Antibiosis in soybean genotypes to Spodoptera cosmioides (Lepidoptera: Noctuidae)

The occurrence of the black caterpillar Spodoptera cosmioides (Lepidoptera: Noctuidae) has been increasing in soybean crops in Brazil leading to yield losses. The objective of this study was to evaluate antibiosis in soybean genotypes to S. cosmioides . The following parameters were evaluated: duration, viability and larval weight; duration and viability of the pre-pupal; duration, weight and viability of pupa; total cycle and sex ratio. The design was completely randomized with 18 treatments (genotypes) and 30 replicates. The genotypes PI 227687, PI227682, IAC 100 and BRS 7270 IPRO showed significant levels of antibiosis against S. cosmioides . These results will be useful for soybean producer or in breeding programs focusing on host plant resistance.


Introduction
The soybean (Glycine max) has great relevance for the Brazilian economy and stands out as the main agricultural product for the country's exports (Follmann et al., 2017). Among the pests that attack the soybean crop, the complex of caterpillars are important and frequently cause economic loss (Lourenção et al., 2010;Bueno et al., 2011;Hoffmann-Campo et al., 2012). Spodoptera cosmioides (Lepidoptera: Noctuidae) has been found in soybean plants, reducing leaf area (Freitas et al., 2018) and feeding on pods Boiça Júnior et al., 2015).
The occurrence of pest has increased in soybean crops in Brazil with the contributing factors being: crop succession presenting food supply for the insect, successive applications of fungicides and insecticides, abundance of host plants and expansion of cultivated areas (Santos et al., 2005(Santos et al., , 2009Souza et al., 2014a).
The control of caterpillars by entomopathogens, predators and parasitoids was important, and kept the pest population below control levels . However, with current agricultural practices, chemical control is the most common method, and insecticides have been unsatisfactory (Constanski et al., 2016). In addition, there are relatively few products registered for control of S. cosmioides in Brazil (Agrofit, 2020) and soybean plants with Bt gene insertion (Bacillus thuringiensis -Bacillacea) are inefficient to control Spodoptera (Bortolotto et al., 2016;Horikoshi et al., 2016). Thus, the study of methods compatible with IPM, that can reduce the use of insecticides are necessary (Sosa-Gómez and Silva, 2010;Constanski et al., 2016).
Plant resistance to insects -PRI, is expressed through different mechanisms such as: antixenosis (non-preference), antibiosis and tolerance. Antixenosis is associated with the presence of trichomes, changes in leaf color or the presence of volatile compounds in the plant (Smith, 2005;Seifi et al., 2013). Antibiosis is manifested mainly by chemical constituents present in the plant and affects the biology and/or physiology of the pest, such as: reduction in larval and pupal weight, prolongation of life cycle, adult deformation, alteration in sex ratio (Souza et al., 2014b;Boiça Júnior et al., 2015;Almeida et al., 2017) and tolerance, which is the ability of the plant to resist or recover from insect damage due to the production of new vegetative or reproductive structures (Smith, 2005;Seifi et al., 2013). RPI is considered an ideal control method because it maintains pest population density below the economic injury level, has no adverse effect on the environment, requires no additional expenses, can be combined with other control tactics, is compatible with IPM and can be used directly by farmers (War et al., 2012;Seifi et al., 2013).
The present study aimed to evaluate antibiosis to S. cosmioides in different soybean genotypes (commercial cultivars and genotypes).

Material and methods
Experiments were carried out at the laboratory of Integrated Pest Management of Federal Goiano Institute, Campus Urutaí, Goiás, Brazil. Laboratory assays were conducted under controlled conditions of temperature (25 ºC ± 2), relative humidity (70% ± 10) and photoperiod (12h).

