Inter- and Mixed Cropping of Different Varieties Improves High-Temperature Tolerance during Flowering of Summer Maize

Abstract

Global warming increases the risk of high-temperature injury to maize. Inter- and mixed-cropping of maize varieties with different genotypes is one way to effectively alleviate the high-temperature injury during the flowering period. However, the mitigation effect of different varieties and intercropping modes on high-temperature injury is still unclear. Based on previous years of field production, Denghai 605, which is more sensitive to high temperatures during the flowering period, was determined as the main test variety, and Zhengdan 958, Dedan 5, Weike 702, and Xianyu 335, which have great genotypic differences, were used as auxiliary varieties. The main test varieties and auxiliary varieties were intercropped and mixed cropped, respectively. Plant height, ear height, leaf area index, population light transmittance, ear characteristics, and yield were measured, and the land equivalent ratio (LER) was calculated. The plant height of Denghai 605 intercropped with Zhengdan 958 and Dedan 5 and mixed with Weike 702 and Xianyu 335 decreased significantly. The population light transmittance of the bottom or middle layer in Denghai 605 increased significantly when intercropped with other varieties. The grain number per ear increased significantly under inter- and mixed cropping with Zhengdan 958 and Weike 702. Except under intercropping with Dedan 5, the yield of Denghai 605 increased significantly, by 8.8–28.0%, under inter- and mixed cropping. Under intercropping with Zhengdan 958 and inter- and mixed cropping with Weike 702 and Xianyu 335, respectively, the group land equivalent ratio was greater than 1.1, indicating that under the combination of these varieties, inter- and mixed cropping effectively reduced the impact of high temperatures during flowering.

1. Introduction

Maize (Zea mays L.) is one of the most important crops in the global and national economy [1,2]. The Huanghuaihai plain is the largest summer-maize-producing area in China with a wheat–maize cropping system. The growing season of summer maize is mainly in the period from June to September, which has the highest temperature in the year and two-thirds of the annual precipitation. In the context of climate change, continuous high-temperature weather occurs frequently [3,4,5], which increases the risk of heat damage to maize [6,7]. The flowering period of summer maize is when it is most sensitive to high temperatures. High temperatures affect male and female ear development, pollen vigor, and grain development [8,9,10]. Maize pollen is more susceptible to high-temperature stress than female ears [11]. Pollen vigor declines quickly when the temperature exceeds 32–35 °C [12,13]. High-temperature stress can cause abnormal pollen development or abortion, resulting in a decrease in the number of pollen grains. In addition, it can cause abnormal division of pollen mother cells, deformation, and shrinkage leading to deformed pollen grains [14,15,16], which in turn affects the percentage seed set, yield, and quality of maize [9,10]. Therefore, it is of great significance to study effective methods to protect maize from the harmful high temperatures during the flowering period.

Single crop varieties are more vulnerable to diseases due to their narrow genetic basis, which is particularly prominent in maize. In addition to the influence of the external environment, there are obvious genotypic differences in the tolerance of maize to high temperatures and heat injury during the flowering period [17,18]. According to the principle of ecological complementation and biodiversity, the temperature–stress resistance of a maize population can be effectively improved by intercropping or mixed cropping of different maize varieties [19,20].

Inter- and mixed cropping among different ecotypes and genotypes of maize or intercropping with other crops could improve the utilization rate of natural resources and increase the yield stability of the crop composite population [21]. The inter- and mixed cropping of maize varieties optimizes the population spatial structure and maintains a higher chlorophyll content and photosynthetic rate [22], which is more conducive to gas exchange and promotes photosynthesis [23,24], increased nutrient absorption and dry matter accumulation [25,26], enhanced crop stress resistance [27,28], and improved yield and quality of maize [29,30]. In the intercropping of multiple varieties of maize, the anthesis and pollination time of the population system are relatively prolonged. When the maize encounters adverse weather events such as continuous rain and high temperatures during the flowering period, the fertilization rate of male flowers in the compound population composed of multiple maize varieties is higher than that in the single variety planting mode, which can effectively improve the bald tip and grain shortage caused by poor pollination, so as to achieve the purpose of increasing and stabilizing yield [21,22,23,24,25,26,27,28,29,30,31,32,33].

