Moderate Nitrogen Management Enhancing Maize Lodging Resistance by Reducing Pathogen Infection and Expansion of Stalk Rot

Abstract

At present, maize production is facing the challenge of balancing stalk lodging caused by stalk rot with maintaining a good grain yield potential. Improving the basal internode properties by optimizing nitrogen (N) management is an effective strategy to reduce the stalk rot incidence and stalk lodging rate, whilst simultaneously achieving a stable grain yield. A two-year field study was conducted to evaluate the basal internode characteristics under natural field conditions and inoculation with Fusarium pseudograminearum, the causative pathogen of stalk rot, and also to measure the ultimate grain yield with four different N fertilizer application rates (0, 120, 180, and 240 kg N ha⁻¹). Rapid injection inoculation and histochemical staining technologies were employed to assess the stalk rot and lodging resistance. The results showed that reducing N rates improved the basal internode qualities (i.e., shorter internode length, larger cross-sectional area, improved internode plumpness and sclerenchyma tissue, and higher lignified degree and vascular number in sclerenchyma tissue) and enhanced the infection resistance. The lodging rate and stalk rot incidence all gradually declined with reduced N rates. Furthermore, pathogen diffusion degree which was indicated by infection index was decreased with reducing N rates. Ultimately, the mechanical strength of the N0, N120, N180, and N240 plants decreased by 5.31%, 5.83%, 5.01%, and 11.21% compared with that of the control, respectively. These results suggested that the stalk quality was improved through optimal nitrogen application, and also that the stalk rot and lodging resistance increased. The grain yield of the N180 plants was superior to that of those receiving the other treatments. Overall, 180 kg N ha⁻¹ is recommended to balance the stalk lodging resistance and grain yield in the research region. Additionally, breeding cultivars with improved stalk quality is likely to be required to counter the escalating lodging risks arising from stalk rot occurrence.

1. Introduction

Maize (Zea mays L.) is one of the most important cereal crops. It is unlikely that further expansions of the agricultural farmland it currently occupies will be needed. Therefore, increasing the yield per unit area is urgently required to satisfy the ever-increasing food, feed, and industrial processing demands [1,2,3]. However, maize stalk lodging has become a critical limiting factor for improving the grain yield in modern production scenarios [4,5]. It reduces the maize yield by approximately 5~25%—up to 75% in severe cases—and leads to a completely diminished yield [6,7]. Lodging also results in grain quality reduction and harvesting difficulties [8]. Furthermore, extreme weather events, including gales and torrential rain, occur frequently in the context of climate change, and are one of the primary external causes of stalk lodging [9]. Hence, enhancing maize lodging resistance has become a key goal for increasing and/or maintaining the grain yield per unit area.

Maize basal internode properties play a vital role in improving the stalk lodging resistance [10,11]. Many researchers have shown that the maize lodging resistance strongly depends on the morphological characteristics, anatomical structure, and mechanical strength of the basal internodes [12,13,14]. A wind tunnel simulation experiment revealed that the plant height and basal internode anatomy traits were key factors for maize stalk lodging resistance [15]. A meta-analysis also indicated that the dry weight of biomass, cellulose and lignin content, and anatomical structure were all identified as important indicators of lodging stress tolerance [16]. The stalk diameter, dry weight, lignin and cellulose content, and bending strength of the basal internodes are all significantly positively correlated with the maize stalk lodging resistance. That is to say, high-quality basal internodes possess a larger stalk diameter, higher dry weight per unit length, thicker sclerenchyma tissues, stronger bending strength, and so on. Thus, improving the basal internode quality could decrease the lodging risk and ensure a stable grain yield.

Lower stalk quality is a direct cause of lodging. However, stalk rot, a devastating soil-borne disease, is an indirect factor causing stalk lodging [17,18]. Stalk rot is recognized by irregular longitudinal brown spots at the basal internodes of the stalk; then, constriction, softening or hardening, and loosening of the stalk interior occur at a later stage of the disease, which is related to the senescence of stalk pith cells [19]. Fusarium (F.) pseudograminearum is the main pathogen causing maize stalk rot in the North China Plain. Conidiums of F. pseudograminearum can infect the plant through the root and/or wounds caused by mechanical injury. Stalk rot leads to vascular bundle damage and then reduces the stalk’s mechanical strength [20]. Therefore, the decomposition of the maize stalk after infection by this pathogen causes a decrease in the stalk quality and thus increases the risk of lodging. Statistically, the incidence of stalk rot in the field reaches 5–30% in China [21]. The causative pathogen spreads and infects plants mainly through the soil; therefore, applying fungicides is ineffective at completely curing stalk rot. A convenient and effective strategy is urgently needed, e.g., crop management optimization for enhancing plants’ resistance and then reducing maize stalk rot occurrence and the lodging rate.

