**5.4 The evaluation of ECB larval damage**

Stalks were dissected at the end of the vegetation period [2], at the beginning of September in 2012 and 2013, and October in 2014 due to the unfavorable environmental conditions. In total, 1080 maize plants were evaluated for tunnel length (cm) [TL].

#### **5.5 Chemical analysis of the maize leaves**

For plant analysis, we took 10 randomly chosen leaves below the ear in the silking stage, from each plot from 1080 maize plants. Leaves were cleaned from dust and other debris, stored in paper bags, and dried at 70°C to decrease in

**83**

*The Role of Irrigation and Nitrogen Fertilization on the Feeding Behavior of European Corn Borer*

elasticity and then further dried at 40°C for 24 hours. Dried leaves were crushed on Retsch Gmbh Germany, SM 100 mill, and from 10 crushed leaves, subsample was taken. Organic carbon was determined by oxidation of dry samples by a wet process at 135°C [37] and nitrogen content by the Kjeldahl method. The C/N ratio

The data were evaluated by analysis of variance after the data were subjected to a normality test using the SAS software [38]. Log transformation (log [n + 1]) was used to normalize the data. Least square means with the Tukey adjustment for multiple comparisons were calculated and reported for significance at the 95% confidence level. Back transformation was done for original values. Data in figures are presented with standard error (SE) bars. Pearson correlation coefficient is used

In the formation of maize reproductive tissues, heat stress occurs at 32.5°C air temperatures, and as a consequence, pollen viability is decreased and pollen tube elongated [14]. We have observed the highest air temperatures (on average 19.95°C) in vegetation season in 2012 with peaks above 33°C in July and August [2] and low natural rainfall (**Figure4**). In 2012, plants undergone drought stress since 22% less precipitation was recorded compared to the multi-year average (62.41 mm; https://meteo.hr). In subse-

**5.7 Influence of irrigation on nitrogen concentration and C/N ratio**

quent years, we noticed over multi-year average precipitations (**Figure4**).

The data on the damage of maize stalks caused by ECB larval feeding were previously analyzed and reported [2]. The greatest damage of maize stalks was observed in 2012, ranging from 49.13 cm (A3) to 79.22 cm (A1) tunnel length per stalk (**Figure 4**). The lowest damage was recorded at the highest level of irrigation (A3). Drought in 2012 probably affected ECB survival and consequently larval damage. Excessive rainfall in 2013 and 2014 could have caused eggs to rinse from maize leaves, thus preventing the penetration of larvae inside the stalk and less

Available nitrogen and water supply are the most important factors for plant growth and quality. Plant nitrogen concentration in maize leaves significantly differed between the irrigation plots and the control plots in 2012. Treatment with the highest nitrogen concentration was also with the highest irrigation level A3 (2.93%); compared to the control plot, it was higher for 8.87%. In the subsequent years, plant nitrogen concentrations were not affected by irrigation treatments (**Figure 5**). Soil moisture level and texture are the major factors influencing the root uptake of nitrogen [2]. Our results revealed that nitrogen uptake was not only increased under irrigation in drought year, which was characterized by high temperature and low rainfall, but also decreased in optimal or extremely wet years with a large amount of rainfall. Some authors reported that nitrogen concentration was increased in the aboveground part of irrigated plants and roots nitrogen concentra-

In 2012, the C/N ratio was the widest in control plots of irrigation (A1) (15:1), and it was significantly higher for 1.17 than in the plots with the highest irrigation level (A3). However, the C/N ratio in 2012 was narrowest comparing to the other 2 years. In subsequent years, there were no statistical differences between irrigation treatments concerning the C/N ratio (**Figure 6**). Due to the decreased nitrogen concentration during drought, the values of the C/N ratio were wider in a drought year (2012) and narrower in the other 2 years. In our study, the greatest damage by

*DOI: http://dx.doi.org/10.5772/intechopen.92598*

to test relationships between variables.

tion decreased in irrigation plots [39].

was calculated.

**5.6 Statistics**

plant damage.

#### *The Role of Irrigation and Nitrogen Fertilization on the Feeding Behavior of European Corn Borer DOI: http://dx.doi.org/10.5772/intechopen.92598*

elasticity and then further dried at 40°C for 24 hours. Dried leaves were crushed on Retsch Gmbh Germany, SM 100 mill, and from 10 crushed leaves, subsample was taken. Organic carbon was determined by oxidation of dry samples by a wet process at 135°C [37] and nitrogen content by the Kjeldahl method. The C/N ratio was calculated.

