Resistance of germplasm enhancement of partially inbred maize lines to fall armyworm and corn earworm

ABSTRACT

Maize (Zea mays L.) is an important crop worldwide. In the southern area of the United States the two major pests of maize are fall armyworm (Spodoptera frugiperda (J.E. Smith)) and corn earworm (Helicoverpa zea (Boddie)). Fall armyworm primarily attacks maize leaves and corn earworm damages the upper part of the ear. An effort to obtain new sources for resistance is conducted through Germplasm Enhancement of Maize (GEM) project. Plants were infested at the whorl growth stage with fall armyworm neonates and evaluated for leaf-feeding damage 14 days after infestation. Plants were evaluated for ear-feeding damage based on natural infestation by corn earworm. Leaf-feeding damage values in Tifton were lower than at Mississippi State. GEMN-0259, GEMS-0311, and GEMS-0312 exhibited similar resistance levels with the resistant check on leaf-feeding damage in Tifton. Ear-feeding ratings in Tifton and Mississippi State were similar. GEMS-0311 showed the most resistance to ear-feeding damage in both environments. GEMS-0311 with resistance to both insects could be used for new germplasm development.

KEYWORDS:

• Host plant resistance
• leaf-feeding damage
• ear-feeding damage
• maize
• environment

Introduction

Maize (Zea mays L.) is an economically important crop, and its benefits range from feed for animals and food for humans to biofuels and compostable containers. As one of the global staple cereal crops, maize has been grown on approximately 200 million hectares (Erenstein et al. 2022). Fall armyworm [Spodoptera frugiperda (J.E. Smith)] (Lepidoptera: Noctuidae) is native to the tropical and sub-tropical regions of the Americas. It is a polyphagous global pest which has been reported to attack 353 plant species from 76 families (Montezano et al. 2018; Overton et al. 2021) including maize. As a major pest of maize in the southern United States, this insect decreases yield potential primarily through leaf-feeding damage (Capinera 2017). On maize, fall armyworm larvae attack leaves from seedling (vegetative stages) to mature plants (reproductive stages) (Davis et al. 1996). Another major pest of maize in North America is corn earworm (Helicoverpa zea (Boddie)) (Lepidoptera: Noctuidae) (Siegfried, Spencer, and Nearman 2000). This pest has more than 100 host plants with maize as its preferred host (Baker 2019; Neunzig 1963). Corn earworm larvae primarily feed on the upper part of the maize ear near the tip (Dicke and Guthrie 1988). As part of maize pest management, host plant resistance is of advantage and a desirable technique. The United States Department of Agriculture, Agricultural Research Service (USDA-ARS), located at Tifton, GA and Mississippi State, MS initiated breeding programs to develop maize germplasm with resistance to the major lepidopterous maize pests in the southern United States more than 50 years ago (Widstrom 1989; Williams and Davis 1997). These research units’ efforts to discover and develop new maize germplasms with resistance to corn earworm and fall armyworm continue today. A continuing need of the ongoing research at both locations is obtaining new sources of germplasm to screen for multiple insect pest resistance. One source of germplasm is the Germplasm Enhancement of Maize (GEM) project. The GEM project is a collaboration among universities, private sectors, non-governmental organizations, and USDA-ARS. It is intended to expand the base of germplasm for commercial maize hybrids in the United States by incorporating useful germplasms from around the world (Anonymous 2023; Rogers et al. 2022). The goal of this research was to evaluate a group of twenty GEM lines for resistance to leaf feeding by fall armyworm and ear feeding by corn earworm.

Materials and methods

Germplasm Enhancement of Maize (GEM) lines

Twenty GEM partially inbred lines were subjected to evaluation for resistance to fall armyworm leaf-feeding damage and corn earworm ear-feeding damage. Ab24E was selected as a susceptible control and Mp708 (Williams, Davis, and Windham 1990) was selected as a resistant control in all environments. The 20 GEM lines and the susceptible (Ab24E) and resistant (Mp708) checks were planted in Tifton, GA, at the University of Georgia Gibbs Research Farm near Tifton, GA (31.5°N, 81.5°W) on 13 April 2021 and 20 April 2022, and at Mississippi State, MS, at the R.R. Foil Plant Science Research Center (33.5°N, 88.8°W) on 19 April 2021 and 20 April 2023. The experiments at both locations were organized in a randomized complete block design with four replications (block as the factor) in Tifton and three replications at Mississippi State. The plots were 4.1 m long and the rows were spaced 0.97 m apart with 20 plants per row. Established maize production practices were followed with supplemental irrigation as needed to ensure normal plant growth.