N is one of the most important nutrients for plant growth and improving grain yield. Moderate N fertilizer application could optimize photosynthate allocation to the basal internodes, improving internode formation and, thus, the stalk quality [22]. Therefore, N application is the most accessible and effective measure that improves the lodging resistance of maize. Many studies have shown that excess N application leads to plant over-growth, increases plant height and internode length, thins internodes, and lowers the stalk filling degree, which increases plants’ susceptibility to stalk lodging [23,24,25]. Furthermore, previous studies have reported that the chemical composition of basal internodes, comprising lignin and cellulose, could be significantly affected by the N application rate, and therefore change the internode toughness and rigidity [26,27]. However, there is limited research on the effects of N application rates on lodging resistance through the regulation of pathogen infection and stalk rot spread as well as the relationship between them.

Based on the above, we hypothesized that moderate N application could increase maize’s lodging resistance by regulating its stalk rot resistance. In this study, we conducted two field trials of different N application rates, examining the basal third internode characteristics under natural field conditions and inoculation with a pathogen in the growing seasons of 2019 and 2020. The morphological characteristics, anatomical structure, basal third internode mechanical strength, and final grain yield were analyzed. Also, the pathogen infection process and its extent were also observed after inoculation. The main objectives of this study were as follows: (1) to analyze the growing dynamics of indicators relating to the mechanical strength of the basal third internodes with different N application rates; (2) to clarify the regulating effects of N application rates on the lodging resistance under natural conditions and inoculation; and (3) to recommend a N application level that balances the lodging resistance and grain yield.

2. Materials and Methods

2.1. Experimental Site Description

The field experiment was established in 2019 and 2020 at Dishang Experimental Station, Hebei province, China (114°72′ E, 37°94′ N; 65 m asl), which represents the agricultural production and climate conditions of the North China Plain in China. The experimental site experiences a warm, temperate zone, semi-humid, and monsoon climate. Figure 1 presents the average monthly precipitation and air temperatures in 2019 and 2020 growing seasons. The soil at the experimental site is light loam, and the soil characteristics at the 0–20 cm soil layer were shown in

Table 1. The soil characteristics at the 0–20 cm soil layer of the experiment site.

Soil Organic Matter (g kg−1)	Total N (g kg−1)	Total K (g kg−1)	Available N (mg kg−1)	Available P (mg kg−1)	Available K (mg kg−1)
16.1	1.27	2.2	75.7	32.1	107.0

. The assay standardized methods of soil organic matter, total N, total K, available N, available P, and available K were the Walkley–Black method, Kjeldahl method, Sodium bicarbonate extraction, Sodium hydroxide melting, Sodium hydroxide extraction, and Ammonium acetate extraction, respectively [28].

2.2. Experimental Material and Design

Experiment 1 was designed to explore the effects of N application rates on maize stalk quality, lodging rate and stalk rot incidence in 2019 and 2020. The maize hybrid Zhengdan 958 (ZD958) was selected as the experimental material, which was applied widely in the research region. The planting density was 7.5 × 10⁴ plants ha⁻¹. The experiment adopted a completely random design. Each treatment was arranged for three replications, and the plot area was 56.3 m² (7.5 m × 7.5 m). Four N fertilizer application rates (0, 120, 180, and 240 kg ha⁻¹) were designed, which were denoted by N0, N120, N180, and N240, respectively. The application rates of the phosphorus (P₂O₅) and potassium (K₂O) for each treatment were 120 kg ha⁻¹ and 150 kg ha⁻¹. The N, P, and K fertilizers were all applied once at sowing according to the general management mode in the research region.