An important strategy to reduce high-temperature stress is to exploit the difference in heat resistance among maize varieties using an inter- and mixed cropping system. However, few studies have considered crucial aspects of this approach, such as maize variety matching, intercropping methods, and mixed cropping ratios. High-temperature and heat damage occurred continuously during the flowering period of maize in Henan from 2013 to 2016. It was found that the yield of Denghai 605 and Xianyu 335 decreased seriously under the high temperatures, while Zhengdan 958, Weike 702, and Dedan 5 had strong heat resistance and stable yield performance. According to the previous results, the high-temperature resistance field experiment of multi-variety inter- and mixed cropping was performed in 2017. In this study, Denghai 605, which has a large planting area and is sensitive to high temperatures, was selected as the main test variety to analyze the effects of inter- and mixed cropping of Denghai 605 with Zhengdan 958, Weike 702, Dedan 5, and Xianyu 335 on the prevention and control of high-temperature and heat damage during the flowering period.

2. Materials and Methods

2.1. Study Location

Our study was located in Shizhuang (114.03° E, 34.15° N), Chencao Township, Xuchang City, Henan Province, which belongs to the north warm temperate monsoon climate zone with abundant heat resources and abundant rainfall. The total area of the experimental field was 0.7 ha, which was mechanized sowing. The heat tolerance evaluation experiment of maize was carried out from 2013 to 2016, and the high-temperature resistance experiment of multi-variety inter- and mixed cropping was performed in 2017. The soil of the experimental field was a fluvo-aquic soil. The soil chemical properties at 0–20 cm depth were as follows: 23.54 g kg⁻¹ soil organic matter, 43.77 mg kg⁻¹ available nitrogen, 202.04 mg kg⁻¹ Olsen potassium, and 11.07 mg kg⁻¹ Olsen phosphorus. The meteorological variables recorded during the summer-maize-growing season at the study site are shown in Figure 1.

2.2. Experimental Design

‘Denghai 605’ (Shandong Denghai Seed Industry Co., Ltd.; Laizhou, Shandong, China) was selected as the main test variety, and ‘Zhengdan 958’ (Henan Academy of Agricultural Sciences Institute of Grain Crops; Zhengzhou, Henan, China), ‘Dedan 5’ (Beijing Denong Seed Industry Co., Ltd.; Beijing, China), ‘Weike 702’ (Zhengzhou Weike Crop Breeding Technology Co., Ltd.; Zhengzhou, Henan, China), and ‘Xianyu 335’ (Tieling Pioneer Seed Research Co., Ltd.; Tieling, Liaoning, China) were chosen as the auxiliary varieties.

Denghai 605 was inter- and mixed cropped with the four auxiliary varieties. A monoculture of each variety was established as the control group. The experimental design and processing codes are summarized in

Table 1. Intercropping and mixed cropping treatments of the maize varieties.

Farming Methods	Variety Combinations	Variety Codes
		Denghai 605	Auxiliary Varieties
Intercropping	Denghai 605||Zhengdan 958 [605||958]	(605)||958	605||(958)
	Denghai 605||Dedan 5 [605||005]	(605)||005	605||(005)
	Denghai 605||Weike 702 [605||702]	(605)||702	605||(702)
	Denghai 605||Xianyu 335 [605||335]	(605)||335	605||(335)
Mixed cropping	Denghai 605 × Zhengdan 958 [605 × 958]	(605) × 958	605 × (958)
	Denghai 605 × Dedan 5 [605 × 005]	(605) × 005	605 × (005)
	Denghai 605 × Weike 702 [605 × 702]	(605) × 702	605 × (702)
	Denghai 605 × Xianyu 335 [605 × 335]	(605) × 335	605 × (335)
Monoculture	Denghai 605	CK605
	Zhengdan 958	CK958
	Dedan 5	CK005
	Weike 702	CK702
	Xianyu 335	CK335

“||“ represents the intercropping of two varieties, and “ד represents the mixed cropping of two varieties. The notation also indicates the variety combination used in the experiment. For example, intercropping of Denghai 605 and Zhengdan 958 is indicated as “605‖958”, “Denghai 605” in the combination is indicated by the form “(605)‖958”, and “Zhengdan 958” is indicated by “605||(958)”.

.

