Plants and aphids
Eight cultivars of genetically unmodified soybeans were studied: ‘Aldana’, ‘Annushka’, ‘Augusta’, ‘Madlen’, ‘Mavka’, ‘Simona’, ‘Violetta’, and ‘Viorica’. These cultivars were selected because they represent various regions of origin in Eastern and Central Europe: Bulgaria (‘Simona’), Lithuania (‘Violetta’), Poland (‘Aldana’, ‘Augusta’, ‘Madlen’, ‘Mavka’), Romania (‘Viorica’), and Ukraine (‘Annushka’) [68], and belong to different maturity groups in these regions: ‘Annushka’ and ‘Augusta’ are very early maturing cultivars, ‘Aldana’, ‘Simona’, ‘Violetta’, ‘Viorica’ – early, ‘Mavka’ – semi-early, and ‘Madlen’ – late maturing cultivars [69]. The seeds were provided by Hodowla Soi Agroyoumis Sp. z o. o. (Kordeckiego 20, 37-420 Rudnik nad Sanem, Poland). Plants were grown in commercial soil in 9 cm diam. plastic pots, in the chamber Sanyo MLR-351H (Sanyo Electronics Co. Ltd.) at 20°C, 65% r.h., and L16:8D photoperiod. The plants were watered regularly and no fertilizers were applied.
The laboratory culture of Acyrthosiphon pisum was maintained on Pisum sativum cv. ‘Milwa’ in the laboratory at 20°C, 65% r.h., and L16:8D photoperiod.
Survival tests
One adult apterous female of A. pisum was placed on a plant at 14 BBCH growth stage (trifoliate leaf on the 4th node unfolded) [70] for 24 hours. After 24 hours, the female and all progeny except one nymph were removed. Each plant was isolated within a plastic cylinder with a fine mesh on top. The development of the nymph was monitored daily. The experiment was replicated 15 times for each soybean cultivar. The tests were conducted in an environmental chamber Sanyo MLR-351H (Sanyo Electronics Co. Ltd.) at L16:D8 photoperiod, 21 ± 1 °C, and 70% r.h.
Aphid probing behavior
The probing and feeding behaviors of A. pisum were monitored using the technique of electronic registration of aphid probing in plant tissues, known as Electrical Penetration Graph (EPG)34. Aphid and plant are made parts of an electric circuit, which is completed when the aphid inserts its stylets into the plant. Weak voltage is supplied in the circuit, and all changing electric properties are recorded as EPG waveforms that can be correlated with aphid activities and stylet position in plant tissues71. In the present study, aphids were attached to a golden wire electrode with conductive silver paint and starved for 1 h prior to the experiment. Probing behavior of A. pisum on soybean cultivars was monitored for 8 h continuously with 4- and 8-channel DC EPG recording equipment. Signals were saved on the computer and analyzed using the PROBE 3.1 software provided by W. F. Tjallingii (www.epgsystems.eu). The following aphid behaviors were distinguished: no penetration (waveform ‘np’ – aphid stylets outside the plant), pathway phase – penetration of non-phloem tissues (waveforms ‘ABC’), derailed stylet movements (waveform ‘F’), salivation into sieve elements (waveform ‘E1’), ingestion of phloem sap (waveform ‘E2’), and ingestion of xylem sap (waveform ‘G’). The E1/E2 transition patterns were included in E2. ‘F’ waveforms occurred sporadically, therefore these events were combined with pathway activities in all calculations and defined as non-phloem activities. The waveform patterns that were not terminated before the end of the experimental period (8 h) were included in the calculations. In sequential parameters, when time to waveforms related to phloem phase (E1 or E2) was calculated, the time from the 1st probe until the end of the recording was used if no phloem phase occurred. In non-sequential parameters, when a given waveform had not been recorded for an individual, the duration of that waveform was given the value of 0.