Experiment 2 was designed with a rapid injection inoculation technology undertaking to evaluate the regulating effects of N application rates on resistance to pathogen expansion of stalk rot. The fifty plants per plot with uniform growth and no pests and diseases were selected to mark using tags at tasseling stage. The main pathogen of F. pseudograminearum causing stalk rot at the research region was inoculated on Potato Dextrose Agar plate medium (200 g potato, 20 g glucose, 20 g agar, 1000 mL water) and cultured for 7 days till the hyphae took over the entire plate. Then, the agar and F. pseudograminearum were mixed with a concentration of 2.0 × 10⁶ mL⁻¹ for inoculation. Before inoculation, a drill with a diameter of 3 mm was used for opening a hole in the middle of the third basal internode above the soil of selected plants at 7 days after silking, into which 0.5 mL of mixture was injected at the four N treatments. Then, petroleum jelly was smeared on the hole to prevent rainwater from flowing into the hole. Healthy maize plants of each N treatment served as controls.

2.3. Measuring Items and Methods

2.3.1. Natural Stalk Lodging Rate and Stalk Rot Incidence

Plants were considered stalk-lodged when they broke at or below the ear-bearing node. The number of stalk lodging plants in each plot was recorded. The stalk lodging rate was calculated by dividing the number of stalk-lodged plants by the total number of plants. The stalk rot incidence was calculated by dividing the number of stalk rot plants by the total number of plants.

2.3.2. Morphological Characteristics of Basal Third Internode

Sampling occurred at V12 (twelve-unfolded leaves), VT (tasseling), R4 (dough stage), and R6 (physiological maturity) in 2019 and added R2 (blister stage) and R5 (dent stage) in 2020. Five representative plants in each plot were sampled to measure mechanical properties of basal third internode and anatomical features.

The lengths of the basal third internodes (L, cm) were measured with a ruler. The major axis (A₁, mm) and short axis (A₂, mm) of the basal third internodes were measured using a digital vernier caliper. The cross sectional area (CSA, mm²) was calculated by Formula (1) [29].

$$CSA=\left({\displaystyle \frac{A_1}{2}}\right)\ast \left({\displaystyle \frac{A_2}{2}}\right)\ast \Pi$$

(1)

The basal third internodes were dried at 75 °C in a drying oven to a constant weight and then weighted using a balance scale (DW, mg). The internode plumpness (mg/cm DW), referring to the dry weight per length of basal third internode, was calculated by Formula (2).

Internode plumpness = DW/L

(2)

2.3.3. Histochemical Staining of Transverse

About 1 cm of tissue was cut from the basal third internodes with a sharp blade at the R2 stage and then put into FAA solution (70% ethanol, acetic acid, 38% formaldehyde) quickly for at least 48 h. To observe the lignified cell walls, safranin O-fast green staining was performed according to standard protocols. The area and number of vascular bundles and the thickness of sclerenchyma tissue were quantified using CaseViewer software 2.3.2 (3DHISTECH Ltd., Budapest, Hungary).

2.3.4. Mechanical Property

The mechanical strength (N) of basal third internodes was determined using a stalk strength tester (Zhengjiang Top Instrument Co., Ltd., Hangzhou, China) at each sampling stage. The test probes (“U” type and pin type) of the tester were vertical and uniform and acted on the middle of the internodes, and then the maximum force required to break or pierce the stem was displayed on the screen of the tester.

2.3.5. Infection Index

After measuring the mechanical strength of the internode, the stalk was longitudinally split to inspect the pith for discoloration, and then the length of discoloration of internodes (DL) was measured with a ruler. The total length of the internode (L) was also recorded (Figure 2). The internode infection index was calculated according to Formula (3) [13].

Infection index (%) = DL/L × 100%

(3)

2.4. Statistical Analysis

Analysis of variance (ANOVA) was performed for the stalk morphological characteristics, mechanical properties, cell wall components, and grain yield using IBM SPSS Statistics 19.0 software (SPSS Inc., Chicago, IL, USA). Comparisons among different treatments were separated using the least significant differences (LSD) test at p ≤ 0.05. The figures in this experiment were plotted using SigmaPlot 14.0 (Systat Software, Inc., SanJose, CA, USA).

3. Results

3.1. The Morphological Parameters of Basal Third Internodes

There were significant differences in the internode length, cross-sectional area, and internode plumpness as growing progressed (Figure 3). The internode length showed a stable trend from V12 with each N treatment (Figure 3a,b). However, the cross-sectional area exhibited an increase with development and reached its maximum at R1, then became stable (Figure 3c,d). The basal third internode plumpness presented a unimodal curve, reached its maximum value at the R4 stage, and then gradually decreased (Figure 3e,f).