It has been proven that in a relay intercropping system, narrow–wide row planting improves the light environment and seed yields of intercrop species [34]. Thus, the narrow–wide row planting pattern with a wide row of 70 cm and a narrow row of 50 cm was used in this experiment. The mechanical precision seeding method was applied to fertilize simultaneously. The seeder sowed two rows concurrently. The five varieties were sown on 11 June. The intercropping sowing design was illustrated in Figure 2. For mixed cropping, varieties were sown according to a 1:1 ratio of the number of seeds. Seedlings were thinned at the three-leaf stage, weeds were chemically controlled, and a commercial compound fertilizer (N-P₂O₅-K₂O: 29-5-6, 750 kg ha⁻²; Luxi, 0303050000001, Liaocheng, Shandong, China) was applied between rows at the jointing stage. Irrigation was applied in accordance with the soil moisture content to ensure that the entire growth period was free from drought stress. Other management measures were identical to those of local farmer practices.

2.3. Measurement of Parameters and Methods

2.3.1. Main Reproductive Assessment Period

The timing of the tasseling, silking, flowering, and pollination stages of maize plants growing in the monoculture area was recorded, which for each stage was expressed as the number of days after sowing.

2.3.2. Population Density Determination

Eleven consecutive plants in a row were selected to measure the spacing of ten plants, and eleven consecutive rows (five wide and five narrow rows each) were selected to measure the spacing of ten rows. The measurements were used to calculate the average plant spacing (from three repetitions). Based on these data, the population density of the intercropping mode was 61,215 plant ha⁻² and that of the mixed cropping mode was 64,815 plant ha⁻².

2.3.3. Main Agricultural Characters Determination

During the tasseling period of maize, five plants of relatively uniform growth were selected for each variety in each treatment area, and the plant height and ear height were measured. The leaf area index was calculated with a length–width coefficient method. The leaf length and maximum leaf width were measured in situ with a ruler. During the tasseling period of the maize, three representative sites were randomly selected in the middle of a narrow row for each treatment. The light transmittance of the middle and bottom layers in the canopy of the maize population was measured with an LAI-2000 plant canopy analyzer (LI-COR, Inc; Lincoln, NE, USA).

2.3.4. Ear Traits and Yield Determination

In the monoculture of the test variety, ten consecutive ears were selected from plants growing in a middle row of uniform growth. For the mixed cropping mode, in a relatively uniform middle row, ten ears were selected from consecutive plants of each of the two combination varieties (note that ears of the same variety were not collected from a row once ten ears had been sampled). For the intercropping treatment, ten consecutive ears were selected from one row (a non-side row) of each variety. Three replicates were collected; thus, a total of 30 ears of each variety in each treatment was sampled. Each variety was sampled 3 times for the determination of ear traits and yield, and 10 consecutive ears were measured for each replicate, for a total of 30 ears. Among them, 3 ears were selected for each repetition (9 ears were selected in total), and ear length, ear thickness, bald length, and grain number per ear were measured. The 100-grain weight and yield were determined by threshing and mixing once we were finished measuring every ten ears.

The ear length and bald length were measured with a ruler, the ear thickness was measured with a vernier caliper at the thickest portion in the middle of the ear, the 100-grain weight was measured with an electronic balance (sensitivity 0.01 g), and grain moisture content was measured using a grain moisture meter.

The population yield was calculated according to the number of grains per ear, 100-grain weight, and population density of each variety in the inter- and mixed cropping combination. The formulas used were as follows:

$${\mathrm{Y}}_{\mathrm{i}}{=(\mathrm{Y}}_{\mathrm{S}1}{+\mathrm{Y}}_{\mathrm{S}2})/2$$

(1)

$$Y_{\mathrm{S}1}{=\mathrm{W}}_{\mathrm{S}1}\times {\mathrm{N}}_{\mathrm{S}1}\times {\mathrm{D}}_{\mathrm{S}}$$

(2)

$${\mathrm{Y}}_{\mathrm{S}2}{=\mathrm{W}}_{\mathrm{S}2}\times {\mathrm{N}}_{\mathrm{S}2}\times {\mathrm{D}}_{\mathrm{S}}$$

(3)

where Y_i is the population yield of the varieties under inter- and mixed cropping mode per unit area, Y_S1 and Y_S2 are the yield of two varieties in the inter-and mixed cropping mode, respectively, W_S1 and W_S2 are the grain weight of the two varieties equivalent to 14% moisture, N_S1 and N_S2 are the number of grains per ear of the two varieties, respectively, and D_S is the population density.

