Resistance of Soybean Genotypes to Anticarsia gemmatalis (Lepidoptera: Erebidae): Antixenosis and Antibiosis Characterization

Abstract Injury by herbivores is a major biotic stress that limits soybean [Glycine max (L.) Merrill] crop production. Among the main soybean insect pests, Anticarsia gemmatalis Hübner is responsible for causing significant economic damage in soybean. The primary management strategy for this insect is chemical control and use of Bt transgenic soybean. Alternative strategies, such as host plant resistance, are considered an efficient and less-aggressive method, especially in association with other strategies as part of an integrated pest management (IPM) approach. In this study, we evaluated 30 soybean genotypes to verify antixenosis expression through oviposition, attractiveness, and food consumption tests. From this, we selected 13 promising genotypes to verify the possible presence of antibiosis. Our results suggest that antixenosis was found in genotypes ‘TMG 133’ RR, ‘TMG 1179’ RR, ‘IAC 19’, ‘IAC 17’, ‘IAC 100’, D75-10169, and IAC 78-2318. By influence on behavior and negative impact on larval viability, antixenosis and antibiosis were indicated for the genotypes IAC 74-2832, ‘IAC 19’, ‘IAC 17’, ‘IAC 100’, and PI 274454. ‘TMG 7062’ IPRO was found to provide antibiosis resistance by negatively affecting larval development and viability. Because of reduced food consumption by larvae, antixenosis was indicated for ‘IAC 24’. These genotypes should be considered in soybean breeding programs focusing on soybean resistance to A. gemmatalis.

Soybean [Glycine max (L.) Merrill] is one of the most important agricultural crops, and it has been increasingly planted worldwide (SOYSTATS 2020). Soybean grain can be used as feed, or it can be processed to produce soybean byproducts, oil, and, more recently, biodiesel (USDA 2019). Soybean productivity can be severely reduced by insect pests that cause significant yield loss (Hoffmann-Campo et al. 2012, Oliveira et al. 2014, Wille et al. 2017. The velvetbean caterpillar, Anticarsia gemmatalis (Hübner), is one of the main soybean insect pests. This species causes defoliation in different soybean-producing regions in the western hemisphere (Buschman et al. 1981, Sosa-Gómez et al. 2010, Bortolotto et al. 2015, Haase et al. 2015. Defoliation can be total, severely affecting yield when infestations are high ).
Traditionally, A. gemmatalis has been controlled with chemical insecticides. However, the extensive use of insecticide sprays can have significant negative effects to nontarget organisms, promote pest outbreaks, and select for insecticide-resistant insect populations (Vieira et al. 2011, Bel et al. 2019. In parallel, genetically modified soybean expressing a Bacillus thuringiensis (Bt) insecticidal protein has taken a prominent position in large-scale management of A. gemmatalis (Bernardi et al. 2012). Although Bt soybean varieties expressing Cry1Ac are high-dose events for this pest (Bernardi et al. 2012), their extensive use without appropriate resistance management plans can compromise their durability. In addition, this technology requires the presence of a refuge (i.e., soybean with no Bt plants), where the use of foliar insecticides should be minimized (Bortolotto et al. 2015). Thus, pest management in the refuge could be conducted by other methods, such as biological control (entomopathogenic virus, fungi, and bacteria), but not biological products based on Bt (IRAC 2021).
Despite the increase in the use of Bt technology, the size of the refuge, limitations of alternative control methods, high cost of seed, and other factors have resulted in many growers seeking alternative management strategies, such as the use of resistant soybean developed in classical breeding programs. Host plant resistance is considered an efficient and less-aggressive method, especially when integrated with other strategies, e.g., biological control. There is a need to identify and incorporate resistant soybean genotypes as pest management tools in integrated pest management (IPM) programs for soybean (Smith and Clement 2012, Canassa et al. 2017, Baldin et al. 2019.