### **5.6 Statistics**

*Pests, Weeds and Diseases in Agricultural Crop and Animal Husbandry Production*

Maize was irrigated by Typhon system (sprinkling irrigation). The range of the system was 20–25 m. Intensity and amount of water were regulated by the selection of nozzles and the speed system movement. Watermark 200SS device was used to determine the start of irrigation and to monitor the soil moisture condition. The

Nitrogen fertilization was applied four times per vegetation. One-third of urea (46% N) was applied in the autumn with the basic soil cultivation, and the twothirds were added pre-sowing. Top dressing was done twice with KAN (calciumammonium nitrate; 27% N). The first top dressing, one sixth of N was done in phase 6–8 leaves, and the last application, one sixth of N was done in phase 8–10 leaves.

Maize genotypes used in this experiment are developed at the Agricultural

**Hybrid Factor FAO group Insect tolerance** OSSK 596 C1 590 + OSSK 617 C2 610 + OSSK 602 C3 620 ++ OSSK 552 C4 580 *—*

*+—Increased tolerance to diseases and insects. ++—Increased tolerance to ECB. – Not specified (Source: http://www.*

Stalks were dissected at the end of the vegetation period [2], at the beginning of September in 2012 and 2013, and October in 2014 due to the unfavorable environmental conditions. In total, 1080 maize plants were evaluated for tunnel length

For plant analysis, we took 10 randomly chosen leaves below the ear in the silking stage, from each plot from 1080 maize plants. Leaves were cleaned from dust and other debris, stored in paper bags, and dried at 70°C to decrease in

× 3 level of nitrogen fertil-

; and

).

= 56 m2

(10 m × 2 rows × 0.7 m = 14 m2

1.Plot size was: factor (a), irrigation, 4 hybrids 56 m2

2.Subfactor (B), nitrogen fertilization, 4 hybrids × 14 m<sup>2</sup>

ization = 168 m2

**5.2 Nitrogen fertilization**

**5.3 The maize genotypes**

**5.1 Irrigation**

;

3.Subsubfactor (C), maize hybrid, 14 m<sup>2</sup>

water quality analysis was satisfactory [35].

Institute Osijek and presented in **Table 2**.

**5.4 The evaluation of ECB larval damage**

**5.5 Chemical analysis of the maize leaves**

**82**

(cm) [TL].

*poljinos.hr) [36].*

**Table 2.** *Maize genotypes.*

The data were evaluated by analysis of variance after the data were subjected to a normality test using the SAS software [38]. Log transformation (log [n + 1]) was used to normalize the data. Least square means with the Tukey adjustment for multiple comparisons were calculated and reported for significance at the 95% confidence level. Back transformation was done for original values. Data in figures are presented with standard error (SE) bars. Pearson correlation coefficient is used to test relationships between variables.

#### **5.7 Influence of irrigation on nitrogen concentration and C/N ratio**

In the formation of maize reproductive tissues, heat stress occurs at 32.5°C air temperatures, and as a consequence, pollen viability is decreased and pollen tube elongated [14]. We have observed the highest air temperatures (on average 19.95°C) in vegetation season in 2012 with peaks above 33°C in July and August [2] and low natural rainfall (**Figure4**). In 2012, plants undergone drought stress since 22% less precipitation was recorded compared to the multi-year average (62.41 mm; https://meteo.hr). In subsequent years, we noticed over multi-year average precipitations (**Figure4**).

The data on the damage of maize stalks caused by ECB larval feeding were previously analyzed and reported [2]. The greatest damage of maize stalks was observed in 2012, ranging from 49.13 cm (A3) to 79.22 cm (A1) tunnel length per stalk (**Figure 4**). The lowest damage was recorded at the highest level of irrigation (A3). Drought in 2012 probably affected ECB survival and consequently larval damage. Excessive rainfall in 2013 and 2014 could have caused eggs to rinse from maize leaves, thus preventing the penetration of larvae inside the stalk and less plant damage.

Available nitrogen and water supply are the most important factors for plant growth and quality. Plant nitrogen concentration in maize leaves significantly differed between the irrigation plots and the control plots in 2012. Treatment with the highest nitrogen concentration was also with the highest irrigation level A3 (2.93%); compared to the control plot, it was higher for 8.87%. In the subsequent years, plant nitrogen concentrations were not affected by irrigation treatments (**Figure 5**). Soil moisture level and texture are the major factors influencing the root uptake of nitrogen [2]. Our results revealed that nitrogen uptake was not only increased under irrigation in drought year, which was characterized by high temperature and low rainfall, but also decreased in optimal or extremely wet years with a large amount of rainfall. Some authors reported that nitrogen concentration was increased in the aboveground part of irrigated plants and roots nitrogen concentration decreased in irrigation plots [39].