The N application rates had significant effects on the internode length, cross-sectional area, and internode plumpness. With decreasing N application rates, both the internode length and cross-sectional area showed a trend of decreasing gradually over the two growing seasons. Compared with those of N240, the internode length and cross-sectional area of the N0, N120, and N180 plants decreased by 12.20%, 7.46%, and 3.81%, and 13.03%, 5.64%, and 1.28%, respectively. It was observed that there was no significant change in the internode length from V12 to R6. The cross-sectional area increased with the N rates, but that of the N180 and N240 plants showed no significant difference during the R1-R6 period. In the V12-R1 period, the internode plumpness tended to decrease with decreasing N application, but in the R1-R6 period, the internode plumpness of the N180 plants was the highest. These results indicate that moderate N application (180 kg ha⁻¹) could reduce the basal internode length, maintain a relatively high stalk diameter, and improve the internode plumpness of maize basal stalks during the filling stage.

3.2. The Anatomical Structure of Basal Third Internodes

The N application rates had significant effects on the vascular bundle diameter per unit visual field of the maize basal third internode (Figure 4). With decreasing N application rates, the bundle diameter per unit visual field showed an increasing trend. That of the plants receiving the N0 treatment was significantly higher than those under other treatments, but there was no significant difference between the plants receiving the N180 and N240 treatments.

The effect of the N application rate on the vascular bundle number in the sclerenchyma and parenchyma of the maize basal third internode was different (

Table 2. Number of vascular bundles under different N application rates.

Years	Treatments	Number of Vascular Bundles in Sclerenchyma	Number of Vascular Bundles in Parenchyma	Total Number of Vascular Bundles
2019	N0	43.14 ± 0.6 c	22.28 ± 1.4 a	65.18 ± 0.5 b
	N120	48.23 ± 1.2 b	20.35 ± 0.7 ab	68.08 ± 0.9 a
	N180	51.32 ± 2.3 b	19.19 ± 0.6 b	70.31 ± 1.2 a
	N240	55.04 ± 0.8 a	16.27 ± 1.1 c	71.42 ± 1.5 a
2020	N0	42.09 ± 0.9 d	26.52 ± 2.1 a	68.51 ± 0.8 a
	N120	46.37 ± 1.1 c	22.37 ± 1.5 b	68.37 ± 2.4 a
	N180	49.06 ± 1.5 b	20.26 ± 1.4 b	69.61 ± 1.6 a
	N240	53.34 ± 1.2 a	15.17 ± 0.9 c	68.57 ± 1.7 a
ANOVA (p-value)
Y (Year)		0.651 ns	0.066 ns	0.199 ns
N (N)		0.036 *	0.025 *	0.494 ns
Y × N		0.142 ns	0.089 ns	0.760 ns

N0, N120, N180, and N240 represent N application rates of 0, 120, 180, and 240 kg N ha⁻¹, respectively. * represent difference at the 0.05 probability level (p < 0.05), and ^ns represent not significant at the 0.05 probability level (p > 0.05). Different lowercase letters denote significant differences between treatments at p < 0.05.

). The number of vascular bundles in the sclerenchyma decreased with decreasing N, while the number of vascular bundles in the parenchyma had the opposite trend. The effect of the N application rate on the total vascular bundle number was inconspicuous, except for that with N0, which was significantly lower than that with the other treatments.

To investigate the basal internode quality in more detail, the anatomical characteristics were measured using paraffin sections (Figure 5). The N application rates had a significant effect on the micro-structure and lignification degree of the maize basal internodes. With increasing N application, the sclerenchyma thickness first increased (from 655.20 μm at N0 to 1074.90 μm at N180) and then decreased (from 1074.90 μm at N180 to 725.80 μm at N240). The lignification degree of the sclerenchyma, parenchyma, and vascular bundle increased with decreasing N. The results showed that moderately reducing the N amount could improve the lignification degree of the maize basal stalks and improve the stalk quality.

3.3. The Mechanical Strength of Basal Third Internodes

The N application levels and growing stages had significant effects on the mechanical strength of the basal third internode (

Table 3. Effect of N application levels on mechanical strength of basal third internodes at V12, R1, R3, R4, R5, and R6 in two growing seasons.

