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Article

Toxic Effects of Bt-(Cry1Ab+Vip3Aa) Maize (“DBN3601T’’ Event) on the Asian Corn Borer Ostrinia furnacalis (Guenée) in Southwestern China

by
Haitao Li
1,
Wenhui Wang
1,2,
Xianming Yang
1,
Guodong Kang
1,
Zhenghao Zhang
1,3 and
Kongming Wu
1,*
1
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
2
Institute of Insect Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China
3
State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(9), 1906; https://doi.org/10.3390/agronomy14091906
Submission received: 18 July 2024 / Revised: 18 August 2024 / Accepted: 23 August 2024 / Published: 26 August 2024
(This article belongs to the Section Pest and Disease Management)
Browse Figures
Versions Notes

Abstract

:
Asian corn borer (ACB), Ostrinia furnacalis, is an important agricultural pest affecting maize production in southwestern China, but knowledge of the toxic effect of Bt maize on the pest has been insufficient until now. In this study, we determined the susceptibility of ACB to Cry1Ab, Vip3Aa, and their complex proteins and evaluated the efficacy of Chinese domestic Bt-(Cry1Ab+Vip3Aa) maize (“DBN3601T” event) against the pest in Yunnan Province of southwestern China. The susceptible bioassay indicated that the LC50 values of the Cry1Ab and Cry1Ab+Vip3Aa proteins expressed by the Bt maize varieties against ACB larvae were 51.42 and 46.85 ng/g, respectively; however, the ACB larva was insensitive to the Vip3Aa protein. The Cry1Ab+Vip3Aa protein contents in V6–V8 leaves, VT tassels, R1 silks, R2 kernels, R3 stalks and R3 cobs of the Bt-(Cry1Ab+Vip3Aa) maize were 114.20, 30.69, 3.77, 8.92, 11.09 and 10.99 μg/g, respectively. The larval feeding test indicated that the Bt-(Cry1Ab+Vip3Aa) maize was more toxic to the early instar larvae, and the survival time of larvae fed on the leaves was the shortest, while it survived the longest on stalks. The identification of maize resistance levels in the field showed that both larval density and plant damage score of Bt-(Cry1Ab+Vip3Aa) maize were significantly lower than those in conventional maize. It is concluded that the Bt-(Cry1Ab+Vip3Aa) maize can be used for control of the ACB in southwestern China.

1. Introduction

Asian corn borer (ACB), Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae) is an important agricultural pest of maize. It is widely distributed in East Asia, Southeast Asia, South Asia, and Oceania and occurs in many provinces in China [1,2]. The number of generations of the ACB varies with latitude and can occur from 1 to 7 generations per year in China [1,2,3,4]. The ACB feeds on 69 crops, including corn, sorghum, soybeans, cotton, and wheat [2]. Early-instar ACB larvae feed mainly on the leaves, silks, and tassels of maize, while later-instar larvae damage the stalks and kernels, causing maize to fall and develop kernel defects [2,5]. The ACB has caused severe yield losses and has led to ear-rot disease in maize, which can reduce maize yield by 10–30% annually [1], posing a long-term threat to food production in the Asian region.
The southwestern region is a major maize-producing area in China. Because of its borders with countries such as Myanmar, Laos, and Vietnam, it has become a source of pests and an annual breeding ground for many pests [6,7,8]. The ACB has long been a major pest in this region [4,9], posing a serious threat to high and stable maize yields. However, due to the burrowing ability of the ACB and frequent rainfall during the ACB outbreak in the southwestern region, it is difficult for chemicals to come into direct contact with the pests, resulting in higher control costs and lower efficacy. Meanwhile, there is an urgent need to develop new control technologies, as long-term and frequent use of chemical pesticides has led to the gradual emergence of resistance in the ACB [10], as well as serious environmental pollution and food safety problems.
Developing and using transgenic insect-resistant maize expressing Bacillus thuringiensis (Bt) represents a new technology for controlling pests. Genetically modified (GM) maize has gradually spread around the world since it was first planted commercially in the United States in 1996 [11]. Statistics show that in 2022, the global area of GM maize reached 66.2 million hectares, an increase of more than 100 times since 1996 [12]. This technology has been successfully applied to a wide range of pests, including lepidopteran and coleopteran pests such as the Spodoptera frugiperda (Smith), Ostrinia nubilalis (Hübner), and the ACB [13,14,15,16]. For example, “Bt11” and “MON810” maize expressing Cry1Ab protein, “TC1507” maize expressing Cry1Fa2 protein, “MIR162” maize expressing Vip3Aa20 protein, and “Bt11×MIR162” maize expressing both Cry1Ab and Vip3Aa proteins have been proven to be effective against Lepidopteran, while “MON863×MON810” maize expressing both Cry3Bb1 and Cry1Ab proteins can control both Lepidopteran and Coleopteran (data from https://www.isaaa.org (accessed on 25 August 2024)), and these maize plants are very effective against target pests.
The Chinese domestic “Ruifeng 125” maize expressing Cry1Ab and Cry2Aj proteins and “DBN3601T” maize expressing Cry1Ab and Vip3Aa proteins have been successively granted safety certificates for production and application [17]. In previous studies, these maize varieties have shown excellent efficacy against major pests in China. For example, “Ruifeng 125” maize is highly effective against the ACB [18]. Chinese domestic “DBN3601T” maize demonstrated up to 90% insecticidal efficacy against the S. frugiperda [19], Mythimna separata (Walker) [20], and Paralipsa gularis (Zeller) [21]. In all of their studies, the insecticidal efficacy of leaves was higher than silks and kernels. There is a difference between the above pests and later-instar ACB larvae, which prefer to feed kernels, cobs, and stalks [2,22,23,24]. Therefore, determining the lethality of different tissues against the ACB is essential in assessing the effectiveness of “DBN3601T” maize.
In recent years, China has been promoting the cultivation of GM. However, few studies have been conducted on the effectiveness of the Chinese domestic “DBN3601T” maize on the ACB in southwestern China. In this study, the virulence of Cry1Ab, Vip3Aa, and their complex proteins against the ACB were determined using bioassays, and the effect of Bt-(Cry1Ab+Vip3Aa) maize on resistance to the ACB was evaluated using tissue bioassays and field resistance identification. The aims were to determine the control of the ACB by Bt-(Cry1Ab+Vip3Aa) maize and to provide scientific and technological support for the development of a Bt maize-based green pest control technological system in southwestern China.