In 2012, the C/N ratio was the widest in control plots of irrigation (A1) (15:1), and it was significantly higher for 1.17 than in the plots with the highest irrigation level (A3). However, the C/N ratio in 2012 was narrowest comparing to the other 2 years. In subsequent years, there were no statistical differences between irrigation treatments concerning the C/N ratio (**Figure 6**). Due to the decreased nitrogen concentration during drought, the values of the C/N ratio were wider in a drought year (2012) and narrower in the other 2 years. In our study, the greatest damage by

**Figure 4.**

*Maize damage caused by ECB larval feeding and agroclimatic conditions 2012–2014.*

**Figure 5.**

*Plant nitrogen concentrations in irrigation treatments presented by years of investigation.*

**85**

**Figure 7.**

*The Role of Irrigation and Nitrogen Fertilization on the Feeding Behavior of European Corn Borer*

ECB was found in 2012 when the narrowest C/N ratio was calculated compared to

Plant nitrogen concentration increased with an increase in fertilization rates,

researchers, and they also obtained similar results [40–43]. Upon herbivores attack, maize plants differently react, and it can be observed as translocation of sugars in stalk and root, increase in nutrition and photosynthesis, and other processes. All these changes can affect the C/N ratio in plant tissue [44]. The significantly highest concentration of nitrogen occurred, as it was expected in fertilization treatments with the highest rates (B3) in all years of research, compared to the control (B1)

The B2 and B3 treatments did not differ statistically in plant nitrogen concentration. The lowest nitrogen concentrations were detected in 2012 in control plots (B1), and compared to the plots with the highest level of fertilization (B3), it was lower for 2.87%, in 2013 for 31.41%, and in 2014 for 22.26% (**Figure 7**). On average, 20.7% higher nitrogen concentration was found in plants in the B3 treatment compared to the control. Nitrogen fertilization increases ear weight and yields [45]. In all years, the greatest damage, tunnel length created by ECB larvae, was in the B3 treatment and the lowest in the control. Our data are similar to previously reported studies [8, 9]. By increasing the level of nitrogen fertilization, the C/N ratio was significantly reduced. Significant differences in C/N ratio were found in all years between the control (B1) and the other two treatments of nitrogen fertilization (B2 and B3), with the exception in 2012, when significantly differed only B1 and B3. The rate of applied nitrogen was not a significant factor for the C/N ratio since the B2 and B3 treatments did not differ statistically. In 2014, the C/N ratio was the widest (15:1) on the control treatment (B1), and it was wider for 5.47 than the B3 treatment. Similar results are obtained in 2013 (23:1) when the C/N ratio was wider for 8.38 and in 2012 (15:1) for 1.27 (**Figure 8**). On average, the widest C/N ratio (21:1) was recorded at the treatment B1, and compared to the B3 treatment, it was

The highest nitrogen concentration in this research and the lowest damage from

ECB larvae were observed in maize hybrid C4 (**Figure 4**) [2]. These results are contrary to the studies who reported a positive relationship between plant nitrogen and ECB damage. The insects' interactions are complex, and other compounds

*Nitrogen concentrations in fertilization treatments presented by years of investigation.*

while the C/N ratio was narrowed. This problem has been studied by many

*DOI: http://dx.doi.org/10.5772/intechopen.92598*

the other 2 years.

(**Figure 7**).

significantly wider for 5.04.

**Figure 6.** *The C/N ratio in irrigation treatments presented by years of investigation.*

*The Role of Irrigation and Nitrogen Fertilization on the Feeding Behavior of European Corn Borer DOI: http://dx.doi.org/10.5772/intechopen.92598*

ECB was found in 2012 when the narrowest C/N ratio was calculated compared to the other 2 years.

Plant nitrogen concentration increased with an increase in fertilization rates, while the C/N ratio was narrowed. This problem has been studied by many researchers, and they also obtained similar results [40–43]. Upon herbivores attack, maize plants differently react, and it can be observed as translocation of sugars in stalk and root, increase in nutrition and photosynthesis, and other processes. All these changes can affect the C/N ratio in plant tissue [44]. The significantly highest concentration of nitrogen occurred, as it was expected in fertilization treatments with the highest rates (B3) in all years of research, compared to the control (B1) (**Figure 7**).