Years	N Rates	Mechanical Strength (N)
		V12	R1	R3	R4	R5	R6
2019	N0	191.1 ± 6.9 c	341.0 ± 10.0 ab	-	375.5 ± 13.8 c	-	269.0 ± 13.8 b
	N120	231.5 ± 12.8 b	337.0 ± 8.6 b	-	389.9 ± 10.1 b	-	287.0 ± 13.4 a
	N180	251.8 ± 11.3 a	347.5 ± 6.9 a	-	415.7 ± 11.1 a	-	295.9 ± 9.4 a
	N240	226.4 ± 10.7 b	321.2 ± 13.4 c	-	364.4 ± 10.4 c	-	259.9 ± 6.4 b
2020	N0	193.1 ± 10.5 d	279.0 ± 12.9 c	321.7 ± 7.8 c	304.7 ± 7.7 c	291.6 ± 13.8 b	233.4 ± 9.5 b
	N120	262.6 ± 5.2 b	328.8 ± 10.2 ab	350.0 ± 12.9 b	374.6 ± 9.2 b	323.6 ± 7.4 a	248.5 ± 10.0 a
	N180	290.6 ± 15.3 a	336.6 ± 3.1 a	363.1 ± 14.7 a	395.0 ± 12.6 a	333.8 ± 10.5 a	254.9 ± 5.7 a
	N240	240.3 ± 3.8 c	303.4 ± 8.9 b	312.6 ± 10.2 c	292.0 ± 8.5 c	283.5 ± 7.6 b	202.3 ± 11.1 c
ANOVA (p-value)
Y (Year)		0.082 ns	0.144 ns	-	0.065 ns	-	0.044 *
N (N)		0.024 *	0.013 *	0.001 **	0.032 *	0.003 **	0.049 *
Y × N		0.065 ns	0.246 ns	-	0.109 ns	-	0.073 ns

N0, N120, N180, and N240 represent N application rates of 0, 120, 180, and 240 kg N ha⁻¹, respectively. ** represent difference at the 0.01 probability level (p < 0.01), * represent difference at the 0.05 probability level (p < 0.05), and ^ns represent not significant at the 0.05 probability level (p > 0.05). Different lowercase letters denote significant differences between the N levels at p < 0.05.

). The mechanical strength showed a trend of first increasing and then decreasing with basal third internode development. The mechanical strength increased to its maximum value at R3–R4, and then decreased gradually. The increasing N application rates contributed to improving the mechanical strength of the basal internode, but excessive N application (such as 240 kg ha⁻¹) had the opposite effect. The average values at the investigated stages were 279.6, 313.0, 328.4, and 282.7 at N0, N120, N180, and N240, respectively (the mean value of two seasons and growing stages). It was observed that N180 was the optimal N application treatment and led to the highest mechanical strength of the basal third internode.

3.4. Stalk Lodging Rate and Stalk Rot Incidence and Relationship Between Them

With the decreasing N application rate, the stalk lodging rate showed a decreasing trend during the two growing seasons (Figure 6a,b). The stalk lodging rate of the N0 plants was significantly lower than that of the plants subjected to the other N treatments. However, that of the N240 plants was significantly higher than that of the plants receiving the other treatments. The stalk lodging rate of the N0, N120, and N180 plants decreased by 85.38%, 65.62%, and 59.11% compared to that of N240 plants (mean value of two seasons and growing stages). By investigating the stalk rot incidence, it was found that its trend was basically the same as that of the stalk lodging rate under different N application rates (Figure 6c,d). Compared with the stalk rot incidence of the N240 plants, that of the N0, N120, and N180 plants was decreased by 74.97%, 54.03%, and 41.95% (mean value of two seasons and growing stages), respectively. The results indicated that the stalk lodging rate and stalk rot incidence of maize could be significantly reduced by reducing the N application rate.

The stalk rot incidence was significantly positively correlated with the stalk lodging rate under natural conditions (Figure 7). According to the coefficient of determination (R²) of the fitting equation over the two growing seasons, the percentage of stalk lodging caused by stalk rot disease reached 70.74%. Moreover, per 0.37% increase in stalk lodging, there was an increase in stalk rot by 1%. This indicated that stalk rot was an important factor affecting maize lodging.