Evaluating GEM lines for resistance to fall armyworm leaf-feeding damage

The fall armyworm used in this study was raised at the USDA-ARS Corn Host Plant Resistance Research Unit, Mississippi State. The larvae were mass-reared using wheat (Triticum aestivum L.) germ-based artificial diet following the standard protocol established by Davis (1987). To maintain colony performance, feral fall armyworms were collected every year from maize fields during the growing season and incorporated into the insect rearing colony. The fall armyworm moths were maintained in a cage where wax paper sheets were placed as an oviposition substrate. The sheets containing egg masses were then removed from the cage and transferred to a rearing room (26.7 ± 1°C, 50–70% relative humidity, 16:8 light: dark hours). Up to 24 hours after egg hatch, first instar larvae (neonates) were added into maize cob grits and mixed well. The larvae-grit mixture was dispensed into the maize whorl utilizing a special dispenser (Wiseman, Davis, and Campbell 1980) using the infestation method described by Davis et al. (1996). When maize plants reached V-6 or V-7 growth stage (L. Abendroth et al. 2009; L. J. Abendroth et al. 2011), all plots within the experiment were infested with fall armyworm neonates: 30 neonates per plant at Tifton and 40 neonates per plant at Mississippi State. Fourteen days following neonates infestation, each plant in a plot was visually rated for leaf-feeding damage using the established rating scale: 0 = no visible damage to whorl leaves; 1 = only pin-hole damage to whorl leaves; 2 = pin-hole and shot-hole lesions on whorl leaves; 3 = a few small (5–10 mm) elongated lesions on whorl leaves; 4 = several whorl leaves with midsize (10–30 mm) lesions; 5 = several whorl leaves with large elongated lesions or small sections eaten away; 6 = several whorl leaves with elongated lesions and large sections eaten away; 7 = many elongated lesions and large sections eaten from whorl leaves; 8 = many elongated lesions and many large portions eaten from whorl leaves; and 9 = many whorl leaves damaged (Davis, Ng, and Williams 1992; Williams, Buckley, and Davis 1989).

Evaluating GEM lines for resistance to corn earworm ear-feeding damage

The ear-feeding damage in this study was evaluated based on natural infestation by corn earworm. The primary maize ear damage of each plant was measured based on the depth of penetration of feeding along the ear in centimeters (Widstrom 1967). Ear-feeding damage rating was taken from maize lines planted in (Tifton, 2022 and Mississippi, 2023).

Data analysis

The mean rating of leaf-feeding damage from each row was calculated across 20 plants. For leaf-feeding damage, data were analyzed by individual year in each location and year, pooled years per location, and pooled over years and locations. Ear-feeding damage was measured on 10 randomly selected ears at pre-harvest. The ear-feeding damage data were then analyzed by locations i.e., Mississippi State in 2023 and Tifton in 2022, Sandra W. Woolfolk, Research Entomologist. Xinzhi Ni, Research Entomologist. Matthew D. Krakowsky, Research Geneticist. Gerald A. Matthews, Biological Science Technician, and pooled over locations. PROC GLM in SAS was used to statistically analyze the rating damage values. Means of the rating values were separated using Fisher’s protected LSD at p ≤ .05 (SAS Institute 2016).