The B2 and B3 treatments did not differ statistically in plant nitrogen concentration. The lowest nitrogen concentrations were detected in 2012 in control plots (B1), and compared to the plots with the highest level of fertilization (B3), it was lower for 2.87%, in 2013 for 31.41%, and in 2014 for 22.26% (**Figure 7**). On average, 20.7% higher nitrogen concentration was found in plants in the B3 treatment compared to the control. Nitrogen fertilization increases ear weight and yields [45]. In all years, the greatest damage, tunnel length created by ECB larvae, was in the B3 treatment and the lowest in the control. Our data are similar to previously reported studies [8, 9]. By increasing the level of nitrogen fertilization, the C/N ratio was significantly reduced. Significant differences in C/N ratio were found in all years between the control (B1) and the other two treatments of nitrogen fertilization (B2 and B3), with the exception in 2012, when significantly differed only B1 and B3. The rate of applied nitrogen was not a significant factor for the C/N ratio since the B2 and B3 treatments did not differ statistically. In 2014, the C/N ratio was the widest (15:1) on the control treatment (B1), and it was wider for 5.47 than the B3 treatment. Similar results are obtained in 2013 (23:1) when the C/N ratio was wider for 8.38 and in 2012 (15:1) for 1.27 (**Figure 8**). On average, the widest C/N ratio (21:1) was recorded at the treatment B1, and compared to the B3 treatment, it was significantly wider for 5.04.

The highest nitrogen concentration in this research and the lowest damage from ECB larvae were observed in maize hybrid C4 (**Figure 4**) [2]. These results are contrary to the studies who reported a positive relationship between plant nitrogen and ECB damage. The insects' interactions are complex, and other compounds

*Pests, Weeds and Diseases in Agricultural Crop and Animal Husbandry Production*

*Maize damage caused by ECB larval feeding and agroclimatic conditions 2012–2014.*

*Plant nitrogen concentrations in irrigation treatments presented by years of investigation.*

**84**

**Figure 6.**

**Figure 5.**

**Figure 4.**

*The C/N ratio in irrigation treatments presented by years of investigation.*

**Figure 8.** *The C/N ratio in nitrogen fertilization treatments presented by years of investigation.*

#### **Figure 9.**

*Nitrogen concentrations in different maize hybrids presented by years of investigation.*

**87**

**Irrigation**

> **A1**

> > **2012**

Rtl;N Rtl;C/N RN;C/N

**2013**

Rtl;N Rtl;C/N RN;C/N

**2014**

Rtl;N Rtl;C/N RN;C/N *length; N, nitrogen; C/N ratio.*

*\*P < 0.05.*

*\*\*P < 0.01.*

**Table 3.** *Correlation coefficients between ECB feeding and nitrogen concentrations and C/N ratio among tested years and treatments.*

−0.96

\*\*

−0.98\*\*

−0.97\*\*

−0.97\*\*

−0.93\*\* *A1—control, A2—from 60 to 100% WFC, A3—from 80 to 100% WFC; B1—control, B2—100 kg N ha−1, B3—200 kg N ha−1; C1—OSSK 596, C2—OSSK 617, C3—OSSK 602, C4—OSSK 552; tl, tunnel* 

−0.96\*\*

−0.96\*\*

−0.98\*\*

−0.98\*\*

−0.97\*\*

0.01

−0.03

−0.05

−0.28

−0.05

−0.07

0.32

−0.26

−0.15\*

0.02

0.01

−0.01

0.03

0.23

0.02

0.14

−0.26

0.22

0.19

−0.05

−0.98

\*\*

−0.99\*\*

−0.98\*\*

−0.99\*\*

−0.95\*\*

−0.99\*\*

−0.98\*\*

−0.99\*\*

−0.99\*\*

−0.99\*\*

−0.11

−0.26

−0.09

−0.05

0.28

−0.11

−0.24

0.10

−0.49\*\*

0.13

0.09

0.27

0.09

0.07

−0.32

0.11

0.23

−0.17

0.47\*

−0.89

\*\*

−0.91\*\*

−0.93\*\*

−0.85\*\*

−0.86\*\*

−0.95\*\*

−0.91\*\*

−0.91\*\*

−0.91\*\*

−0.89\*\*

0.01

−0.15

−0.01

0.36\*

−0.18

0.30

0.19

−0.08

0.24

−0.11

−0.04

0.16

0.07

−0.43

0.09

−0.26

−0.16

0.09

−0.21

0.07

\*\*

**A2**

**A3**

**B1**

**B2**

**B3**

**C1**

**C2**

**C3**

**Nitrogen fertilization**

**Maize hybrids**

*The Role of Irrigation and Nitrogen Fertilization on the Feeding Behavior of European Corn Borer*

−0.14

*DOI: http://dx.doi.org/10.5772/intechopen.92598*

**C4**

**Figure 10.** *The C/N ratio in different maize hybrids presented by years of investigation.*


*The Role of Irrigation and Nitrogen Fertilization on the Feeding Behavior of European Corn Borer DOI: http://dx.doi.org/10.5772/intechopen.92598*