3.5. Infection Process and Infection Index Under Different N Rates

The disease spots showed a gradual increasing trend as the infection progressed. Different N application rates had significant effects on the stalk rot severity, which was indicated by the infection index (Figure 8). As the infection time advanced, the infection index also increased. Decreasing the N application rate could help reduce the infection index. Compared with the infection index of the N240 plants, that of the N0, N120, and N180 plants was, on average, reduced by 24.51%, 15.16%, and 11.04%. This result indicated that reducing the N application levels appropriately could alleviate the degree of stalk rot infection.

3.6. The Mechanical Strength of Basal Third Internodes Inoculated by Pathogen Under Different N Rates

Compared with the mechanical strength of the non-inoculated stalks, that of the inoculated stalks exhibited a decreasing trend (Figure 9). Also, as the infection duration increased, reductions in the strength intensified gradually. Compared with the controls, the reduction in the stalk mechanical strength decreased from 2.70% to 10.78% (mean value of N application rates) from 7 to 42 days after inoculation. The different N application rates had significant effects on the stalks’ mechanical strength after inoculation. Increasing the N application rate contributed to improving the stalk mechanical strength, but excessive N, for example, 240 kg ha⁻¹, had the opposite effect. The fluctuations in the stalk mechanical strength in the plants receiving the N240 treatment were significantly higher than those receiving the other N treatments (average of each sampling period). The mechanical strength of the N0, N120, N180, and N240 plants decreased by 5.31%, 5.83%, 5.01%, and 11.21% compared with that of the control, respectively. From 7 to 21 days after inoculation, the mechanical strength of the plants receiving the N0 treatment was not significantly different from that of the control plants, while that of the plants receiving the other N treatments was significantly reduced. The results showed that moderately reducing the N application rate could reduce the decreasing fluctuations in the stalk mechanical strength, slowing down the decline in the stalk quality in the case of infection.

3.7. Grain Yield Under Different N Application Levels

The N application rates had significant effects on the maize yield and its constituent factors (

Table 4. Effect of different N application rates on grain yield and yield components.

Years	N Rates	Harvested Ear Numbers (104 hm−2)	Kernel Number per Ear	1000-Kernel Weight (g)	Grain Yield (kg ha−1)
2019	N0	7.14 ± 0.12 a	412.94 ± 11.42 c	296.93 ± 8.92 c	8155.07 ± 94.27 c
	N120	7.13 ± 0.08 a	422.85 ± 9.34 b	312.86 ± 9.29 b	8572.72 ± 42.03 b
	N180	7.31 ± 0.01 a	444.53 ± 9.12 a	319.73 ± 4.26 a	9193.28 ± 54.31 a
	N240	7.24 ± 0.02 a	447.19 ± 5.83 a	310.48 ± 5.87 b	9058.65 ± 71.50 a
2020	N0	7.21 ± 0.13 a	443.95 ± 13.44 c	270.91 ± 3.86 c	8089.86 ± 92.57 c
	N120	7.29 ± 0.09 a	484.30 ± 6.23 b	291.47 ± 7.31 b	8419.88 ± 81.46 b
	N180	7.44 ± 0.04 a	495.72 ± 4.87 a	302.91 ± 5.35 a	9040.59 ± 95.66 a
	N240	7.31 ± 0.08 a	498.93 ± 5.40 a	300.22 ± 6.86 a	8943.01 ± 62.55 a
ANOVA (p-value)
Y (Year)		0.047 *	0.015 *	0.052 ns	0.431 ns
N (N)		0.055	0.044 *	0.034 *	0.042 *
Y × N		0.073 ns	0.125 ns	0.082 ns	0.236 ns

N0, N120, N180, and N240 represent N application amounts of 0, 120, 180, and 240 kg ha⁻¹, respectively. * represent difference at the 0.05 probability level (p < 0.05), and ^ns represent not significant at the 0.05 probability level (p > 0.05). Different lowercase letters denote significant differences between the N levels at p < 0.05.