Results and discussion

Fall armyworm leaf-feeding damage

The means for fall armyworm leaf-feeding damage sustained by GEM lines and the controls are listed in Table 1. In general, maize lines are classified as resistant, moderately resistant, and susceptible when the leaf-feeding damage rating values are 0–3, 4–6, and 7–9, respectively. In Tifton environment, the overall leaf-damage rating in 2022 was significantly higher than in 2021 (p = .0007). The rating values among lines were significantly different in both 2021 (p = .0003) and 2022 (p < .0001) at Tifton as well as for means combined over both years (p < .0001). The mean damage rating values in 2021 and 2022 were 2.19 and 2.93, respectively. The GEM lines damage rating values in Tifton for combined years were between 2.93 (GEMN-0292) and 3.13 (GEMS-0255). These GEM lines exhibited better than moderate resistance to fall armyworm leaf-feeding damage in Tifton environments. No interaction between lines and evaluation year was found (p = .1572). In the combined analysis, three GEM lines (GEMN-0259, GEMS-0311, GEMS-0312) did not differ significantly from the resistant control (Mp708) in the extent of leaf feeding damage (Table 1). The GEM project is a cooperative breeding undertaking between public and private sectors with the goal to improve the genetic diversity of maize lines (Rogers et al. 2022). The GEM project typically breeds tropical maize with temperate adapted maize to develop new germplasm for maize breeders to enhance traits of interest. The female (donor) parent of GEMN-0259 which was Saint Croix 1 pedigree (PI 484,036) was evaluated in previous studies for leaf-feeding damage by fall armyworm. One study showed that this pedigree was susceptible to fall armyworm leaf-feeding damage with 7.7 rating value at 14 days (Williams and Davis 1997) while the other study showed that Saint Croix 1 was moderately resistant to fall armyworm leaf-feeding damage with 6.2 rating value at 14 days (Abel, Coates, and Scott 2019). The authors of both studies suggested that Saint Croix 1 pedigree could be a potentially useful source of resistance to fall armyworm. The results of the prior research support the results of this study where GEMN-0259 was the top line that exhibited resistance to fall armyworm and its female parent was Saint Croix 1.

Table 1. Fall armyworm leaf-feeding damage sustained by 20 GEM lines, susceptible line, and resistant line evaluated at Tifton, GA, and Mississippi State, MS, and multiple environments.

Germplasm line*	FAW leaf-feeding damage***
	Tifton	Mississippi State	Multiple environments
Ab24E**	3.63 a	6.35 abc	4.80 a
GEMS-0255	3.13 ab	6.52 ab	4.58 ab
GEMN-0254	3.00 bc	6.55 a	4.52 ab
GEMN-0232	2.88 bcd	6.31 abcd	4.35 abc
GEMS-0293	2.63 bcdef	6.64 a	4.34 abc
GEMS-0245	2.57 bcdef	6.69 a	4.33 abc
GEMS-0320	2.50 cdef	6.42 ab	4.31 abc
GEMN-0124	2.56 bcdef	6.57 a	4.28 abc
GEMN-0271	2.69 bcde	6.18 abcd	4.18 abcd
GEMS-0295	2.38 def	6.57 a	4.17 abcd
GEMN-0137	2.81 bcd	5.82 abcde	4.09 bcd
GEMS-0321	2.38 def	6.22 abcd	4.02 bcd
GEMN-0259	2.13 efg	6.45 ab	3.98 bcd
GEMN-0135	2.56 bcdef	5.42 cde	3.79 cd
GEMN-0270	2.44 cdef	5.55 bcde	3.77 cd
GEMS-0243	2.94 bcd	5.97 abcd	3.76 cd
GEMS-0312	2.06 fg	6.03 abcd	3.76 cd
GEMN-0104	2.36 def	5.35 de	3.74 cd
GEMS-0311	2.19 ef	5.72 abcde	3.70 cd
GEMS-0294	2.44 cdef	5.37 de	3.69 cd
GEMN-0292	2.50 cdef	4.93 e	3.54 d
Mp708**	1.56 g	3.26 f	2.22 e
Mean	2.56	5.97	4.01
LSD (0.05)	0.28	0.98	0.69

*Twenty GEM lines were evaluated for fall armyworm resistance.