**Table 3.**

*Correlation coefficients between ECB feeding and nitrogen concentrations and C/N ratio among tested years and treatments.*

*Pests, Weeds and Diseases in Agricultural Crop and Animal Husbandry Production*

*Nitrogen concentrations in different maize hybrids presented by years of investigation.*

*The C/N ratio in nitrogen fertilization treatments presented by years of investigation.*

*The C/N ratio in different maize hybrids presented by years of investigation.*

**86**

**Figure 10.**

**Figure 9.**

**Figure 8.**

may have an impact on larval feeding such as phenols, carbohydrates, and other components [46]. The highest nitrogen concentrations on average were determined in 2012, while the lowest was found in 2014 (**Figure 9**). In both years, a statistically significant difference occurred between C2 and C4 hybrid. On average, hybrid C4 had the highest nitrogen concentration, and it was higher from 2.75 to 9.45% than observed in other hybrids.

The C/N ratio was found to be significantly different among several hybrids only in 2012. The hybrid C1 had significantly wider value than the hybrid C4 (**Figure 10**). On average, no significant difference occurred between the hybrids; however, the widest C/N ratio was measured for hybrid C2 (18:1).

The relationship between nitrogen concentration and C/N ratio was strong negative in all years and all treatments. The relationship between tunnel length in stalks caused by the ECB larvae and nitrogen concentrations was weak or moderately strong but inconsistent over the years of investigation. Plants require carbon and nutrients for growth. If nutrients are limited, plants tend to accumulate more carbohydrates that can be immediately used. When the ratio of carbon is increased to nutrients, some carbohydrates can be incorporated into secondary metabolism of plant. Secondary metabolites have a defensive role in plants [47]. Carbon, water, and mineral nutrient allocation in a plant depend on genotype and plant environment [48]. The concentration of secondary metabolites increases with drought stress [49]. Nitrogen fertilization leads to a high concentration of nitrogen in plant tissue and a lower concentration of secondary metabolites, but drought stress limits nitrogen adsorption, and such plants are not attractive to herbivores. Our investigation did not give strong evidence that nitrogen concentration and the C/N ratio impact the feeding behavior of ECB larvae in maize stalks (**Table 3**).

### **6. Conclusion**

Nitrogen uptake was increased in irrigation treatments in drought year characterized by high temperatures and a small amount of rainfall. Decreased plant nitrogen concentrations were observed in optimal or extremely wet years with a large amount of rainfall. By increasing the level of nitrogen fertilization, the C/N ratio was significantly reduced. The highest nitrogen concentration in this research and the lowest damage from ECB larvae were observed in maize hybrid C4. The relationship between nitrogen concentration and C/N ratio was strongly negative. We found a weak or moderately strong relationship between damage caused by the ECB larva and nitrogen concentration. Our results indicate that maize damage caused by ECB is negatively affected by plant nitrogen concentrations only when plants are under drought stress. However, the relationship between ECB larval damage and plant nitrogen concentration depends on the nitrogen fertilization rates. We did not find strong evidence for this hypothesis and did not prove that plant nitrogen concentration or more quality plants would be more damaged by European corn borer. Further studies, in controlled environments, are needed since our results were inconsistent over the years and indicate the great impact of agroclimatic conditions (drought) on the potential of ECB to create damage.

#### **Acknowledgements**

The research was financed by the Ministry of Science, Education, and Sports of the Republic of Croatia (Project: 079-0790570-2208).

**89**

**Author details**

Ankica Sarajlić1

and Ivana Majić1

\*, Emilija Raspudić1

2 Agricultural Institute Osijek, Osijek, Croatia

provided the original work is properly cited.

Section of Entomology and Nematology, Osijek, Croatia

\*Address all correspondence to: ankica.sarajlic@fazos.hr

, Zdenko Lončarić1

1 Faculty of Agrobiotechnical Sciences Osijek, Department of Phytomedicine

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Marko Josipović2

*The Role of Irrigation and Nitrogen Fertilization on the Feeding Behavior of European Corn Borer*

All authors saw and approved this book chapter. We warrant that the chapter is the authors' original work and is not under consideration for publication elsewhere. All coauthors agree that the corresponding author will be responsible for the submission. We warrant that all authors have contributed significantly to the work.

*DOI: http://dx.doi.org/10.5772/intechopen.92598*

**Conflict of interest**

*The Role of Irrigation and Nitrogen Fertilization on the Feeding Behavior of European Corn Borer DOI: http://dx.doi.org/10.5772/intechopen.92598*