2.3.5. Calculation of the Land Equivalent Ratio

The land equivalent ratio is the ratio of the income of two or more mixed crops (varieties) to the income of each crop in the same farmland. The land equivalent ratio (LER) was calculated using the following formula:

$$\mathrm{LER}=\frac{{\mathrm{Y}}_{\mathrm{i}}}{{\mathrm{Y}}_{\mathrm{ii}}}$$

(4)

$${\mathrm{Y}}_{\mathrm{ii}}={(\mathrm{Y}}_{\mathrm{c}1}+{\mathrm{Y}}_{\mathrm{c}2})/2$$

(5)

where Y_ii is the average yield of the two varieties and Y_c1 and Y_c2 are the yields of the two varieties grown in the monoculture method, respectively.

2.4. Determination of Maize Flowering Period and High-Temperature Stress Threshold

The date of the onset of flowering for each variety was recorded from plants growing in the monoculture. Flowering of the entire ear of summer-sown maize usually lasts 7–10 d; therefore, the onset of tasseling is the date for the start of flowering, and the date of the end of flowering is 10 d after tasseling. The maximum temperature ≥ 35 °C was used as the critical threshold of high-temperature stress during the flowering period [17,35].

2.5. Statistical Analysis

Data processing and graphing were performed using Microsoft Excel 2016 and GraphPad Prism 8. All data were expressed as the mean ± standard deviation (SD), and n refers to the number of samples in each group. Statistical analyses were performed using SPSS 17.0 (SPSS, IBM Corp., Armonk, NY, USA). Analysis of variance (ANOVA) was performed to determine the significance of differences between treatments. Means for different treatments were compared using the Bonferroni test at the significance level α = 0.05.

3. Results

3.1. Differences in the Onset of Flowering and the Occurrence of High Temperatures

As shown in Figure 3, Dedan 5 was the first variety to start flowering (54 d after sowing) and Xianyu 335 was the last variety to flower (57 d after sowing). Two main periods of high temperatures were recorded in 2017. The first period was from 3 to 6 August, in which the highest temperature exceeded 35 °C for 4 consecutive days. The second period was from 9 to 11 August, in which the highest temperature exceeded 35 °C for 3 consecutive days. The first high-temperature period coincided with the beginning of tasseling of the varieties. The second high-temperature period occurred 2–6 d after tasseling and had a greater impact on flowering and pollination.

3.2. Plant Height and Ear Height

As shown in Figure 4, aside from the increase in plant height of Denghai 605 intercropped with Xianyu 335, the plant height of Denghai 605 showed different degrees of decline in other inter- and mixed cropping modes. Among these treatments, intercropping with Zhengdan 958 and Dedan 5 and mixed cropping with Weike 702 and Xianyu 335 resulted in a significant decrease in the plant height of Denghai 605. The plant heights of Zhengdan 958, Dedan 5, and Weike 702 decreased under inter- and mixed cropping with Denghai 605, and the plant heights of the mixed cropping modes all decreased significantly. Given that Dedan 5, Weike 702, and Zhengdan 958 have genetically similar parents, the trends for changes in plant height under inter- and mixed cropping with Denghai 605 were generally similar. However, the plant height of Xianyu 335 increased significantly under inter- and mixed cropping with Denghai 605.

As shown in Figure 5, the changes in ear height of most varieties under the inter- and mixed cropping modes were similar to the observed changes in plant height. The main difference was that the ear height of Zhengdan 958 did not decrease significantly under the mixed cropping mode. The ear height of Weike 702 under intercropping was significantly lower than that of the monoculture. The ear height of Xianyu 335 increased significantly under the inter- and mixed cropping modes, and the ear height under intercropping exceeded that observed in the mixed cropping mode.

3.3. Leaf Area Index

As shown in Figure 6, except for a nonsignificant difference in leaf area between Denghai 605 and Weike 702, the leaf area index of Denghai 605 decreased significantly under the other inter- and mixed cropping modes. Under the intercropping mode, the leaf area index of the four auxiliary varieties showed no significant change compared with that of the monoculture. Under the mixed cropping mode, the leaf area index of Zhengdan 958 and Dedan 5 mixed with Denghai 605 decreased significantly compared with that of the monoculture, whereas the leaf area index of Weike 702 and Xianyu 335 mixed with Denghai 605 increased significantly compared with that of the monoculture.

3.4. Population Light Transmittance

As shown in Figure 7, under the intercropping mode, the mid-canopy-level population light transmittance of Denghai 605 and Zhengdan 958 increased significantly compared with that of each monoculture. The mid-canopy-level population light transmittance between Denghai 605 and the other three varieties was not significantly affected by intercropping. Under the mixed cropping mode, the population light transmittance of the mid-canopy of the three varieties was significantly lower than that of the Denghai 605 monoculture.