**Ab24E line served as a susceptible control and Mp708 line as resistant control.

***Fall armyworm leaf-feeding damage sustained by maize lines planted in four different environments (two years for each location) was rated on a scale of 0 (no damage) to 9 (extensive damage) 14 days after maize plants were infested with neonates: 30 neonates in Tifton, GA in 2021 and 2022, 40 neonates in Mississippi State, MS in 2021 and 2023. Values in a column followed by the same letter do not differ according to Fisher’s protected LSD at p ≤ .05.

Leaf-feeding damage ratings in the Tifton environments were higher than those in Mississippi. The overall leaf-feeding damage rating in 2022 in Tifton was significantly higher than the ratings in 2021 (p < .0001). In Mississippi, the rating values among lines differed significantly in both 2021 (p = .0002) and 2023 (p < .0001) as well as for means combined over both years (p < .0001). The mean overall rating values in 2021 and 2023 were 5.18 and 6.78, respectively. The GEM lines rating values in Mississippi for combined years were between 4.93 (GEMN-0292) and 6.69 (GEMS-0245). These GEM lines exhibited moderate resistance to fall armyworm leaf-feeding damage in Mississippi environments. All GEM lines sustained significantly greater damage than Mp708 over the two years. Significant interaction (p = .0125) was also found between maize lines and year of evaluation.

Among the multiple environments with two years for each of the two locations , i.e., at Tifton in 2021 and 2022, Mississippi in 2021, and 2023 (Table 1), the overall fall armyworm leaf-feeding damage rating values significantly differed (p < .0001) across environments. The ratings among lines also differed significantly (p < .0001) as did the interaction between lines and environments (p < .001). Nine of the GEM lines (GEMS-0255, GEMN-0254, GEMN-0232, GEMS-0293, GEMS-0245, GEMS-0320, GEMN-0124, GEMN-0271, and GEMS-0295) did not differ from the susceptible check in the amount of damage sustained. None of the GEM lines exhibited a level of resistance to fall armyworm feeding equivalent to that exhibited by Mp708. The rating values at Tifton were lower compared to values at Mississippi State indicating that the level of resistance to fall armyworm feeding in Tifton was higher than in Mississippi State. Different methods of neonates calibration prior to field infestation could affect the difference of the rating values between Tifton and Mississippi State. Numbers of neonates infested in Tifton were lower (30 neonates per plant) than at Mississippi State (40 neonates per plant). Given the presence of natural enemies (predators) on maize seedlings under the southern climate (Ni et al. 2011, 2014), there might be fewer surviving fall armyworm larvae causing damage in Tifton (2021 and 2022) than at Mississippi State (2021 and 2023).

Corn earworm ear-feeding damage

The GEM lines significantly (p < .0001) differed in ear-feeding damage at Tifton in 2022 and GEMN-0137 exhibited the highest rating value at 5.78. On the other hand, at the Mississippi State location in 2023, the maize lines did not differ in ear-feeding damage (p = .2091) where GEMS-0320 exhibited the highest rating value at 5.44. For combined environments, the overall rating values among maize lines did not differ (p = .7075) when Tifton 2022 and Mississippi 2023 data were combined (Table 2). The mean ear-feeding damage rating values for Tifton 2022 and Mississippi 2023 environments were similar at 4.03 cm and 4.15 cm, respectively. The GEM lines ratings for combined environments were between 2.35 cm (GEMS-0311) and 5.38 cm (GEMN-0137). There was significant (p < .0001) difference in ear damage rating among lines. No significant (p = .2241) interaction was found between maize lines and the environments. It is worth noting that corn earworm infestation and damage on maize ears are prevalent in the maize fields in the southern states. The infestation rate could be approximately 90% or higher on maize plants of some transgenic Bt-maize hybrids in recent years, particularly in 2023 (data not presented). Bt maize are widely planted worldwide to manage lepidopteran maize pests including corn earworm. In their 3-year study Dively et al. (2023) reported a widespread resistance across many states in the Unites States. Their studies showed a high percentage of corn earworm infestations and injury to Bt-maize containing Cry1Ab, Cry2Ab2, and Cry1A.105 Cry toxins in comparison to non-Bt maize. The purpose of the current evaluation was to evaluate GEM lines primarily for fall armyworm but also to see if any of the lines showed any resistance to ear-feeding damage by corn earworm, and to identify germplasm lines with multiple insect resistance. Further evaluations will be needed by including additional control lines that exhibit resistance to corn earworm. GEMS-0311 appeared to exhibit the most resistance to ear-feeding damage in both environments in addition to fall armyworm resistance in both locations (Table 2).