). Among the yield’s components, the ear numbers exhibited little change with alterations in the N rates. However, the kernel number per ear and 1000-kernel weight increased with an increase in N application rates, while those of the N180 and N240 plants had no significant difference. Compared with the kernel number per ear and 1000-kernel weight of the N0 plants, those of the N120, N180, and N240 plants increased by 5.87%, 9.73%, and 10.41% (average of two years), respectively. The grain yield of the N0 and N120 plants was reduced by 9.76% and 5.61%, respectively, compared with that of N240. Ultimately, the grain yield performance of the plants receiving the N180 and N240 treatments exhibited no significant difference but was significantly higher than that of the N0 and N120 plants. The results showed that moderately reducing N could lead to a higher yield by improving the three components of grain yield together. N180 was the optimal N fertilizer application strategy for maintaining a relatively higher grain yield.

4. Discussion

4.1. Moderately Reducing N Optimized Basal Internodes Morphogenesis and Achieved Stable Grain Yield

The evidence from the 2-year field experiment demonstrated that a moderately reduced N application strategy could improve the basal internode quality and increase the lodging resistance. In terms of morphological characteristics, previous studies have shown that the internode length, stalk diameter, and filling degree of the basal internode are pivotal indicators of the stalk mechanical strength [14,29]. Significantly decreasing the basal internode length and increasing the stalk diameter and filling degree could contribute to improving plants’ lodging resistance [30]. This research is fully consistent with former results. In this study, based on the morphological characteristics of basal third internode, we estimated that the internode length, cross-sectional area, and internode filling plumpness increased with increasing N application rates. However, excessively high N application rates (such as over 180 kg ha⁻¹) also significantly increased the internode length, which reduces the lodging resistance. There was no significant difference between the N180 and N240 plants regarding the cross-sectional area and internode plumpness. That is to say, the cross-sectional area and plumpness of the basal third internode could reach a high level, and a suitable internode length could be maintained under the application of 180 kg N ha⁻¹; continuing to increase N was redundant and wasteful.

Regarding anatomical characteristics, it has been reported that the sclerenchyma cells in the mechanical tissues under the epidermis are very important for mechanical strength [31,32]. Also, a previous study indicated that stabilizing the cell structure, preventing intracellular space expansion, and reinforcing the cell wall were key targets to increase the stalk rot resistance of maize [33]. In this study, the micro-structure observations of the transverse sections of the basal third internode revealed that the sclerenchyma tissue width increased with increasing N application, but excessive N application led to a reduced sclerenchyma tissue thickness under the application of 240 kg ha⁻¹. A noteworthy aspect of the anatomical traits was that the degree of lignification of the sclerenchyma tissues and vascular bundles increased with decreased N application rate. Vascular bundles play a vital role in mechanical support. Therefore, the vascular bundles in plant stalk tissues provide hardness and toughness and can be regulated by cultivation management. It might be that mechanical support was sufficiently regulated by moderate nitrogen application rates.

Previous studies have shown that mechanical strength is a pivotal indicator of maize lodging resistance [34,35]. Appropriate N application could promote maize growth and the development of basal stalks, thus improving the maize lodging resistance. In this study, the basal internode mechanical strength increased with increased N application rates, but excessive N application, i.e., at 240 kg ha⁻¹, had the opposite effect on the mechanical strength. In real-life production, the balance between lodging resistance and grain yield should be considered. It is generally believed that the risk of lodging is higher under high-yielding conditions and is much more pronounced under excessive N application. Previous studies have demonstrated that crop lodging is only observed in plots where higher grain yields are obtained [36,37]. The higher the N application rate, the higher the lodging risk [38]. This study is fully in agreement with these findings. Increasing the N application contributed to improving the grain yield; however, an excessive N application rate carried with it a higher lodging risk. In this study, the grain yield of the N180 and N240 plants showed no significant difference, but the lodging resistance of the N180 plant was significantly higher than that of the N240 plant. Although the N0 and N120 plants had a lower lodging rate and stalk rot incidence, their grain yield was also reduced significantly. Therefore, optimizing N fertilizer management could improve the morphological traits and dry matter accumulation of basal internodes and thus increase their mechanical strength and enable a higher grain yield to be achieved. A moderate N application rate of 180 kg ha⁻¹ is recommended for maize production under the tested environmental conditions, with the yield performance and lodging resistance considered simultaneously.