2. Materials and Methods

2.1. Collection and Rearing of the ACB

ACB larvae were collected from Simao District, Pu’er City, Yunnan Province, China (22°49′13.17″ N, 100°36′25.68″ E) on 23 October 2022 and brought back to the laboratory to be reared on artificial diets [25,26]. ACB adults were placed in a 3000 mL plastic box (diameter 19.5 cm × high 8.8 cm) and fed 10% honey water. After laying eggs, the egg masses on the walls of the plastic box were cut off and placed in 50 mL centrifuge tubes. Both larvae and adults were reared in the laboratory at a temperature of 26 ± 1 °C, a relative humidity of 60% ± 10%, and a photoperiod of 16L:8D.

2.2. Maize Variety for Testing

The recipient conventional maize line for both Bt-Vip3Aa maize (“DBN9501” event) and Bt-(Cry1Ab+Vip3Aa) maize (“DBN3601T” event) was “Luodan566”, while it was “Qiushuoyu6” for the Bt-Cry1Ab maize (“DBN9936” event). The aforementioned maize lines were provided by DBN Group, Beijing. All Bt maize events were planted in Jiangcheng County, Pu’er City, Yunnan Province, China (22°40′54.48″ N, 101°39′4.79″ E). The expression of the insecticidal protein in Bt maize was determined following the methods of Wang et al. [27].

2.3. Determination of ACB Susceptibility to Insecticidal Proteins Expressed by the Bt Maizes

Bt protein samples from Bt maize leaves were diluted with an artificial diet [28]. The dilution concentrations of Cry1Ab protein were 169.13, 84.56, 42.28, 21.14 and 10.57 ng/g; those of Cry1Ab+Vip3Aa protein were 224.95, 112.48, 56.24, 28.12 and 14.06 ng/g; those of Vip3Aa protein were 1591.20, 795.60, 397.80, 198.90 and 99.45 ng/g (ng/g: nanograms of Bt protein per gram of artificial diet). The corresponding quantity of the negative control samples was mixed with the artificial diet as a control.
The above-diluted diet (5 g) was poured into 24-well plates (diameter 17 mm/well), and an ACB neonate (with 12 h) was randomly placed in each well. There were 24 larvae per replicate and 3 replicates per concentration, giving a total of 72 larvae. The toxic diet was replaced after 7 days, and the number of dead larvae was assessed after 14 days by gently touching the larvae with a brush. Larvae that could not move normally or weighed less than 0.1 mg were considered dead. We weighed the surviving larvae and calculated the mortality and growth inhibitory rates. Weighing the ACB neonates was challenging due to their extremely low mass, which was consistently below 0.1 mg. All tests were conducted in the laboratory under the same rearing condition outlined in Section 2.1.

