Experimental conditions, plant material, and test insects
Experiments were performed from December 2012 to May 2013 in a greenhouse and laboratory of the Department of Plant Health of the São Paulo State University, School of Agriculture and Veterinarian Sciences, Jaboticabal, state of São Paulo, Brazil. Laboratory bioassays were conducted under environmentally controlled conditions (25 ± 2ºC temperature; 70 ± 10% relative humidity; 12L:12D h photoperiod).
Soybean plants of the resistant genotype PI 227687 and of the susceptible genotype IGRA RA 626 RR were chosen as they were previously screened for resistance to lepidopteran larvae (Souza et al. 2012, Souza et al. 2014) and were reported to vary in the levels of antixenosis-resistance in function of leaf age and plant growth stage (Boiça Júnior et al. 2015). Plants were grown in 5-L pots filled with soil (dystrophic red latosol) (Centurion et al. 1995), bovine manure, and sand at 3:1:1 ratio, and kept in a greenhouse under ambient temperature and lighting until used in assays. The plants were watered daily. Larvae of A. gemmatalis and S. frugiperda were obtained from colonies maintained in the laboratory since September 2011. VBC and FAW larvae were fed on artificial diet (Greene et al. 1976) and the adults on 10% honey solution.
Four independent experiments were conducted to investigate the effects of leaf age and plant stage on expression of soybean resistance to each insect species, as described below. The first and second experiments compared the development of VBC and FAW fed on younger leaves from the upper part and older leaves from the lower part of plants. The third and fourth experiments compared the insects’ development on leaves from soybeans at vegetative and reproductive growth stages. Experiments 1 and 2 were conducted simultaneously using the same batch of soybean plants and under the same environmental conditions, and the same was performed for Experiments 3 and 4. These standardized conditions allowed to compare and discuss the results regarding the effects of leaf age and plant stage on expression of soybean resistance to the specialist VBC and generalist FAW.
Experiment 1: Effects of soybean leaf age on VBC development
The first experiment evaluated the influence of leaf age on expression of antibiosis-resistance to VBC. Plants of the resistant and susceptible soybean genotypes were used at the V5 stage (Fehr and Cavines 1977). The “younger leaves” consisted of the first and second trifoliates from the plant apex and the “older leaves” consisted of the third and fourth trifoliates from the same V5 plants. Neonates of VBC were collected from the rearing colony and transferred into 9-cm-diameter Petri dishes lined with moistened filter paper using a fine paintbrush. One larva was placed per dish, which was allowed to feed on either younger or older leaflets of the resistant or susceptible genotypes during the larval stage. Leaflets in the dishes were changed every three days from neonates to third instars, and then replenished daily until pupation.
The experiment was arranged in a completely randomized design, and treatments consisted of a 2 x 2 factorial. The four treatments consisted of combinations of two soybean genotypes (PI 227687 and IGRA RA 626 RR) and two leaf ages (younger and older leaves). Five replications were used per treatment, with five individual larvae per Petri dish considered as a replication. Duration of the larval and pupal stages, larval and pupal survival, and larval and pupal weights of VBC were recorded. Larval weights were recorded from 10-d-old larvae and pupal weights in 24-h-old pupae.
Experiment 2: Effects of soybean leaf age on FAW development
The second experiment evaluated the influence of leaf age on expression of antibiosis to FAW. The same treatments, experimental design, number of replicates, and environmental conditions of Experiment 1 were employed, except that larval weights of FAW were recorded in 12-d-old larvae.
Experiment 3: Effects of soybean plant stage on VBC development
The third experiment compared the antibiosis-resistance expression to VBC at vegetative and reproductive soybean plant stages. V5-stage plants were used for the vegetative stage, while R3-R4-stage plants were used for the reproductive stage (Fehr and Cavines 1977). For this experiment leaves were collected from the upper part (younger leaves) of soybean plants at both growth stages and then offered to VBC larvae in Petri dishes. For vegetative-stage plants, the first/second trifoliates from the plant apex were used, whereas the first/second/third trifoliates from the apex were collected from reproductive-stage plants.
The experiment was arranged in a completely randomized design, and the four treatments consisted of a 2 x 2 factorial consisting of combinations of two soybean genotypes (PI 227687 and IGRA RA 626 RR) and two plant stages (vegetative and reproductive). Five replications were used per treatment, with five individual larvae per Petri dish considered as a replication. The same biological parameters of VBC in Experiment 1 were recorded in this experiment.
