. This is probably related to the increase in GP, as Dalla Marta et al. [35] reported an association between protein content increase and increase in yellow pigment content.

Jandaroi was selected in its breeding for its high dough strength reflected in the MPT (3-5 min), low RBD (<50) and very high GI (>90) superior to DBA Lillaroi [25]. While there was little effect of Nrate on GI in the 2020 season for both varieties, in 2021 increasing N application reduced GI slightly in Jandaroi (95 to 85) but to a much larger extent for DBA Lillaroi (92 to 76). The FCR inoculum however significantly reduced GI in both varieties in 2020 but had no effect in 2021, (Figure S1). The season was too wet and temperatures mild during grain filling which limited the expression of whiteheads and hence yield loss and many quality traits being unaffected from FCR infection in 2021. The RBD in both varieties in 2021 increased with increasing N in both varieties (Jandaroi from 27 to 46; DBA Lillaroi from 27 to 53) but not in 2020. These data indicate a weaking of dough strength with increasing N application consistent with the decrease in GI. The reason for the seasonal differences and effect of N is complex and may depend on other conditions like water availability and other environmental changes as a genotype’s dough properties are affected by both genotypic and environmental factors [24,36]. Giuliani et al., [18] found in their N application study in durum wheat that while GI was affected by N in one year, there was no change in another season. Other studies in wheat have shown a decrease in gluten strength with N fertilization in hard white winter wheats due to a preferential increase of gliadins over glutenins [37]. The negative influence of a higher Gli/Glu ratio on common wheat dough rheological properties is well known [38,39]. Increasing N led to an increase in the Gli/Glu ratio in both varieties with different responses between varieties (Figure 12B). The regression of Gli/Glu on RBD and GI (Figure 15) show that as gliadin percentage increases the dough becomes weaker. In Jandaroi the increase in Gli/Glu did not result in much change in GI whereas in DBA Lillaroi there was a much stronger relationship suggesting that changes in Jandaroi Gli/Glu are much less likely to result in large shifts in GI compared to DBA Lillaroi supporting the findings of Saint Pierre et al. [37] but with qualification. This relationship is more complex and related to genetic differences in varieties ability to retain gluten strength despite changing protein composition. However, both varieties showed a similar change in RBD, increasing with an increase in Gli/Glu but that did not translate to much reduction in GI for Jandaroi. While mixograph parameters and the gluten index tests are correlated, they measure different aspects of dough properties because they are fundamentally different methods to evaluate dough [40]. Horvat et al., [41] showed in common wheat that increasing nitrogen level led to higher grain protein content with changes in the gliadin to glutenin ratio to a lesser extent which was variety specific.

Figure 15. Linear models showing fitted values (line) and observed (triangles) between Gli/Glu ratio and RBD (A) and between Gli/Glu ratio and GI(B) according to variety.

Figure 15. Linear models showing fitted values (line) and observed (triangles) between Gli/Glu ratio and RBD (A) and between Gli/Glu ratio and GI(B) according to variety.

5. Conclusions

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