2.4. Determination of Insecticidal Protein Expression in Different Tissues of Bt Maize and Tissue Bioassays

For the determination of Bt protein expression and the laboratory tissue bioassays, Bt-(Cry1Ab+Vip3Aa) maize and conventional maize (“Luo Dan 566”) were collected from the field for the leaves (V6–V8), tassels (VT), silks (R1), kernels (R2), stalks and cobs (R3). The expression of the insecticidal protein in Bt maize was determined following the methods of Wang et al. [27].
Bioassays of different tissues: The leaves, tassels, silks, cobs, and stalks were cut into small fragments (approximately 1.5 cm) with scissors and put into 24-well plates (or plastic containers). The ACB neonates were reared in 24-well plates. The plates were sealed with parafilm to prevent the larvae from escaping. The third-instar larvae were individually reared in plastic containers (diameter 3.2 cm × high 3 cm) with holes, each containing 25 mL. In each container, 1–4 portions of the above tissues were placed and accessed by the first- or third-instar larvae. In each larval stage, Bt maize variety and maize tissue constituted a treatment, and each treatment was repeated 3 times for a total of 180 larvae. All tests were conducted in the laboratory under the same rearing conditions outlined in Section 2.1. Fresh maize tissues were replaced daily, and larvae were checked for survival that could not move normally were considered dead. The number of larvae killed in the different treatments was recorded, and the mortality and corrected mortality rates were calculated.

2.5. Field Evaluation

The experiment was conducted with Bt-(Cry1Ab+Vip3Aa) maize (“DBN3601T” event) and conventional maize (“Luo Dan 566”) in Jiangcheng County, Pu’er City, Yunnan Province (22°40′54.48″ N, 101°39′4.79″ E). Each plot was 3 m × 3 m with three replicates per treatment. A completely randomized block design was adopted for all treatments. The maize was planted with a spacing of 32 cm between plants, with row spacing of 40 cm for narrow rows and 70 cm for wide rows. Each plot was separated by at least 2 m. After sowing, all plots were covered with a 5 m × 5 m × 3.5 m 20 mesh cage. During the V6–V8 or silk stage, we selected 50 uniformly growing plants from each plot. ACB neonates were introduced to the leaves (or silks during the silk stage), with 20–30 larvae per plant. The number of ACB larvae and the plant damage scores were assessed on days 7, 11, 15, and 19 after artificial infestation with larvae [29,30]. The number of ACB larvae and the degree of maize damage were assessed by cutting plants on day 20 after artificial infestation.

2.6. Data Analysis

Corrected mortality, growth inhibitory rates, and control efficacy were calculated as follows:
Corrected mortality (%) = (treatment group mortality − control mortality)/(1 − control mortality) × 100
Growth inhibitory rate (%) = [(body mass increase in control group − body mass increase in treatment group)/body mass increase in control group] × 100
Control efficacy (%) = [(Larval density of conventional maize − Larval density of Bt maize)/Larval density of conventional maize] × 100
The susceptibility of the ACB to Cry1Ab and Cry1Ab+Vip3Aa proteins was analyzed by probit regression using SPSS 26.0 (IBM, Armonk, NY, USA), which obtained LC50, LC95, GIC50, and GIC95 values with 95% fiducial limits (FLs), chi-square (χ2), standard error slopes (slopes ± SE) and degrees of freedom (df). A significant difference between the values of LC50, LC95, GIC50, and GIC95 for the Cry1Ab and Cry1Ab+Vip3Aa proteins was obtained if their 95% fiducial limits did not overlap. One-way ANOVA was used to analyze the contents of Cry1Ab, Vip3Aa, and Cry1Ab+Vip3Aa proteins expressed by the different tissues of Bt-(Cry1Ab+Vip3Aa) maize and the corrected mortality rate and mean survival time of the ACB larvae feeding on Bt-(Cry1Ab+Vip3Aa) maize. Duncan’s multiple-range test for multiple comparisons and the Log-Rank test were used in GraphPad Prism 10.1 to analyze the survival curves of the ACB feeding on the different tissues of Bt-(Cry1Ab+Vip3Aa) maize. Mann–Whitney U-tests were used to test for differences in the number of larvae per 100 plants, plant damage scores, number of holes per plant, number of tunnels, and mean tunnel length between Bt-(Cry1Ab+Vip3Aa) maize and conventional maize in the evaluation of insect resistance at the whorl and silk stages using SPSS 26.0.