Under intercropping of the other four varieties, the population light transmittance of the lower canopy increased significantly. The population light transmittance of the lower canopy under all mixed cropping modes showed no significant change compared with that of the Denghai 605 monoculture.

3.5. Effects of Inter- and Mixed Cropping Modes on Ear Traits

The effects of the different inter- and mixed cropping modes on ear morphology were mainly manifested as changes in ear length (

Table 2. Ear traits of summer maize varieties under different inter- and mixed cropping modes.

Variety	Ear Length /cm	Ear Thickness /cm	Bald Length /cm	Grain Number per Ear	100-Grain Weight/g
CK 605	18.4 ± 0.8	5.1 ± 0.6	1.7 ± 0.6	503 ± 4	32.8 ± 0.3
(605)||958	21.5 ± 1.1 *	5.1 ± 0.5	0.9 ± 0.3	615 ± 33 *	34.5 ± 0.3 *
(605)||005	17.8 ± 0.5	5.2 ± 1.1	1.8 ± 0.2	488 ± 32	34.1 ± 0.5 *
(605)||702	19.7 ± 2.6	5.1 ± 0.9	1.5 ± 0.2	551 ± 59 *	34.9 ± 0.7 *
(605)||335	18.6 ± 1.1	5.0 ± 1.5	1.2 ± 0.4	523 ± 5	34.3 ± 0.7 *
(605) × 958	20.8 ± 0.6 *	4.8 ± 0.9*	1.4 ± 0.4	546 ± 16*	33.6 ± 0.4
(605) × 005	18.5 ± 0.7	5.1 ± 0.4	1.7 ± 0.4	516 ± 20	34.8 ± 0.7 *
(605) × 702	19.3 ± 0.6	5.1 ± 0.5	1.3 ± 0.4	560 ± 13 *	34.2 ± 0.8 *
(605) × 335	18.8 ± 0.9	4.9 ± 0.9	2.2 ± 0.4	488 ± 24	33.6 ± 0.1
CK958	16.9 ± 0.5	5.4 ± 1.6	0.3 ± 0.1	523 ± 49	33.5 ± 0.3
605||(958)	17.4 ± 0.5	5.3 ± 0.3	0.6 ± 0.2	531 ± 12	34.2 ± 1.2
605 × (958)	17.1 ± 0.5	5.2 ± 0.9	0.9 ± 0.3 *	528 ± 59	33.6 ± 0.5
CK005	15.7 ± 1.0	5.0 ± 0.6	0.0 ± 0.0	504 ± 20	30.9 ± 0.3
605||(005)	15.3 ± 0.3	5.1 ± 0.6	0.1 ± 0.1	557 ± 7 *	30.0 ± 0.3
605 × (005)	15.4 ± 0.3	5.1 ± 0.9	0.0 ± 0.0	533 ± 33	30.5 ± 0.1
CK702	17.6 ± 0.8	5.4 ± 0.1	1.2 ± 0.3	524 ± 30	35.1 ± 1.0
605||(702)	19.7 ± 0.4 *	5.5 ± 0.3*	0.6 ± 0.3 *	577 ± 34 *	37.3 ± 0.6 *
605 × (702)	19.4 ± 0.8 *	5.3 ± 0.3	0.8 ± 0.2	565 ± 20 *	35.3 ± 0.5
CK335	16.2 ± 0.9	5.2 ± 0.5	2.0 ± 1.1	426 ± 11	34.5 ± 0.6
605||(335)	18.9 ± 0.8 *	5.1 ± 1.2	1.7 ± 0.5	526 ± 46 *	35.4 ± 0.5
605 × (335)	18.5 ± 0.8 *	5.2 ± 0.2	2.3 ± 0.3	529 ± 42 *	35.4 ± 0.3

* Significant at the 0.05 probability level; Values are the mean ± SD (n = 9 for lines 2-5 and n = 3 for line 6).

). The ear length of Denghai 605 increased significantly under inter- and mixed cropping with Zhengdan 958. The ear lengths of Weike 702 and Xianyu 335 under inter- and mixed cropping with Denghai 605 increased significantly compared with that of the monoculture. The number of grains per ear of Denghai 605 increased significantly under inter- and mixed cropping modes with Zhengdan 958 and Weike 702. Moreover, under inter- and mixed cropping, the number of grains per ear of the four auxiliary varieties increased to varying degrees compared with that of the corresponding monoculture. Specifically, intercropping of Dedan 5 and inter- and mixed cropping of Weike 702 and Xianyu 335 significantly increased the number of grains per ear. In addition, 100-grain weight Denghai 605 increased significantly under intercropping with the four varieties and under mixed cropping with Dedan 5 and Weike702. The 100-grain weight of the auxiliary varieties showed no significant differences except for that of Weike 702 under intercropping with Denghai 605.