Plant resistance can be divided into three categories: antibiosis, antixenosis, and tolerance (Panda and Khush 1995, Smith 2005, Baldin et al. 2019. Plants expressing antibiosis negatively affect colonizing insect biology by interfering with their development, reproduction, and survival. Negative impacts on various life-history traits of target pests are commonly caused by host plants expressing antibiosis. Antixenosis affects insect behavior and reduces host colonization by chemical, physical, and morphological factors. Tolerance is the ability of plants to resist or overcome from injury caused by a pest, without affecting the pest's biology or behavior (Painter 1951, Panda and Khush 1995, Smith 2005, Baldin et al. 2019. Plants exhibiting high levels of antixenosis can cause negative effects on insect development, like that of antibiosis. Thus, it is essential to differentiate antibiosis and antixenosis categories by using specific insect feeding studies (Beach and Todd 1988, Smith 2005, Baldin et al. 2015, Coelho et al. 2020. Although some studies have evaluated soybean resistance to A. gemmatalis (Beach and Todd 1988;Hoffmann-Campo et al. 1994;Fugi et al. 2005;Piubelli et al. 2005;Franco et al. 2014Franco et al. , 2017, there are few studies characterizing resistance of a large number of soybean genotypes (with high genetic variability). Therefore, to identify new resistance sources, this study characterized the expression of antibiosis and antixenosis in 30 soybean genotypes under laboratory and greenhouse conditions.

Materials and Methods
The initial assays were performed with 30 soybean genotypes ( Table  1) that were selected to represent wide genetic variability and include sources of resistance against other Lepidoptera species, other insect orders such as stinkbugs and whitefly, and commercial genotypes never previously tested. The plants were grown in a greenhouse and evaluated under greenhouse and laboratory conditions in 2018 and 2019 in Botucatu, São Paulo, Brazil. Polyethylene pots (2 liters) containing a mixture of soil, sand, manure, and substratum (1:1:1:1) were kept in a greenhouse free from insect infestation. All plants received recommended crop fertilization (Malavolta 2006) and other necessary cultural practices (e.g., irrigation, thinning, and cleaning). Leaves from plants at V4/V5 developmental stages (Fehr and Caviness 1977) were used in all experiments.

Rearing A. gemmatalis
A colony of A. gemmatalis was established from soybean fieldcollected larvae and pupae in the municipality of Botucatu, in the state of São Paulo. After emergence, the insects were identified and the colony was maintained under laboratory conditions (T: 25 ± 2°C, photoperiod of 12:12 h, RH: 60 ± 10%) at the Department of Crop Protection, São Paulo State University, Botucatu, São Paulo, Brazil. To reinvigorate the colony, insects were continually introduced until the colony population reached the level necessary to initiate the bioassays. The rearing methodology and bean-based artificial diet were adapted from the protocols of Greene et al (1976) and Parra (2001). Insects from this colony were used for the antixenosis bioassays. For the antibiosis bioassay, A. gemmatalis eggs were commercially acquired (Pragas.com, Piracicaba, Brazil).

Antixenosis Bioassays
Three different types of bioassays (oviposition preference, attractiveness, and food consumption) with 30 soybean genotypes were initially conducted. The objective was to verify the possible expression of antixenosis and select the most promising resistant genotypes for the subsequent phase of the study.
For the oviposition bioassay, five plants (five replicates) were arranged in a completely randomized design and infested with two females per plant. Egg number was counted 2 d after insect release by visual observation of the whole plant.
For attractiveness and free-choice food consumption bioassays, two fourth-instar larvae/genotype (Boiça Júnior et al. 2015) were released in arenas (23 cm of diameter), following a complete randomized block design. Each arena with 30 genotypes represented one block, totaling 10 blocks. To avoid contact among larvae and leaf tissues, discs (6.9 cm 2 ) were enclosed with a plastic micro arena (50 ml) partially covered with organdy fabric to allow volatile flow. Before the tests, larvae were starved for 2 h. The mean number of larvae on leaf discs was calculated 120 min after release.