3. Results

3.1. Susceptibility of the ACB to Bt Insecticidal Proteins Expressed by the Bt Maize

The expression of Cry1Ab insecticidal proteins in the leaves of “DBN9936” and “DBN3601T” events was 67.65 and 68.93 μg/g (μg/g: micrograms of Bt protein per gram of dried maize tissue), respectively. The expression of Vip3Aa insecticidal proteins in the leaves of “DBN9501” and “DBN3601T” events was 19.89 and 21.06 μg/g, respectively. The sensitivity of the ACB to Bt proteins was determined using the above proteins.
The susceptibility of Cry1Ab and Cry1Ab+Vip3Aa proteins to the ACB is shown in Table 1. The LC50 of the Cry1Ab and Cry1Ab+Vip3Aa proteins against the ACB was 51.42 and 46.85 ng/g (ng/g: nanograms of Bt protein per gram of artificial diet), respectively, while the LC95 was 190.15 and 217.97 ng/g, respectively, which were not significantly different. Because the Vip3Aa protein was nearly non-toxic to the ACB larvae, we could not obtain any LC50 and LC95 values. The GIC50 of the Cry1Ab and Cry1Ab+Vip3Aa proteins against the ACB were 0.63 and 0.30 ng/g, respectively, while the GIC95 was 14.55 and 14.99 ng/g, respectively. There were no significant differences between the GIC50 and GIC95 of the two insecticidal proteins. Likewise, the GIC50 and GIC95 of the Vip3Aa protein against the ACB could not be achieved.

3.2. Expression of Insecticidal Proteins in Different Tissues of Bt-(Cry1Ab+Vip3Aa) Maize

Bt-(Cry1Ab+Vip3Aa) maize in different tissues of Cry1Ab, Vip3Aa and the total protein expressions were significantly different (Cry1Ab: F5, 12 = 186.58, p < 0.001; Vip3Aa: F5, 12 = 54.58, p < 0.001; total protein: F5, 12 = 1045.95, p < 0.001) (Table 2). The expression of Cry1Ab protein was higher than that of Vip3Aa protein in all tissues. The expression of Cry1Ab protein in different tissues was in the following order: leaves > tassels > stalks and cobs > kernels > silks. Vip3Aa protein was expressed as follows: leaves > tassels and kernels > cobs and stalks > silks. Total protein was expressed as follows: leaves > tassels > stalks, cobs, and kernels > silks.
Tissue bioassays in the laboratory showed significant differences in the insecticidal effect of the different tissues of Bt-(Cry1Ab+Vip3Aa) maize against ACB larvae (first-instar larvae: F5, 12 = 3.61, p < 0.05; third-instar larvae: F5, 12 = 56.09, p < 0.001), with the highest lethality in the leaves. First-instar larvae feeding on the leaves, tassels, kernels, and silks had a corrected day 3 mortality rate of 100.00%, which was significantly higher than that of the cobs at 88.23% but not significantly different from that of the stalks at 94.57% (Figure 1A). The corrected mortality of third-instar larvae feeding on different tissues of Bt-(Cry1Ab+Vip3Aa) maize leaves on day 5 were in the order of leaves > tassels > kernels > stalks, and there was no significant difference in the corrected mortality for feeding on silks and cobs. The corrected mortality from feeding on all tissues, except the leaves and tassels, was less than 40%; the highest corrected mortality was 91.64% for feeding on the leaves; the lowest corrected mortality was 1.69% for feeding on the stalks (Figure 1B).
The survival curves of first- and third-instar larvae of the ACB feeding on different tissues of Bt-(Cry1Ab+Vip3Aa) maize showed a decreasing trend; there was a highly significant difference in the survival curves of ACB larvae feeding on different tissues of Bt-(Cry1Ab+Vip3Aa) maize (first-instar larvae: χ2 = 521.0, df = 5, p < 0.001; third-instar larvae: χ2 = 722.8, df = 5, p < 0.001). The decreasing trend in the survival curve of first-instar larvae feeding on the leaves was significantly greater than that of other tissues, and the decreasing trend of feeding on the stalks was the smallest (Figure 2A). The decreasing trend of third-instar larvae feeding on different tissues was in the following order: leaves > tassels > kernels > silks and cobs > stalks (Figure 2B).
The mean survival times of ACB larvae feeding on different tissues of Bt-(Cry1Ab+Vip3Aa) maize differed (first-instar larvae: F5, 12 = 15.56, p < 0.001; third-instar larvae: F5, 12 = 57.04, p < 0.001). The shortest mean survival time was observed in larvae feeding on the leaves, while the longest mean survival time was observed in larvae feeding on the stalks. The mean survival time of first-instar larvae feeding on Bt-(Cry1Ab+Vip3Aa) maize leaves was 1.28 days, which was significantly lower than that of other tissues, while that of larvae feeding on the stalks was 2.57 days. There were no significant differences in the survival time of first-instar larvae feeding on the silks, kernels, and cobs. The mean survival times of third-instar larvae feeding on different tissues of Bt-(Cry1Ab+Vip3Aa) maize were as follows: leaves < tassels < kernels, cobs, and silks < stalks. The mean survival time of larvae feeding on the leaves and stalks was 3.74 and 9.13 days, respectively (Figure 2C).