3.6. Effect of Inter- and Mixed Cropping Modes on Yield

The yield of Denghai 605 increased significantly under inter- and mixed cropping with the auxiliary varieties except for intercropping with Dedan 5, with the yield increase ranging from 8.8% to 28.0% (

Table 3. Yield of summer maize varieties under different inter- and mixed cropping modes.

Variety	Yield /kg ha−2	Yield Variety Compared with Monoculture/%
CK605	9075 ± 63	—
(605)||958	11,615 ± 641 *	28.0
(605)||005	9328 ± 609	2.8
(605)||702	10,426 ± 405 *	14.9
(605)||335	9878 ± 96 *	8.8
(605) × 958	10,587 ± 664 *	16.7
(605) × 005	9967 ± 383 *	9.8
(605) × 702	10,549 ± 381 *	16.2
(605) × 335	9988 ± 493 *	10.1
CK958	10,812 ± 318	—
605||(958)	11,028 ± 760	2.0
605 × (958)	9846 ± 387 *	−8.9
CK005	9622 ± 379	—
605||(005)	9842 ± 806	2.3
605 × (005)	9983 ± 624	−7.7
CK702	10,722 ± 520	—
605||(702)	12,861 ± 747 *	19.9
605 × (702)	12,068 ± 361 *	12.6
CK335	9144 ± 597	—
605||(335)	10,800 ± 606 *	18.1
605 × (335)	10,738 ± 595 *	17.4

* Significant at the 0.05 probability level; Values are the mean ± SD (n = 3).

). When inter- and mixed cropped with Zhengdan 958, the yield of Denghai 605 had the largest increase of 28.0% (intercropping) and 16.7% (mixed cropping). This increase was then followed by an increase of 14.9% (intercropping) and 16.2% (mixed cropping) when inter- and mixed cropped with Weike 702.

The yield of the auxiliary varieties increased under inter- and mixed cropping with Denghai 605. The yield of Weike 702 and Denghai 605 increased by 19.9% (intercropping) and 12.6% (intercropping), respectively, and that of Xianyu 335 and Denghai 605 increased by 18.1% (intercropping) and 17.4% (intercropping), respectively. However, the yield of Zhengdan 958 decreased significantly after mixed cropping with Denghai 605.

3.7. Population Yield and Land Equivalent Ratio

The land equivalent ratio was consistently more than 1.0 under inter- and mixed cropping of Denghai 605 and four auxiliary varieties, indicating that the different inter-and mixed cropping methods were beneficial to increase the population yield (

Table 4. Population yield and land equivalent ratio of summer maize varieties under different inter- and mixed cropping modes.

Inter-and Mixed Cropping Patterns	Population Yield /kg ha−2	Average Yield of Monoculture/kg ha−2	Land Equivalent Ratio	Population Yield Variety Compared with Denghai 605 Monoculture/%	Population Yield Variety Compared with Other Varieties’ Monoculture/%
605||958	11,572	9944	1.16	24.8	4.7
605||005	9585	9349	1.03	5.6	−0.4
605||702	11,644	9899	1.18	28.3	8.6
605||335	10,339	9110	1.13	13.9	13.1
605 × 958	10,217	9944	1.03	12.6	−5.5
605 × 005	9975	9349	1.07	9.9	3.7
605 × 702	11,309	9899	1.14	24.6	5.5
605 × 335	10,363	9110	1.14	14.2	13.3

). Intercropping with Zhengdan 958 and inter- and mixed cropping with Weike 702 resulted in a population yield exceeding 11,000 kg ha⁻². Under intercropping with Zhengdan 958 and mixed cropping with Weike 702 and Xianyu 335, the population yield increased significantly, and the land equivalent ratio was greater than 1.1. Inter- and mixed cropping with Dedan 5 resulted in the lowest increase in population yield.

Comparing the population yield with the monoculture yield of Denghai 605, the population yield increased by more than 20% under intercropping of Denghai 605 with Zhengdan 958 and inter- and mixed cropping with Weike 702. The yield of Denghai 605 increased by 10%, compared with that of the monoculture, under mixed cropping with Zhengdan 958 and inter- and mixed cropping with Xianyu 335.