Experiment 4: Effects of soybean plant stage on FAW development
The fourth experiment compared the antibiosis-resistance expression to FAW at vegetative and reproductive growth stages. The same growth stages of soybean were used for the vegetative (V5) and reproductive (R3-R4) stages as in Experiment 3. However, the leaf age/position within plants that were used in the bioassay with FAW was different from that of VBC; here the third/fourth trifoliates were collected from vegetative-stage plants and the fourth/fifth/sixth trifoliates from reproductive-stage plants and then offered to FAW larvae in Petri dishes. Different methodologies between VBC and FAW bioassays were used because of the varying results for each insect species; a clearer distinction in the levels of antixenosis-resistance in reproductive-stage soybeans to FAW was obtained in previous evaluation using older leaves collected from the lower part of plants relative to younger leaves from the upper part of plants (Boiça Júnior et al. 2015).
Other conditions of this experiment such as treatments, experimental design, and replication were similar to those of Experiment 3, except that larval weights of FAW were recorded in 12-d-old larvae.
Concentrations of nutrients and flavonoids in younger and older leaves of soybeans at vegetative and reproductive stages
To give insights on possible chemical mechanisms underlying the antibiosis-resistance to VBC and FAW in soybean in function of leaf age and plant stage as observed in the bioassays, plants of both genotypes were further cultivated to collect leaves for analysis of nutrients and flavonoids. For this, resistant and susceptible soybeans were grown in two small field plots consisting of four 5-m-long rows of plants spaced 0.5 m apart, with stands of 10-12 plants per row meter. Soil type in the field plots was the same as that used for potted plants, and was fertilized once with bovine manure before seeds sowing. Field-grown plants were watered by drip irrigation 2-3 times a week. Preventive insecticide and fungicide applications were applied to soybean plants to prevent damage by biotic stressors and minimize unintended alterations in plant biochemistry due to induced defense responses.
When plants reached the V5 stage, 30 plants were randomly selected for each genotype. “Younger leaves” were collected from the first/second trifoliates (upper part) from the plant apex, and “older leaves” were collected from the third/fourth trifoliates (lower part) of the same plants. The leaves were stored in paper bags and taken to the laboratory. When the set of plants of the other field plot reached the R3-R4 reproductive stage, 30 plants of each genotype were randomly selected, and “younger leaves” were collected from the first/second/third trifoliates (upper part) from the plant apex, and “older leaves” from the fourth/fifth/sixth trifoliates of the same plants.
Analysis of nutrients was performed in the Department of Soils and Fertilizers of the School of Agriculture and Veterinarian Sciences. In the laboratory, leaves of each soybean leaf-age and plant-stage categories collected from the 30 plants were pooled together for each genotype in order to reduce potential between-plant variability. The leaves were rinsed with demineralized water + 0.5% neutral detergent solution, and rinsed consecutively four times with demineralized water. Next, they were oven-dried at 60ºC for 48h and milled to a fine powder through a rotary-knife mill (Thomas Wiley Mill, model Te 650, Swedesboro, NJ, USA) with 1 mm mesh size. The macronutrients N, P, K, Ca, Mg, and S, and the micronutrients B, Cu, Fe, Mn, and Zn were quantified following the methodology of Miyazawa et al. (1999). A single sample consisting of leaflets pooled together from 30 plants of each treatment combination was analyzed in triplicate.
Quantitative analysis of flavonoids was performed in the Department of Chemistry of the Federal University of São Carlos, in São Carlos, state of São Paulo, Brazil. The flavonoids rutin, isoquercitrin, daidzin, daidzein, hesperidin, naringin, naringenin, and hesperidin in younger and older leaves of the resistant and susceptible soybeans at vegetative and reproductive stages were quantified using high performance liquid chromatography - mass spectrometry (HPLC-MS) following the methodology developed by Perlatti et al. (2016).
Statistical analysis
Data of biological parameters of VBC and FAW were checked for normality of residuals and homogeneity of variance using the Kolmogorov-Smirnov and Levene tests, respectively. FAW larval and pupal weights data were not normally distributed and were square root-transformed to meet assumptions of the analysis of variance (ANOVA). Data were subjected to two-way ANOVA, with genotype, leaf age/plant stage (depending on the experiment), and their interactions as main effects. Means of treatments were separated by Tukey honestly significant difference (HSD) test (α = 0.05). Statistical analysis was performed in Statistica v.7 (Statsoft 2004).