Aphids for EPG experiments were 2-3 days old (2-3 days after the final moult) viviparous apterous A. pisum. The plants of G. max used in the bioassays were at 14 BBCH growth stage (trifoliate leaf on the 4th node unfolded)70. Each aphid was given access to a freshly prepared plant. Each plant-aphid set was considered as a replication and was tested only once. The number of replications for each plant cultivar/aphid combination was 24. However, only the replications that included complete 8-hour recording were kept for analysis, which were: ‘Aldana’, n=16; ‘Annushka’, n=16; ‘Augusta’, n=22; ‘Madlen’, n=16; ‘Mavka’, n=13; ‘Simona’, n=16; ‘Violetta’, n=15, ‘Viorica’, n=18. Recordings that terminated due to aphid falling from the plant or where EPG signal was unclear were discarded from analysis. All bioassays started at 10:00–11:00 h MEST (Middle European Summer Time). Aphids show distinct diurnal feeding activity, with peak activity during day time, independently of host plants34,71-72.
High-performance liquid chromatography of flavonoids
The dried soybean leaves, of different botanical varieties, i.e., ‘Annushka’, ‘Aldana’, ‘Augusta’, ‘Madlen’, ‘Mavka’, ‘Simona’, ‘Violetta’ and ‘Viorica’ (1.2 g of each) were homogenized in an aqueous ethanol solution (80%) using a Diax 900 homogenizer. The resulting suspension was centrifuged (12000 rpm, 10 min) and the supernatant solution was collected in a graduated flask and the pellet was reconditioned. This operation was repeated three times, and the obtained extracts were combined. The homogenization procedure in combination with the extraction was carried out in such a way that the final volume of the extract was 100 ml. From the prepared ethanol extracts, 10 ml was taken and evaporated to dryness in a rotary evaporator under reduced pressure at 40 °C. The dry extracts were dissolved in 100% methanol to a volume of 1 ml. Resulting methanolic extracts containing flavonoids compounds were analyzed by HPLC–ESI-MS/MS.
The content of: ampelopsin, apigenin, daidzein, glycitein, hesperetin, hesperidin, isorhamnetin, kaempferol, luteolin, naryngin, quercetin, rutin, taxifolin was determined. The selection of the flavonoid spectrum for analysis was based on literature data73-77.
Individual pure flavonoids were purchased from Sigma–Aldrich (Poland). Ethanol, HPLC gradient grade methanol and acetonitrile were supplied by Merck (Germany). Formic acid was purchased from Sigma-Aldrich (Poland). Stock standard solutions of individual flavonoids (50 mg/1) were prepared by dissolving appropriate amounts of solid reagents in methanol. Mixed working standard solutions of flavonoid compounds at 20, 10, 5, 2.5 and 1 mg/1 or lower concentrations were prepared by appropriate dilutions of stock standard solutions.
The chromatographic analysis was carried out with a Shimadzu LC system, comprising a LC20-AD binary pump, a DGU-20A5 degasser, a CTO-20AC column oven and a SIL-20AC autosampler, connected to a 3200 QTRAP hybrid triple quadrupole (Applied Biosystem, MDS SCIEX, USA) with electrospray ionization source (ESI) operated in negative-ion mode. Phenolic compounds were separated on a Phenomenex Luna C-18 column (100 × 2.0 mm × 3.0 µm) with a pre-column, both maintained at 30 °C. A 7,4 mmol/l solution of formic acid (pH 2.8, eluent A) and acetonitrile (eluent B) were used. The mobile phase was delivered at 0.2 ml/min in a linear gradient mode as follows: 0–2 min 10% B, 30 min 60% B, 40 min 100% B, 55 min 10% B. Flavonoids were identified by comparing their retention times and m/z values of precursor and resulting fragmentation product ions in their MS and MS/MS spectra, respectively, to those obtained for respective standard solutions analyzed under the same conditions. The quantification of flavonoids was done using calibration curves obtained in the SRM (single reaction mode) mode78-79.
Statistical analysis
All statistical calculations were performed using StatSoft, Inc. (2014) STATISTICA (data analysis software system), version 12. Parameters of aphid performance were analyzed using Kruskal-Wallis test and post-hoc multiple comparisons of mean ranks for all groups (Dunn’s test). EPG parameters describing aphid probing behavior were calculated manually and individually for every aphid and the mean and standard errors were subsequently calculated using the EPG analysis Excel worksheet created by the authors especially for this study. Data thus obtained were analyzed by Kruskal-Wallis test and post-hoc multiple comparisons of mean ranks for all groups (Dunn’s test). Additionally, the relationships among all the traits were estimated on the basis of correlation coefficients. The graphic distribution of cultivars, described by means of the 24 observed traits, was obtained by means of the principal components analysis (PCA). Correlation and PCA analyses were done in GenStat 18.