Spodoptera cosmioides rearing
A Spodoptera cosmioides colony was established from larvae provided by the São Paulo State University (UNESP), laboratory of Plant Resistance to Insects in Jaboticabal, São Paulo, Brazil. The larvae were fed with artificial diet (Greene et al., 1976) and raised in plastic pots (300 mL) in a group of four larvae up to the pupal stage. The pupas were sexed and placed in a PVC cage (20 cm Height x 20 cm in diameter) for emergence and mating of adults.
The adults were fed on 10% honey solution and the diet was changed every two days. Ovipositions were removed daily and placed in plastic pots (500 mL) until larva hatching.

Plant material
The genotypes used in the studies were obtained from the soybean breeding program at the Brazilian Agricultural Research Corporation -EMBRAPA, National Soybean Research Center (Londrina, Parana, Brazil) (Table 1).
Plants were grown in 5L pots filled with substrate (3:1:1 -soil, sand and organic bovine manure) and fertilized according crop requirements (Sousa and Lobato, 2004). The soybean plants were kept in the greenhouse under environmental conditions and irrigated daily using the same volume of water per pot to avoid water stress. Soybean plants were used in the assays 30 days after sowing.
Antibiosis in soybean genotypes to Spodoptera cosmioides Newly hatched S. cosmioides larvae were individualized in plastic containers (100 mL) containing moistened filter paper and fed with leaves from soybean genotypes. The larvae remained in the plastic container until the pupal stage when feeding was interrupted. The emerged adults were individualized and caged to measure the longevity and food was not provided for the insect. The following biological parameters were evaluated: a) larval: duration and viability of the larval stage and weight of larvae at ten days; b) pre-pupal: duration and viability; c) pupal: duration, weight of pupa at 24 hours and viability; d) adult: longevity and e) total cycle: period and viability and sex ratio. A completely randomized design with 18 treatments (genotypes) and 30 replicates was adopted. Each repetition was considered to be an individualized larva in the plastic container.

Statistical analysis
The results were submitted to multivariate analysis of variance (MANOVA). There was a significant effect (P < 0.05) of genotypes when the means were compared by the Scott Knott test at 5% probability (R Development Core Team, 2017, Scott Knott Package). A cluster analysis was performed using the Hierarchical Cluster Analysis -UPGMA method based on the Mahalanobis generalized distance to group cultivars by their level of resistance (R Development Core Team, 2017, Biotools
The cultivars BRS 7470 IPRO and ANTA 82 RR presented the highest larval weight. The genotypes IAC 100, BRS GO 7460, BMX Desafio, NA 5909,   (Table 3). By hierarchical grouping analysis -UPGMA was observed the influence of soybean genotypes in the biological parameters of S. cosmioides (Fig. 1) This was supported by the canonical variable analysis -CVA (Fig. 2). Both multivariate methods provided similar results regarding the resistance groupings. The genotype PI 227687 that appears isolated in the CVA analysis, also appears in group V in the UPGMA, influencing the S. cosmioides pupal period. IAC 100, which appears isolated in the CVA analysis, also appears in group IV in the UPGMA analysis, influencing the total viability and larval weight of S. cosmioides. The susceptible cultivar BMX Desafio also appears isolated in CVA analysis and pupal weight was the main characteristic that defined this degree of resistance.
The first canonical variable explained 31.6% of the total parameters evaluated for the characteristics described in the soybean genotypes and this component was influenced by pupal period and the total cycle. The second component explained 30% of the variability and was influenced by larval period, total viability, larval weight and pupal weight.