For the no-choice food consumption, two larvae/genotype were isolated in Petri dishes (8 × 2 cm) with one leaf disc of each genotype, ten replicates per genotype, in a completely randomized design. In free-and no-choice tests, all replicates were visually observed until at least one of the leaf discs in any of the treatments had larval feeding reaching approximately 90%, at which time the study was concluded. The nonconsumed food (cm 2 ) was measured with a leaf area meter (LI-COR LI-3100, LI-COR, Lincoln, NE).

Life-History Traits of A. gemmatalis Feeding on Selected Genotypes
After initial bioassays, the 11 genotypes most resistant to A. gemmatalis were selected for further assessment. Genotype selection was based on higher resistance levels expressed in the previous bioassays, primarily selecting genotypes that were promising in two or more tests. Genotypes 'Conquista' and 'Coodetec 208' were included as susceptible commercial genotypes (Silva et al. 2012), totaling 13 genotypes. Based on other studies (Gómez et al. 2018, Bel et al. 2019, Coelho et al. 2020, first-instar larvae were used to initiate the experiment. However, to reduce stress caused by manipulating newly hatched neonates, 48-h-old larvae were transferred from the artificial diet and placed into individual Petri dishes (8 × 2 cm) lined with moist filter paper containing leaf disks (6.9 cm 2 ) from each genotype. Each Petri dish containing one larva, corresponding to one replicate (total of 50 replicates) in a completely randomized design.
For further resistance evaluation and to discriminate antixenosis and antibiosis, each individual's food consumption (cm 2 ) was recorded. Leaf disks were replaced daily, and nonconsumed food was measured with the leaf area meter.

Statistical Analyses
Data were subjected to analysis of variance (ANOVA). Normality and homogeneity of the data were verified with Shapiro-Wilk and Levene tests (Winer et al. 1991). For the antixenosis study, data were compared using Scott-Knott test (P < 0.05; Scott and Knott 1974). For the antibiosis study, 'genotype' was considered a fixed effect and the pairwise t-test comparisons were conducted by least squares means (LS-means; P < 0.05), using the SAS 'Proc Glimmix' procedure (SAS Institute 2001).

Discussion
Some of the soybean genotypes evaluated in this study significantly affected A. gemmatalis behavior and biological parameters, characterizing expression of two resistance categories, antixenosis and antibiosis (Smith 2005). Even though a previous study indicated a few promising soybean genotypes with resistance to A. gemmatalis (Fugi et al. 2005), in this study, for the first time, we assessed the behavior and development of A. gemmatalis immature and adult stages across a wide selection of soybean germplasm with high genetic variability.
In practical terms, the number of eggs laid in a field is particularly important because eggs will initiate a pest infestation. Results from the initial antixenosis bioassays in the greenhouse revealed differences among the soybean genotypes for the number of eggs laid by A. gemmatalis. Other studies have also indicated differences in the number of eggs laid among soybean genotypes by other insect species. In a no-choice test, PI 274453 and L1-1-01 had the fewest eggs laid by Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae) (Schlick- Souza et al. 2018). In a multiple-choice oviposition test with 15 soybean genotypes, 'IAC 19' and UX-2569-159 had the lowest number of eggs laid by Bemisia tabaci (Gennadius) biotype B (Hemiptera: Aleyrodidae), indicating antixenosis. In the same study, PI 229358 was the least attractive and least used for oviposition. The antixenosis expressed by these genotypes to whitefly was related to characteristics of their trichomes (lower density and inclined; Valle et al. 2012, which could also explain the low number of eggs laid on these genotypes in our study.