Comparing the population yield with the monoculture yield of the auxiliary varieties, the population yield was higher than that of the Xianyu 335 monoculture, with the yield increased by 13.1% (605||335) and 13.3% (605 × 335), respectively.

4. Discussion

The maize growing season is in summer, with high temperatures and humidity, which brings the increasing risk of high-temperature stress [36]. High-temperature stress can cause abnormal pollen development or abortion, resulting in a decrease in the number of pollen grains; in addition, it can cause abnormal division of pollen mother cells, deformation, and shrinkage leading to deformed pollen grains [14,15]. High temperatures during flowering causes shrinkage of pollen grains and depression of the germination pore, which significantly reduces pollen vitality; the higher the temperature, the greater the reduction of pollen vitality [37]. High-temperature stress can also thicken the anther wall and hinder dehiscence, resulting in the release of fewer pollen grains and lower vitality [38]. Pollen metabolic activity is associated with the starch content of the pollen grain; a significant decrease in the starch content causes a corresponding decrease in pollen metabolic activity [17,39]. Distinct differences in pollen viability are observed among maize hybrids, which are derived from genetic differences among the parents [18,40]. Different varieties differ in their gene sources, and thus heat tolerance can differ significantly. Varieties that produce a greater number of branches, full glume, and higher number of pollen grains usually show stronger resistance to high-temperature stress [21,41]. Sowing two or more varieties with different genotypes increases the probability of cross pollination as well as the yield [42]. Therefore, changing single cropping to inter- and mixed cropping among multiple varieties without increasing cost is one of the new strategies to improve the high-temperature tolerance of maize during the flowering period by making full use of the heat tolerance differences of different genotypes.

Inter- and mixed cropping, which creates a multi-level and multi-functional composite group through different combinations of crops and varieties [43], can improve the canopy structure of the population, improve efficiency in the use of light energy and land area, and overcome the harmful impacts of diseases, insects, and grasses on monocultures, so as to increase the yield per unit area [44,45]. Intercropping forms a wavy canopy, while mixed cropping forms a concave–convex canopy of crops in order to change plane light into three-dimensional light in the upper part of the population crops. It has been shown that the photosynthetic potential of intercropping during the big bell mouth period was 76% and 78% higher, and the field light transmittance was 54.0% higher than that of the single cropping, respectively [46]. The photosynthetic intensity of the intercropping population increased by 37.2% and 28.8% compared with that of the monoculture, and the light energy utilization rate in the whole growth period increased by 58.6% compared with that of the monoculture during the jointing stage and filling stage, respectively. In addition, the yield of Jundan 20 and Dedan 5 increased by 5.6% and 7.9% compared with their monoculture after intercropping treatment, respectively [47,48]. In our study, the results showed that the light transmittance of the bottom population increased after intercropping Denghai 605 with Dedan 5, Weike 702, and Xianyu 335, and the light transmittance of the middle layer increased after intercropping with Zheng Dan 958, which is consistent with the previous findings.

The advantages of inter- and mixed cropping among different maize varieties mainly reflect the contemporary heterosis [49], including resistance complementarity and fertility complementarity. Complementary resistance means that inter- and mixed cropping of two varieties that differ in disease resistance effectively improves the disease resistance of the population after planting [27,45]. Sterility complementary refers to the similarity of the male–female interval between two varieties. The pollen of each variety is used to extend the duration of pollination and fertilization, which can enhance the utilization of heterosis in maize cross-pollination and increase yields by enhancing the number of grains per ear [50]. It had been demonstrated that intercropping was beneficial to the increase of grain number per ear and grain weight compared with monoculture, and the number of grains per ear in the intercropping of Yedan 12 and Yedan 13 increased by 9.28% and 15.66%, respectively, compared with that of the monoculture [51]. In addition, the average number of grains per ear of free pollination in different combinations increased by 40.7 grains, and 100-grain weight increased by 1.1 g, which manifested the existence of heterosis among different varieties [52]. Making full use of flowering and pollen complementary advantages of varieties and improving fertilization and seed setting rate while avoiding high temperature stress is one of the focuses of our study. The flowering period of Zhengdan 958, Weike 702, and Denghai 605 were basically the same. After inter-and mixed cropping, the flowering and pollination of Denghai 605 were compensated, and the grain number per ear was significantly increased compared with that of single cropping. It indicated that the selection of varieties with a consistent flowering period was helpful to give full play to the advantages of inter-and mixed cropping planting mode. The number of grains per panicle of Xianyu 335, a variety with a small amount of pollen and intolerant to high temperatures during the flowering period, also increased significantly after inter-and mixed cropping with Denghai 605 compared with that of single cropping. This shows that inter-and mixed cropping can make better use of heterosis of maize varieties.