3.3. Field Evaluation for Resistance Level of the Bt Maize to ACB

Leaf-feeding resistance: When ACB larvae infested maize plants for 4–19 days, the number of larvae on Bt-(Cry1Ab+Vip3Aa) maize was 0.00–0.67 larvae per 100 plants, which was significantly lower than the number of larvae on conventional maize, 206.67–356.00 larvae per 100 plants (p < 0.05). Bt-(Cry1Ab+Vip3Aa) maize was 99.63–100% effective against the ACB (Figure 3A). After infestation by the ACB, Bt-(Cry1Ab+Vip3Aa) maize had only a small number of damaged leaves, with a damage score of 1.00–1.25; meanwhile, conventional maize was severely damaged, with some leaf veins broken by larval feeding, with leaf damage score of 8.03–8.95. The damage score of Bt-(Cry1Ab+Vip3Aa) maize was significantly lower than that of conventional maize (p < 0.05) (Figure 3B).
Silk resistance: After infestation by the ACB, the number of ACB larvae on Bt-(Cry1Ab+Vip3Aa) maize was 0.00 larva per 100 plants, which was significantly lower than the number of larvae on conventional maize, 12.67–240.00 larvae per 100 plants (p < 0.05). Bt-(Cry1Ab+Vip3Aa) maize was 100% effective against the ACB (Figure 3C). Bt-(Cry1Ab+Vip3Aa) maize was not damaged by the ACB, while the silks, kernels, and stalks of the ears of conventional maize were all damaged. A small number of ears of conventional maize were severely damaged, resulting in mold growth, and the plant damage score of conventional maize gradually increased. The plant damage score of Bt-(Cry1Ab+Vip3Aa) maize was 1.00–1.04, which was significantly lower than that of conventional maize, 3.49–6.14 (p < 0.05) (Figure 3D).
The results from cutting the maize plants showed that 20 days after larvae infested the maize at the whorl stage, the number of ACB borer holes, the number of tunnels, and the length of the tunnels in Bt-(Cry1Ab+Vip3Aa) maize were 0 (p < 0.05). This was significantly lower than the average of 4.14 borer holes, 0.52 tunnels, and 1.21 cm tunnel length per plant in conventional maize. The above values were also 0 in Bt-(Cry1Ab+Vip3Aa) maize after 20 days of larval infestation in maize at the silk stage and were significantly lower than the average of 1.43 borer holes, 1.19 tunnels, and 5.43 cm tunnel length per plant in conventional maize (p < 0.05).

