*3.1. Weather Conditions*

The weather regime, in terms of the maximum (Tmax) and minimum air temperature (Tmin) and rainfall, during both experiments, is presented in Figure 2. The maximum and minimum temperatures increased from February to May, as typically occurs in Mediterranean environments. The environmental conditions di ffered between the two experimental years, with di fferences in the amount and distribution of precipitation during the growing season, as well as di fferences in temperature. In general, 2015–2016 (Exp 2) was considered to be a drier growing season compared to 2014–2015 (Exp 1).

**Figure 2.** Precipitation and air temperature (Tmin and Tmax) during Exp 1 ((**A**), 2014–2015) and Exp 2 ((**B**), 2015–2016). The arrows indicate the main phenological stages: S = sowing; A = anthesis.

#### *3.2. Temporal and Genotypic E*ff*ects*

Charles, Grace, Traveler, Fortuna, KWS Asta and RGT Planet were exclusively infected with *Pyrenophora teres* f. *teres* (net form net blotch—NFNB), whereas the cultivar Zhana was exclusively infected with *Rhynchosporium secalis* (leaf scald). NFNB occurred at all developmental stages and in both experiments, whereas leaf scald was consistently observed after the onset of the stem elongation phase (Figure 3). Although the disease severity tended to be higher in Exp 1 (disease dispersal from the infected barley seed) compared to Exp 2 (disease dispersal from the infected barley debris left after harvest) during the tillering phase for the malt barley, after the onset of the stem elongation stage, it was more pronounced in Exp 2. The same trend was also observed concerning leaf scald. The initial seeds from the malt barley varieties studied in Exp 1 presented different infection levels due to the occurrence of different disease severities in the collection sites (i.e., Charles DS = 33%, Grace DS = 26.5%, Fortuna DS = 17.8%, KWS Asta DS = 18.6% and Zhana = 6.7%). Interestingly, the disease severity in Exp 1 followed to a grea<sup>t</sup> extent the differences in the initial seed infection levels (Figure 3).

**Figure 3.** Malt barley cultivars' susceptibility to *Pyrenophora teres* f. *teres* (net form net blotch—NFNB) and *Rhynchosporium secalis* (leaf blotch and scald) at different developmental phases during both experiments. The numbers in the brackets refer to the Zadoks scale. Broad lines are medians, square open dots are means, boxes show the interquartile ranges, and whiskers extend to the last data points within 1.5 times the interquartile ranges. *p*-values of ANOVA and permutation tests are given. Groups not sharing the same letter are significantly different according to least significant difference (LSD) test (*p* < 0.05).

In general, infections by NFNB were more severe compared to leaf scald during all the tested developmental phases for the malt barley (Figure 3).

#### *3.3. The Area under Disease Progress Curve (AUDPC)*

The area under disease progress curve (AUDPC) in Exp 2 was not significantly affected either by the nitrogen rate or the interaction cultivar x nitrogen (Table 1). However, the analysis of variance for AUDPC indicated that a significant degree of genotypic variation existed among the studied malt barley cultivars in both experiments. The AUDPC values were lower in Exp 1 compared to Exp 2. Charles and Grace presented the highest values in Exp 1 and Exp 2, respectively (Figure 4).


**Table 1.** ANOVA summary for grain yield (GY), grain protein content (GPC), maltable (% grains > 2.2 mm), AUDPC and disease severity during the onset of stem elongation (DSSE) and grain filling (DSGF) phases.

\*, \*\* and \*\*\*: F values significant at the *p* < 0.05, *p* < 0.01 and *p* < 0.001 probability levels, respectively. ns stands for non-significant effect. a AUDPC: Area under disease progress curve.

**Figure 4.** Malt barley cultivars' susceptibility to *Pyrenophora teres* f. *teres* (net form net blotch—NFNB) and *Rhynchosporium secalis* (leaf blotch and scald) based on the area under disease progress curve (AUDPC). Broad lines are medians, square open dots are means, boxes show the interquartile ranges, and whiskers extend to the last data points within 1.5 times the interquartile ranges. *p*-values of ANOVA and permutation tests are given. Groups not sharing the same letter are significantly different according to LSD test (*p* < 0.05).

#### *3.4. Epidemiology Assessment When Nitrogen Rate and Genotype Are the Main Sources of Variation*

The distribution patterns of disease severity were analyzed by using hotspot and cluster and outlier analysis in ArcGIS 10x for three different crop developmental periods: (1) tillering (20–21Z), (2) stem elongation (30–31Z) and (3) milk development (71–73Z). Cluster and outlier analysis was used to identify clusters of disease-infected areas with the cluster types of HH, HL, LL and LH. LH represents a cluster of low values surrounded by high values, while HL is a cluster of high values surrounded by low values. In addition, LL and HH were statistically significant (*p* < 0.05) clusters of low and high disease severity values, respectively.

During the onset of the tillering phase, two experimental plots presented significant positive z scores, demonstrating significant clusters of intense disease severity. They were located on the western part of the field, and both of them included Traveler with nitrogen rates of 100 and 140 kg/ha, respectively (Figure 5). RGT Planet with a nitrogen rate of 100 kg/ha was also marked as a hotspot but less intense, though presenting a lower z-score (Figure 5). Note that lower z-scores indicate less intense clustering. The local Moran's I spatial analysis indicated only one High–Low outlier in the western part of the field. Indeed, Traveler with a rate of 100 kg N/ha was considered as an outlier since it presented high values of disease severity surrounded by lower surrounding values.