Table 2. Ear-feeding damage sustained by 20 GEM lines, susceptible line, and resistant line evaluated at Tifton, GA in 2022 and Mississippi State, MS in 2023, and combined environments.

Germplasm line**	Ear-feeding damage*
	Tifton 2022	Mississippi State 2023	Combined environments
Ab24E***	8.59 a	5.65	7.33 a
GEMN-0137	5.78 b	4.84	5.38 b
Mp708***	5.64 b	4.64	5.21 bc
GEMS-0294	4.98 bc	4.47	4.76 bcd
GEMN-0135	4.65 bc	4.90	4.76 bcd
GEMS-0320	4.03 bcd	5.44	4.63 bcd
GEMS-0255	4.40 bcd	4.69	4.53 bcdc
GEMN-0292	4.38 bcd	4.25	4.33 bcdef
GEMN-0232	4.44 bcd	3.93	4.22 bcdef
GEMS-0243	4.13 bcd	-	4.13 bcdef
GEMN-0259	3.98 bcde	4.14	4.05 bcdef
GEMS-0321	3.98 bcd	3.50	3.77 bcdefg
GEMS-0312	4.20 bcd	3.18	3.76 bcdefg
GEMS-0245	3.20 cdef	4.25	3.65 cdefg
GEMS-0295	3.29 cdef	3.99	3.59 cdefg
GEMN-0254	3.36 cdef	3.70	3.51 defg
GEMN-0271	3.48 cdef	3.40	3.44 defg
GEMN-0104	2.97 cdef	4.07	3.44 defg
GEMN-0270	3.20 cdef	3.57	3.36 defg
GEMS-0293	2.54 def	3.54	2.97 efg
GEMN-0124	1.98 ef	3.72	2.72 fg
GEMS-0311	1.57 ef	3.39	2.35 g
Mean	4.03	4.15	4.08
LSD (0.05)	2.03	NS	1.65

*Ear-feeding damage sustained by maize lines was measured based on the depth of penetration (cm) of feeding along the ear. Values in a column followed by the same letter do not differ based on Fisher’s protected LSD at p ≤ .05. NS = not significant.

**Twenty GEM lines were evaluated for ear-feeding damage.

***Ab24E line served as a susceptible control and Mp708 line a resistant control. Ab24E and Mp708 were checks for ratings of fall armyworm damage, but not for corn earworm. Both are susceptible to corn earworm.

Conclusion

Among the 20 GEM partially inbred lines evaluated, GEMN-0259, GEMS-0311, and GEMS-0312 were found to exhibit similar resistance levels compared to Mp708 (resistant check) to leaf-feeding damage by fall armyworm. In addition to fall armyworm leaf-feeding damage resistance, GEMS-0311 also exhibited resistance to corn earworm ear-feeding damage. With further evaluations, these newly identified germplasm lines could potentially be utilized as a new source for multiple insect resistance in maize under the warm temperate/sub-tropical climate. These lines could be used as additional genetic resources for resistance not only to multiple insects and diseases, but also to produce breeding crosses for resistance with maize lines that possess desirable agronomic traits.

Acknowledgments

The authors would like to thank Dr. Paul Williams for his invaluable advice, and to Penny Tapp, Marley Martin, and Patrick Tranum for their excellent technical assistance in insect rearing and field trials. We would also like to express our gratitude to Dr. Xueyan Shan (Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, Mississippi State University) and Dr. Xiangbing Yang (USDA-ARS, Subtropical Horticulture Research Unit, Miami, FL) for their constructive review of the earlier version of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

Funding for this research was provided by the United States Department of Agriculture – Agricultural Research Service (USDA-ARS).