4. Discussion

Our research results showed that the ACB was much sensitive to both the Cry1Ab and Cry1Ab+Vip3Aa insecticidal proteins but insensitive to Vip3Aa and insecticidal protein contents in different tissues of Bt-(Cry1Ab+Vip3Aa) maize were in the following order: leaves > tassels > stalks, cobs, kernels > silks. The laboratory tissue bioassay results showed that the six tissues were highly effective against first-instar ACB larvae. The efficacy of the six tissues in the control of third-instar larvae was in the following order: leaves > tassels > kernels > silks, cobs, and stalks. All first-instar ACB larvae feeding on the six tissues died within 7 days, and all third-instar larvae died within 17 days. Field infestation trials showed that both larval density and plant damage degree of Bt-(Cry1Ab+Vip3Aa) maize were significantly lower than conventional maize. The above results indicate that Bt-(Cry1Ab+Vip3Aa) maize has a strong insecticidal effect and can effectively control the number of ACB larvae.
Previous studies have shown that the LC50 of Cry1Ab proteins was 0.05–0.37 μg/g against the ACB populations in the Huanghuaihai summer corn and northeastern corn regions of China [31]. The LC50 of Cry1Ab protein at 51.42 ng/g in this study was consistent with such a report. However, He et al. showed that the LC50 of Cry1Ab protein against the ACB was 0.10–0.81 μg/g in 10 different populations [32], which suggested that there were different sensitivities for geographical populations of ACB [33,34]. In contrast with the ACB, S. frugiperda and M. separata were more sensitive to Vip3Aa [35,36,37], which would also weaken the efficacy of Bt-(Cry1Ab+Vip3Aa) maize in controlling the ACB. In contrast to another drilling pest, P. gularis [21], third-instar ACB larvae feeding on Bt-(Cry1Ab+Vip3Aa) maize stalks had the lowest corrected mortality of 1.69% for 5 days and the longest survival time, suggesting that Bt-(Cry1Ab+Vip3Aa) maize stalks are more ineffective against ACB. All of the above will affect the effectiveness of Bt-(Cry1Ab+Vip3Aa) maize in providing long-term control of ACB and increasing the risk of resistance management against ACB. In addition, the insecticidal protein contents in Bt maize are one of the important factors affecting its insecticidal efficacy. A positive correlation between the insecticidal protein contents in different tissues of Bt maize and their effectiveness against the ACB larvae was confirmed in the laboratory bioassay [38,39]. Similarly, the higher the expression level of insecticidal protein in the tissues of Bt-(Cry1Ab+Vip3Aa) maize, the better the insecticidal efficacy against M. separata [20]. This study was generally consistent with the previous works, but some differences existed. For example, the expression of insecticidal proteins in kernels did not significantly differ from that in the stalks and cobs. Still, the insecticidal efficacy of kernels against third-instar larvae of the ACB was significantly higher than that of the stalks and cobs. This may be due to the environment’s influence and the maize’s weight on which the larvae are fed, although the exact reasons need to be investigated further. Combined with field infestation trials and previous studies, Bt-(Cry1Ab+Vip3Aa) maize showed excellent control of the S. frugiperda, M. separata, and P. gularis [19,20,21], indicating that the Chinese domestic Bt-(Cry1Ab+Vip3Aa) maize has a wide range of application value and can effectively control a wide range of pests in the southern Yunnan region.
Over the past 20 years, GM maize has been widely used to control lepidopteran pests such as the ACB and the S. frugiperda, with excellent results in many countries. For example, planting “TC1507” maize in the Philippines significantly reduced ACB infestation, with a 99% reduction in the number of tunnels and tunnel lengths and an increase in yield of 2.08 tons of maize per hectare [40]. Additionally, planting GM maize in Vietnam achieved more than 99% control of ACB, with a 30.8% increase in yield [41,42]. Planting GM maize reduces damage by targeting pests, indirectly reducing the number of pests on conventional maize. Studies have shown that large-scale planting of GM maize in the United States has also reduced O. nubilalis infestations on conventional maize, thereby increasing the economic benefits for conventional maize farmers [43]. While GM maize has brought enormous benefits, it has also led to several resistance events. For example, “TC1507” and “MON810” maize were planted in the United States and Brazil, respectively, to control the S. frugiperda, but after a few years, the S. frugiperda developed resistance to the GM maize [44,45]. In Canada, “TC1507” maize expressing Cry1F protein was planted in 2002, and 12 years later, the O. nubilalis was found to have developed practical resistance [46], while in the Philippines, monitoring of areas planted with Bt maize for three years showed that the ACB displayed an early warning of resistance [47]. The emergence of resistance events will lead to the failure of GM maize to control pests in the field, seriously threatening its sustainable cultivation; therefore, several actions have been taken to delay the development of pest resistance.
At present, the high-dose/refuge strategy is the most widely used resistance management strategy across the world. “High dose” means that the contents of the insect-resistant maize Bt protein kill 100% of sensitive homozygous individuals (ss) and 95% of sensitive heterozygous individuals (sr) in the target pest population [48]. This strategy is key to a resistance management strategy for GM maize. Planting Bt maize that does not meet the high dosage requirements will lead to a series of resistance events. For example, in the United States, the planting of the Bt maize “TC1507” against the S. frugiperda and “Bt11” and “M89034” against Helicoverpa zea (Boddie) resulted in practical resistance because the high-dose criterion was not met [49]. A refuge is the planting of non-Bt maize around Bt maize to allow the target pests to grow and develop normally. The main types of refuges being used at this stage are seed mixture, structural, and natural refuges. High-dose/refuge applications, which effectively extend the life of Bt maize, have been used successfully in many countries with greater efficacy. For example, Bt maize is grown commercially in the United States and Spain, where strict refuge strategies have been developed and implemented [50,51,52]. Local O. nubilalis populations have not developed resistance to Bt maize. In most parts of China, where small-scale farming is predominant, the use of “natural refuges” has also delayed the development of Helicoverpa armigera (Hübner) resistance to transgenic cotton [53]. Important factors in determining the type of refuges are the farming pattern and pest occurrence characteristics of the country or region. For example, in countries where mechanized farming is predominant, the use of structural refuges is highly operational, whereas, in countries with small-scale farming, structural refuges are more difficult to promote and less likely to be implemented. The size of the refuges in the northern and southern United States varies depending on the pest generation, and the distance between the refuges varies in response to the occurrence characteristics of lepidopteran and coleopteran pests.
This study showed that China already can develop and cultivate Bt maize. Given the increasing maize pest problems in recent years and the invasion of S. frugiperda, appropriate resistance management strategies should be formulated early. The commercial cultivation of transgenic maize should be promoted to facilitate the development of high-quality maize production in China.