**Figure 5.** Composite hotspot analysis (Gi z-score) and cluster pattern analysis (local Moran's I) of disease severity (caused by *Pyrenophora teres* f. *teres* and *Rhynchosporium secalis*) assessed at different developmental stages of malt barley. A georeferenced arrangemen<sup>t</sup> of the experimental area showing the distribution of the cultivar and N-fertilizer treatments is also presented. The abbreviations stand for Gr = Grace, Zh = Zhana, Tr = Traveler and Pl = Planet.

During the stem elongation phase, hotspots increased in number and continued to be present in the western part of the field. The analysis identified three hotspots with very high z-scores (Grace with 60 kg N/ha; Traveler with 100 kg N/ha; and Traveler with 140 kg N/ha, one with a high (RGT Planet with 0 kg N/ha) and one with a moderate z-score (Grace with 60 kg N/ha). Although Zhana with 60 and 100 kg N/ha was surrounded by hotspots, it presented low values of disease severity. The local Moran's I spatial analysis confirmed the abovementioned results by characterizing these plots as Low–High outliers, indicating low values of disease severity compared to the surrounding plots. The analysis also identified a statistically significant (*p* < 0.05) cluster of increased disease severity, which coincided with two of the hotspots (Traveler and Planet in the western side) determined with the Getis–Ord G\* statistic (Figure 4).

Two Grace plots with 140 kg of N/ha were identified as hotspots of the highest z-scores during milk development and were followed by RGT Planet without nitrogen application. The local Moran's I spatial analysis again identified two Zhana plots (i.e., with nitrogen rates of 0 and 100 kg/ha) as spatial outliers, since they presented low disease severity in a neighborhood of high values (Figure 5).

#### *3.5. Quantifying the E*ff*ects of the Rate of Nitrogen Application and the Distance from the Nearest Hotspot on Crop Disease Severity*

Commonality analysis (CA) served to quantify the relative contributions of the rate of nitrogen application (kg/ha) and the distance from the nearest hotspot to crop disease severity. It is a method of partitioning variance that can discriminate the synergistic or antagonistic processes operating among predictors. Commonalities represent the percentage of variance in the dependent variable that is uniquely explained by each predictor (unique effect) or by all possible combinations of predictors (common effect), and their sum is always equal to the R<sup>2</sup> of the multiple linear regression. The distance from the nearest hotspot (m) and the quantity of applied nitrogen (kg/ha) explained 10 to 74% of the variance in disease severity (Table 2).


**Table 2.** Commonality coefficients including both unique and common effects, along with % total contribution of each predictor variable or sets of predictor variables to the regression effect.

a Refers to the distance from the nearest hotspot (m); b Refers to the rate of nitrogen application (kg/ha).

Examining the unique effects, it was found that for the period of the stem elongation phase, the distance from the nearest hotspot (m) was the best predictor of disease severity for all the study cultivars, uniquely explaining 16.8 to 45.5% of its variation. This amount of variance represented 38.76 to 97.65% of the R<sup>2</sup> effect (Table 2). On the contrary, during the onset of the grain filling phase the variation in disease severity was best explained by either the nitrogen rate (i.e., Traveler and Grace) or the distance from the nearest hotspot (m) (i.e., RGT Planet and Zhana) (Table 2).

#### *3.6. E*ff*ect of N and Genotype on Grain Yield and Quality Characters*

Disease severity was clearly not influenced by the N rate during the vegetative phase (i.e., stem elongation phase) of the malt barley. On the contrary, during the grain filling phase, the experimental data demonstrated a tendency for a positive relationship between the disease severity and the rate of nitrogen application (Figure 6); however, this tendency was not expressed in a statistically significant way according to ANOVA (Table 1).

**Figure 6.** The effect of nitrogen rate on disease severity (caused by *Pyrenophora teres* f. *teres* and *Rhynchosporium secalis*) assessed at different developmental stages of the studied malt barley varieties (Zhana, Grace, Traveler and RGT Planet). Broad lines are medians, square open dots are means, boxes show the interquartile ranges, and whiskers extend to the last data points within 1.5 times the interquartile ranges. *p*-values of ANOVA and permutation tests are given.

The grain yield was significantly affected by the cultivar and by the interaction cultivar x nitrogen (Table 1), and varied from 0.84 to 4.26 t ha−1. Grace and Traveler were the only cultivars that presented significant relationships between the grain yield and disease severity (Figure 7). In particular, Traveler showed a marginal, statistically significant negative relationship between the grain yield and disease severity, only for the period of tillering (Figure 7). Concerning Grace, the grain yield showed a negative, significant direct relationship with disease severity for the period of grain filling (milk development) and, on the contrary, presented a moderate, positive association with disease severity for the period of the tillering phase (Figure 7).

**Figure 7.** Relationship between grain yield and disease severity (caused by *Pyrenophora teres* f. *teres* and *Rhynchosporium secalis*) assessed at different developmental stages of malt barley, when the main source of variation is the nitrogen rate. The numbers in the brackets refer to the Zadoks scale. \* At *p* ≤ 0.05; \*\* at *p* ≤ 0.01; ns = non-significant.

Although the grain protein content was significantly affected by the N rate, the proportion of the maltable grain size fraction (% grains > 2.2 mm) seemed to be unaffected (Table 1). The relationship among the disease severity, maltable grain size fraction and grain protein content is shown in Figure 8.

**Figure 8.** Relationship of disease severity (caused by *Pyrenophora teres* f. *teres* and *Rhynchosporium secalis*) with grain protein content and maltable grain size fraction (>2.2 mm) at grain filling phase when the main source of variation is the nitrogen rate. \*\* At *P* ≤ 0.01; ns = non-significant.