Discussion
Antibiosis is the category of resistance that occurs when the insect feeds on the plant, negatively affecting the insect's biology. The cause of resistance can be chemical and/or morphological plant defensive factors. The reduction of body size and weight, prolonged periods of development and reduced fecundity are characteristics of antibiosis (Lara, 1991;Smith, 2005;Seifi et al., 2013). The results showed that soybean genotypes influenced the biological parameters and presented different degrees of resistance to S. cosmioides.
Genotypes BRS 7270 IPRO, PI 227687 and IAC100 extended the larval period of S. cosmioides in relation to BRS 8482, BRS GO 7460, NA 5909 and NS 7447 IPRO. The larval viability was lower in BMX Desafio, BRS GO 7460, NA 5909, PI 227682 and PI 227687. The prolongation of the larval period may be associated with the presence of chemical compounds which confers antibiosis and/or antixenosis (Silveira et al., 1997;Boiça Júnior et al., 2015). Defenses in plant resistance results from physical tissue strength, structural barriers (trichomes), presence of allelochemicals such as alkaloids, ketones, glucosinolates, isoflavonoids, terpenoids and organic acids (War et al., 2012).
The pupal period of S. cosmioides was also affected by soybean genotypes. The longest period was observed in pupae from PI 227687 and the lowest viability in BMX Desafio, BRS 7270 IPRO, BRS GO 7460, IAC 100, NA 5909, NS 7447 IPRO and PI 227682 and PI 227687. The chronic effects of antibiosis often cause larval and pupal mortality (Smith, 2005). S. cosmioides fed on IAC 100, PI 227682 and PI 227687 did not reach the pupal stage (Boiça Júnior et al., 2015). This finding characterizes antibiosis in these soybean genotypes to S. cosmioides.
The lowest larval weight of S. cosmioides was observed on BMX Desafio, BMX Potência, BRS 397, BRS GO 7460, BRS GO Jataí, IAC 100, M 7739 IPRO, NA 5909 and PI 227682. This lower weight may be due the presence of secondary metabolites present in these genotypes. The flavonoids (rutin and genistin) were identified in PI 274454, PI 227687, and IAC-100 genotypes, and these substances play a defense role in soybean plants and are deterrents, conferring resistance to insects (Piubelli et al., 2005). The flavonoid rutin prolonged the larval period, reduced the larval and pupal weight and decreased the pupal viability of S. frugiperda .
Genotypes IAC 100 and PI 227682 have been reported to negatively affect insect biology. Boiça Júnior et al. (2015) found that PI227682 and IAC100 reduced the larval weight and viability of S. cosmioides. Piubelli et al. (2005) observed larval weight reduction in A. gemmatalis fed on plants with leaf extract of IAC 100 and PI 227687.
Larvae fed on PI-227687, IAC 100 and BRS 7270 IPRO had their life cycles extended. Probably these genotypes were not adequate to supply the nutritional demands for S. cosmioides. This characteristic is common in insects that have unbalanced feeding or that ingest Figure 1 Dendrogram resulting from the multivariate grouping analyses, using the UPGMA method, based on the Mahalanobis distance, from the LP (Larval period -days), PP (Pupal period -days), LW (Larval weight -mg), PW (Pupal weight -mg), LC (Life cycle -days) and TV (Total viability -percentage) in soybean genotypes for resistance to Spodoptera cosmioides (Lepidoptera: Noctuidae). Urutaí, Goiás, Brazil. inappropriate metabolites (Panizzi and Parra, 2009). The pest life cycle prolongation is desirable in plant resistance to insects, since the insect will have fewer generations, reducing population density generating a consequent reduction in the damage to agricultural crops (Lara, 1991;Baldin et al., 2019).
Both CVA and UPGMA analyses separated the soybean genotypes into different levels of resistance to S. cosmioides and can be used as a complement to univariate methods in selection of plant resistance to insects (Pitta et al., 2010). The least suitable genotypes for S. cosmioides were found to be PI 227687, PI 277682 and IAC 100. Thus, the high percentage of mortality in the larval phase can be attributed to the possible effects of secondary metabolites characterizing antibiosis. The cultivar BRS 7270 IPRO can be used by soybean producers in combination with other control tactics in integrated pest management of S. cosmioides.

Conclusions
Genotypes PI 227687, PI 226782, IAC 100 and BRS 7270 IPRO show antibiosis to S. cosmioides. The cultivar BRS 7270 IPRO needs to be further evaluated under field conditions to verify the level of resistance to S. cosmioides. These results will be useful for soybean producer or in breeding programs focusing on host plant resistance.