5. Conclusions

The ACB larvae were very sensitive to the toxins expressed by the Bt-(Cry1Ab+Vip3Aa) maize in the laboratory. The field trial showed that both larval density and plant damage score of Bt-(Cry1Ab+Vip3Aa) maize were significantly lower than those in conventional maize. This study provides new technological support for the management of pests using Bt maize in southwestern China.

Author Contributions

Conceptualization, K.W. and H.L.; methodology, H.L., W.W., X.Y., G.K. and K.W.; software, H.L.; formal analysis, H.L.; investigation, H.L. and Z.Z.; resources, K.W.; data curation, H.L.; writing—original draft preparation, H.L.; writing—review and editing, K.W., W.W., G.K. and X.Y.; visualization, H.L.; supervision, K.W.; project administration, K.W.; funding acquisition, K.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Modern Agricultural Industry Technology System Construction Fund of China (CARS-02).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We acknowledge DBN Group for providing seeds.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Agronomy 14 01906 g001
Figure 1. Corrected mortality of ACB larvae of various stages on different tissues of Bt-(Cry1Ab+Vip3Aa) maize. (A) Corrected mortality of first-instar larvae on different tissues on day 3. (B) Corrected mortality of third-instar larvae on different tissues on day 5. Different lowercase letters indicate significant differences in the corrected mortality of larvae of the same instar stage on different maize tissues (one-way ANOVA, Duncan’s test, p < 0.05).
Figure 1. Corrected mortality of ACB larvae of various stages on different tissues of Bt-(Cry1Ab+Vip3Aa) maize. (A) Corrected mortality of first-instar larvae on different tissues on day 3. (B) Corrected mortality of third-instar larvae on different tissues on day 5. Different lowercase letters indicate significant differences in the corrected mortality of larvae of the same instar stage on different maize tissues (one-way ANOVA, Duncan’s test, p < 0.05).
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Agronomy 14 01906 g002
Figure 2. Survival curves and average survival time of first- or third-instar larvae on different tissues of Bt-(Cry1Ab+Vip3Aa) maize. (A) Survival curves of first-instar larvae on different tissues. (B) Survival curves of third-instar larvae on different tissues. (C) Average survival time of first- or third-instar larvae on different tissues. Different lowercase letters in the figure indicate significant differences in the average survival time of the same instar larvae on different tissues (one-way ANOVA, Duncan’s test, p < 0.05).
Figure 2. Survival curves and average survival time of first- or third-instar larvae on different tissues of Bt-(Cry1Ab+Vip3Aa) maize. (A) Survival curves of first-instar larvae on different tissues. (B) Survival curves of third-instar larvae on different tissues. (C) Average survival time of first- or third-instar larvae on different tissues. Different lowercase letters in the figure indicate significant differences in the average survival time of the same instar larvae on different tissues (one-way ANOVA, Duncan’s test, p < 0.05).
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Figure 3. Number of ACB larvae and leaf damage score after ACB infestation of different maize plants. (A) The number of larvae per 100 plants on Bt-(Cry1Ab+Vip3Aa) maize and conventional maize at the whorl stage. (B) Leaf damage score of Bt-(Cry1Ab+Vip3Aa) maize and conventional maize at the whorl stage. (C) The number of larvae per 100 plants on Bt-(Cry1Ab+Vip3Aa) maize and conventional maize at the silk stage. (D) Plant damage score of Bt-(Cry1Ab+Vip3Aa) maize and conventional maize at the silk stage. * A significant difference in the number of larvae on maize per 100 plants or leaf/plant damage score between different maize plants on the same survey date (Mann–Whitney U-test, p < 0.05).