4.2. Moderately Reducing N Enhanced Resistance to Pathogen Infection and Reduced the Lodging Rate

One reason for reduced lodging rates with moderate N application is the reduction in stalk rot incidence under natural conditions. N is involved in plant developmental processes but is also an energy source required for pathogen infection [39]. There is evidence that N fertilizer increases the stalk rot severity, especially if at an excessive dosage. When plants are supplied with excessive N, pathogens are able to reproduce at a high rate and invade plant tissues. The main symptom of stalk rot is that the internodes become soft and the pith becomes hollow; thus, the plant is susceptible to lodging under adverse weather conditions [40]. Hence, maintaining a moderate N supply to prevent or alleviate stalk rot and stalk lodging needs more attention in maize management. This study’s findings are in full agreement with this statement. In the current study, the maize stalk rot prevalence and lodging rate with four different N application rates were evaluated under natural conditions. The stalk rot and lodging rate all decreased with reduced N application rates. In addition, the stalk rot incidence was significantly positively correlated with the stalk lodging rate under natural conditions. Based on the coefficient of determination (R²) of the fitting equation, the percentage of stalk lodging induced by stalk rot was 70.74%. Moreover, per increase in stalk lodging of 0.37%, the stalk rot increased by 1%. This indicated that stalk rot was a very important factor affecting stalk lodging. Therefore, reducing stalk rot infection through moderate nitrogen management is an effective strategy for reducing the lodging rate.

4.3. Moderately Reducing N Improved Resistance to Pathogen Expansion and Maintained Relatively High Stalk Mechanical Strength

Another contributing factor to enhancing lodging resistance through moderate N application is the resistance to pathogen spread with inoculation. This study utilized a rapid injection inoculation technique to evaluate how N application rates regulated the resistance to stalk rot spread under field conditions. In this research, the lesion area spread from the parenchymatous tissues to the sclerenchyma tissues with the inoculation treatment, which can be explained by the parenchymatous tissues being more susceptible to infection and the sclerenchyma tissues having a degree of resistance against pathogen spread during the inoculation process. This finding was consistent with previous research and indicated that a higher proportion of the sclerenchyma tissue functioned similarly as a hard, protective defense against pathogen spread [41]. The infected internodes softened, thus reducing their mechanical strength, which eventually led to stalk lodging. The infection index reflects the degree of longitudinal spread of the pathogen. Moderately reducing the N application increased the pathogen spread resistance, which could be expressed as a reduction in the infection index.

This research also indicated that, compared with the control, the inoculation treatment reduced the mechanical strength of the basal third internodes. Moderate N application could mitigate the degree of mechanical strength loss and reduce the lodging risk. In addition, stalk rot resistance is related to the degree of lignification of the internode. A previous study has shown that a higher content of cell wall structural carbohydrates, such as lignin and cellulose, in the internodes leads to stronger stalk rot resistance [42]. These carbohydrates can act as structural barriers against the adaptation of plants to disease stress [43]. Therefore, increasing the stalk quality, sclerenchyma tissue proportion, and lignification could increase the stalk rot resistance, which can be achieved through moderate N application, at 180 kg ha⁻¹.

This study has clarified the regulatory effects of moderate N application on improving properties related to lodging and also enhancing resistance to infection and expansion of stalk rot. However, in-depth research is required to reveal the defense mechanism of N’s regulation of stalk rot and lodging resistance. For example, plant cell walls are changeable structures, the elements of which can be altered in response to stalk rot. This will trigger adaptive responses (such as in plant metabolism processes, hormone levels, signaling pathways, and so on) during stalk development and infection, which is worthy of elucidation in the future.

5. Conclusions

This study systematically analyzed the regulating effects of N rates on lodging resistance of maize under natural field conditions and after inoculation. The results indicated that moderately reducing the N application to 180 kg ha⁻¹ improved the morphological characteristics (such as decreasing the internode length and increasing the cross-sectional area and filling degree of the basal internode), reinforced the anatomical structure (such as increasing the degree of lignification of the internodes and sclerenchyma tissue proportion), and enhanced the mechanical strength of the maize plants. These changes all contributed to increasing the lodging and stalk rot resistance compared to with excessive N application, at 240 kg ha⁻¹. Also, reducing N alleviated the fluctuations in the decline in mechanical strength after inoculation. A moderate 180 kg ha⁻¹ N treatment could balance the stalk lodging resistance and grain yield and, overall, is recommended for increasing the maize stalk lodging and stalk rot resistance and for maintaining a higher grain yield performance in the present experimental region. This research might provide a theoretical foundation for reducing stalk rot and increasing lodging resistance through optimal N management.