In this study, micro arenas were used to avoid contact among larvae and leaf disks, so larvae were attracted exclusively by volatile compounds released by each genotype. In this context, regarding the total mean of larvae attracted over the time, IAC 74-2832, 'IAC 17', 'TMG 133' RR, 'KS-4202', PI 274454, 'IAC  (-) = insects did not turn into the instar. (-*) = Consumption was low, and the leaf area meter did not register the value. LS-means with the same letter in the column are not significantly different (P ˃ 0.05). 'Coodetec 208' and 'Conquista' were considered the susceptible checks. 100', 'Dowling', and 'TMG 1179' RR had the least number of larvae (i.e., most repellant), indicating antixenosis, which could be governed by a chemical factor present in the genotypes. In a similar study testing the influence of different genotypes, 'IAC 19', 'IAC 18', 'IAC 23', L1-1-01, PI 274453, PI 229358, PI 171451, 'IAC 100', 'IAC 24', 'IAC 17', and IAC 74-2832 were classified as repellents for C. includens (Schlick-Souza et al. 2018). The lower attractiveness or level of colonization of an insect to a genotype suggests the presence of factors that inhibit the acceptance by the specific material, and consequently feeding and/or oviposition, which usually occurs in plants exhibiting antixenosis (Smith 2005, Baldin et al. 2019. Volatile compounds expressed by resistant plants are common defenses; however, in crops, their potential has not been fully realized for the biological control of pests. In addition to their effect on pests, certain volatiles attract natural enemies (predators and parasitoids), so both types of effects should be considered when devising strategies to improve biological control in refuge areas (Stenberg et al. 2015).
Food consumption was low for 'TMG 1179' RR, D75-10169, 'TMG 7062' IPRO, and the 'IAC' genotypes in the free-and no-choice tests, indicating antixenosis. Similar results were observed in another study where 'IAC 100' and IAC 74-2832 were rejected by A. gemmatalis larvae based on a consumption index (Hoffmann-Campo et al. 1994). Genotypes D75-10169, 'IAC 100', IAC 78-2318, and 'IAC 19' were also less attractive and less consumed by Piezodorus guildinii (Westwood) (Hemiptera: Pentatomidae) . Low consumption may indicate the presence of deterrents, the lack of a feeding stimulant, or leaf hardness, which are all known to decrease feeding (Reynolds et al. 1984). However, antixenosis and antibiosis commonly overlap in resistance bioassays, making it difficult to interpret the two resistance categories in isolation (Panda andKhush 1995, Smith 2005). Thus, it is important to evaluate other parameters through larval development, with the purpose to exclude or to confirm the additional presence of antibiosis.
The capacity of resistant plants to reduce larval consumption also has implications in conjunction with insecticide use. Chrysodeixis includens reared on resistant soybean leaves (PI 227687) were significantly more susceptible to acephate (two times) than when they were reared on susceptible leaves (Rose et al. 1988). This enhanced efficacy could also reduce insecticide use and be particularly important and directly affect resistance management in a crop system.
Larvae that fed on 'TMG 7062' IPRO were unable to transform into third instar because the insecticidal protein (Cry1Ac) was present in these Bt plants. In general, the 'IAC' genotypes showed a similar response. Most larvae fed 'IAC' genotypes had relatively short instar duration, but required more time (days) to complete the larval phase because of an additional instar extending the cycle. The total larval period (14.53-19.86 d) had a difference of 5.33 d between low and high mean, indicating a high degree of antibiosis and/ or antixenosis. A previous study conducted with P. guildinii testing 17 soybean genotypes also indicated antibiosis resistance in some genotypes, especially 'IAC 100' and 'IAC 19', where 'IAC 100' significantly prolonged nymphal development (Silva et al. 2013). (-) = insects did not turn into fifth-instar larvae and pupae. LS-means with the same letter in the column are not significantly different (P ˃ 0.05). 'Coodetec 208' and 'Conquista' were considered the susceptible checks. Prolongation of the immature phase may result from intake of deleterious compounds typically present in antibiotic genotypes that inhibit insect development. However, genotypes with high levels of antixenosis can also cause this effect, either by hampering insect feeding, nutritional improprieties, or poor nutritional quality of host (Painter 1951). In practical terms, the prolongation of the larval cycle increases the exposure of an insect to natural enemies or other mortality factors.