In previous studies, extensive research has been performed on the physiological and ecological effects of maize intercropping. The yield-increasing benefits of maize in inter- and mixed cropping have been discussed in detail. In terms of morphological structure, the maize inter- and mixed cropping system facilitated the formation of a three-dimensional canopy structure and different intercropping row ratios and planting densities brought differences in light distribution, which improved the ventilation and light transmittance and increased CO₂ concentration in the growth space [53,54,55]. In terms of physiology, inter- and mixed cropping mode increased the chlorophyll content, leaf area index, and photosynthetic rate of maize [19,22]. Meanwhile, it enhanced the antioxidant enzyme activity and Rubisco’s carboxylation efficiency and improved soil quality and nutrient absorption [23,25,55]. In terms of stress resistance, intercropping of different genotypes of maize profoundly strengthened the resistance to the disease [27], and the appropriate cultivar collocation effectively reduced the lodging resistance of the population. This study focused on the mitigation effect of multi-variety- inter- and mixed cropping modes on population resistance to high temperatures during the flowering period, and demonstrated that the tolerance to high temperatures during flowering was significantly enhanced by choosing reasonable variety-matching and row-spacing ratios, which were hardly studied in the past.

Previous studies have fully confirmed that the yield-increasing effect of inter- and mixed cropping was the result of multiple compound effects. However, the mechanism for increasing the yield is extremely complicated, as it is affected by diverse factors, such as genotype, phenotype, population structure, population physiology, field microclimate, and soil microecology [44]. Therefore, no simple combination of varieties will necessarily increase production, and in-depth prior analysis is required. For the selection of maize varieties, the consistency of variety traits, plant type, plant height, resistance, and other characteristics should be considered so as to increase the yield of dominant varieties, stabilize the yield of other varieties, and finally, increase the population yield. This study demonstrated that the land equivalent ratio of Denghai 605 inter- and mixed cropped with four other auxiliary varieties was more than 1.0, among which the LER intercropping with Zhengdan 958 and inter- and mixed cropping with Weike 702 and Xianyu 335, respectively, was greater than 1.1, further confirming the yield-increasing effect. Based on the previous studies, the technical key point is that the heat resistance of the combined varieties should be complementary and the growth period should be consistent. The flowering period especially should be consistent if possible. In addition, varieties with basically the same plant height and plant type should be selected if possible. If there are large differences in plant height or plant type, the 2:2 or 2:4 row ratio intercropping mode can be adopted, which is for making use of the spatial advantages of high-stalk varieties without reducing the yield of short-stalk varieties and having strong complementarity among varieties in heat resistance [56,57,58]. The production technology of complementary resistance enhancement of maize varieties has been listed as the main technology in 2021 by the Ministry of Agriculture and Rural Affairs of the People’s Republic of China and issued and implemented as the agricultural industry standard in China [59].

5. Conclusions

In this study, Denghai 605, which produces low amounts of pollen and is intolerant of high temperatures during flowering, was used as the main test variety, and Zhengdan 958, Dedan 5, Weike 702, and Xianyu 335 were used as auxiliary varieties to determine the effect of inter- and mixed cropping of maize on high-temperature tolerance during flowering and on yield. The population light transmittance of the bottom or middle layer of crops increased in the intercropping of Denghai 605 with other varieties, which was conducive to the formation of an efficient canopy structure. The number of grains per ear increased after intercropping Denghai 605 with Zhengdan 958 and Weike 702, indicating that inter- and mixed cropping enhanced pollen vigor and improved tolerance to high temperatures during the flowering period. Moreover, the yield increased significantly by 8.8–28.0% after inter- and mixed cropping Denghai 605 with other varieties (except intercropping with Dedan 5). Among them, the LER was greater than 1.1 when Denghai 605 was intercropped with Zhengdan 958 and inter- and mixed cropped with Weike 702 and Xianyu 335, which indicated that under the combination of these varieties, inter- and mixed cropping effectively reduced the impact of high temperatures during flowering and improved the maize population yield.