Figure 3. Number of ACB larvae and leaf damage score after ACB infestation of different maize plants. (A) The number of larvae per 100 plants on Bt-(Cry1Ab+Vip3Aa) maize and conventional maize at the whorl stage. (B) Leaf damage score of Bt-(Cry1Ab+Vip3Aa) maize and conventional maize at the whorl stage. (C) The number of larvae per 100 plants on Bt-(Cry1Ab+Vip3Aa) maize and conventional maize at the silk stage. (D) Plant damage score of Bt-(Cry1Ab+Vip3Aa) maize and conventional maize at the silk stage. * A significant difference in the number of larvae on maize per 100 plants or leaf/plant damage score between different maize plants on the same survey date (Mann–Whitney U-test, p < 0.05).
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Table 1. Lethal and growth inhibitory concentrations of insecticidal proteins expressed by different Bt maize against ACB larvae.
Table 1. Lethal and growth inhibitory concentrations of insecticidal proteins expressed by different Bt maize against ACB larvae.
Inhibition.
Effect		Bt Event
(Protein)		NLC50/GIC50
(95%FL) ng/g		LC95/GIC95
(95%FL) ng/g		Slope ± SEχ2df
LCDBN9936
(Cry1Ab)		36051.42
(45.11–58.90) a		190.15
(150.32–261.01) a		2.90 ± 0.2515.8313
DBN9501
(Vip3Aa)		360>1591.20————
DBN3601T
(Cry1Ab+Vip3Aa)		36046.85
(40.36–54.20) a		217.97
(168.01–311.21) a		2.46 ± 0.2215.3813
GICDBN9936
(Cry1Ab)		3600.63
(0.01–1.66) A		14.55
(9.90–19.26) A		1.20 ± 0.233.1013
DBN9501
(Vip3Aa)		360>1591.20————
DBN3601T
(Cry1Ab+Vip3Aa)		3600.30
(0.00–1.30) A		14.99
(718–22.01) A		0.97 ± 0.223.3113
N: total number of larvae analyzed in each event; LC50/95: concentration of the protein (μg/g) required to kill 50% (95%) of the larvae over an observation period of 14 days; GIC50/95: growth inhibitory concentration of the protein (µg/g) required to cause 50% (95%) growth inhibition over an observation period of 14 days. SE: standard error; χ2: chi-square; df: degrees of freedom. The same lowercase letter in the same column indicates no significant difference in LC50 (or LC95) values; the same uppercase letter in the same column indicates no significant difference in GIC50 (or GIC95) values (overlapping 95% fiducial limits).
Table 2. Content of Bt proteins in the different tissues of Bt-(Cry1Ab+Vip3Aa) maize.
Table 2. Content of Bt proteins in the different tissues of Bt-(Cry1Ab+Vip3Aa) maize.
TissueCry1Ab μg/gVip3Aa μg/gTotal Bt Protein μg/g
V6–V8 leaf82.35 ± 8.39 a31.86 ± 6.91 a114.20 ± 3.22 a
VT tassel27.11 ± 3.32 b3.59 ± 0.11 b30.69 ± 3.41 b
R1 silk3.17 ± 1.21 e0.60 ± 0.29 d3.77 ± 0.96 d
R2 kernel5.45 ± 0.54 d3.48 ± 0.01 b8.92 ± 0.53 c
R3 Stalk9.55 ± 1.57 c1.55 ± 0.70 c11.09 ± 2.09 c
R3 cob9.15 ± 1.83 c1.84 ± 0.69 c10.99 ± 1.79 c
Note: Different lowercase letters in the same column indicate significant differences in the Bt protein content between tissues (one-way ANOVA, Duncan’s test, p < 0.05).
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Li, H.; Wang, W.; Yang, X.; Kang, G.; Zhang, Z.; Wu, K. Toxic Effects of Bt-(Cry1Ab+Vip3Aa) Maize (“DBN3601T’’ Event) on the Asian Corn Borer Ostrinia furnacalis (Guenée) in Southwestern China. Agronomy 2024, 14, 1906. https://doi.org/10.3390/agronomy14091906

AMA Style

Li H, Wang W, Yang X, Kang G, Zhang Z, Wu K. Toxic Effects of Bt-(Cry1Ab+Vip3Aa) Maize (“DBN3601T’’ Event) on the Asian Corn Borer Ostrinia furnacalis (Guenée) in Southwestern China. Agronomy. 2024; 14(9):1906. https://doi.org/10.3390/agronomy14091906

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Li, Haitao, Wenhui Wang, Xianming Yang, Guodong Kang, Zhenghao Zhang, and Kongming Wu. 2024. "Toxic Effects of Bt-(Cry1Ab+Vip3Aa) Maize (“DBN3601T’’ Event) on the Asian Corn Borer Ostrinia furnacalis (Guenée) in Southwestern China" Agronomy 14, no. 9: 1906. https://doi.org/10.3390/agronomy14091906

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