Genotype 'IAC 100' has significant levels of the flavonoids rutin and genistin, which suggests these compounds may be involved in resistance observed for A. gemmatalis, similar to that documented for P. guildinii (Bentivenha et al. 2018). These compounds were also related to resistance against A. gemmatalis in another study where leaf extracts of 'IAC 100' were offered to larvae (Piubelli et al. 2005).
However, chemical and morphological analysis of the other genotypes is needed to fully understand the role of various resistance factors.
Pupal weight was also significantly affected by the 'IAC 19','IAC 24','IAC 100',and 'IAC 17'. Larvae fed 'IAC 19','IAC 100',or 'IAC 17' weighed 0.12 g,and 0.13 g for those fed 'IAC 24', while pupae from the susceptible genotypes 'Coodetec 208' and 'Conquista' weighed 0.19 and 0.21 g, respectively. Anticarsia gemmatalis fed PI 229358, 'IAC 17', 'IAC 24', and 'IAC PL-1' in a biological development study did not show significant differences in pupal weight (Fugi et al. 2005); however, in another study, A. gemmatalis pupal weight was significantly reduced when larvae were fed 'IAC 17' (Gazzoni and Tutida 1996). The reduction in larval and pupal weights may be associated with low reserves acquired during the larval period because of a reduction in the food's nutritional value. Together, these effects might compromise the performance of the insect in the reproductive stage by reducing the number of copulations, oviposition, and fertility (Smith and (-) = insects did not turn into adult. LS-means with the same letter in the column are not significantly different (P ˃ 0.05). 'Coodetec 208' and 'Conquista' were considered the susceptible checks. (-) = insects did not turn into pre-pupae, pupae, and adult. LS-means with the same letter in the column are not significantly different (P ˃ 0.05). 'Coodetec 208' and 'Conquista' were considered the susceptible checks. Clement 2012, Smith and Chuang 2014), which are generally associated with the expression of resistance.
In our study, the 'IAC' genotypes caused significant changes in A. gemmatalis life cycle (i.e., larval through pupal developmental time). Similarly, 'IAC 100' was found to provide resistance by extending the life cycle of Spodoptera eridania (Cramer) (Lepidoptera: Noctuidae) , and H. armigera larvae fed 'IAC 17' had a higher numerical mean life cycle duration (Coelho et al. 2020). Genotypes that result in the longest life cycle durations usually have low nutritional quality, which can influence insect development (Pereyra and Sánchez 2006). The increase in life cycle duration affects directly on population dynamics and may determine the success of the species on the host plant. Another consequence is fewer insect generations per year (Green et al. 2002, Rajapakse andWalter 2007).
As expected, the adult parameters were severely affected by the genotypes. Differences were observed in the preoviposition period (1.85-5.50 d) and oviposition period (4.75-13.54 d). These results could be a consequence of reduced nutrition of resistant plants during the larval period that translates into deficiencies, quantitative, or qualitative nutrient imbalances in the adult phase ). Significant effects of the genotypes were also observed by the low number of eggs laid by A. gemmatalis fed 'IAC 24', 'IAC 17','IAC 100',. Egg production can be affected by physical or chemical differences in the food, by the amount of food ingested during insect development, and by the nutritional value that each host can offer to the insect (Umbanhowar and Hastings 2002). Negative effects on fecundity, as well as in the number of progeny, are common for insects feeding on genotypes expressing antibiosis (Panda andKhush 1995, Smith 2005).
Planting resistant materials in a refuge area, e.g., could be a good strategy to reduce the number of foliar insecticide sprays, since insects in the refuge would feed less, have lower viability, and cause reduced or no economic impact. Thus, resistant cultivars would be agronomically desirable and compatible with other IPM strategies (Stenberg 2017).
The resistance found in some of the evaluated genotypes increases their importance as sources of resistance to A. gemmatalis, and possibly other insect pests. Our study provides baseline information which can be useful for developing management strategies for this species in soybean, especially in South America. In the future, further trials must be carried out under field conditions to validate whether the genotypes are effective in suppressing A. gemmatalis populations.