A colony of YPMs was established and had been maintained for about 25 generations on maize in climate incubators (RTOP-B, Zhejiang Top Instrument Co., Ltd.) at 23 ± 1℃, RH 75 ± 2%, 16L/8D photoperiod, and 3, 500 lux light intensity (Guo et al. 2021). Adult moths were provided with 5%–8% honey solution after emergence. Apples covered with gauze pieces were provided for the oviposition of mated YPM females in the cage.
The P. digitatum isolated from orange fruits were purified according to the method described in Guo et al. 2022. PDA medium (7 mm diameter) with fully grown P. digitatum was also prepared and incubated at 30℃ for 6 d before behavioral assays.
Fungi non-infected apples (NIA): Apple (Malus pumila, Red fuji variety) fruits with uniform size (7-9 cm diameter) and shape were bought from the supermarket of Beijing University of Agriculture (Beijing, China) and were stored at 4℃ in a refrigerator for further experiments.
Mechanically damaged apples (MDA): Apple fruits with uniform size were firstly sterilized using 75% alcohol for 1 min, and then 1% sodium hypochlorite for 3 min, at last washed with sterilized distilled water under the horizontal-laminar airflow clean bench. Two holes (7 mm diameter) at the opposite sides of each apple were punched and immediately stuffed using sterilized fungus-free potato dextrose agar medium.
P. digitatum-infected apples (PDA): Apple fruits with uniform size were firstly sterilized using 75% alcohol for 1 min, and then 1% sodium hypochlorite for 3 min, at last washed with sterilized distilled water under the horizontal-laminar airflow clean bench. Two holes were punched as MDA, and then stuffed using potato dextrose agar medium with fully grown P. digitatum.
After treatments, each apple was placed into a sterilized plastic box (25 × 18× 12 cm) and incubated at 30℃ for 2, 4, 6, and 8 d before used for following behavioral assays. Considering to the fact that the apples infected by P. digitatum for 8 d or longer would become rotten in the following days, the apples infected for 6 d were, therefore, used in the later experiments.
Oviposition behavioral experiments
To test the effects of P. digitatum on the oviposition behavior of mated YPM females, four treatments (NIA, MDA, PDA, and PPD) were simultaneously offered in a wood-frame cage (35 cm × 27 cm × 25 cm) with plastic gauzes on side walls to allow the oviposition of mated YPM females according to the methods reported in Guo et al. (2022). Each experiment was replicated 20 times with a total number of 200 females. The egg numbers on each sheet were counted separately and the data were statistically treated on the basis of average number of eggs by 10 females.
Four-arm olfactometer experiments
A four-arm olfactometer was used to test behavioral responses of mated YPM females to the odors from NIA, MDA, PDA, and PPD according to the method described in Guo et al. (2022). The behavioral response was classified as a choice if the moth passed over 1/3 length of the arm associated with one of four odors and stayed there for more than 30 sec. Conversely, no-choice was assigned if the tested moth remained in the common arm for 3 min. A total of 211 individual moths were tested. The selection rate in the four-arm olfactometer experiment was defined as the number of females that made a selection for the odor divided by the total number of females that made a selection for any odors offered simultaneously.
VOCs collection and analysis
Five apples of each treatment (NIA, MDA, PDA) and correspondingly similar size of PPD that were placed into a 48.2 cm × 59.6 cm oven bag respectively (Reynolds Kitchens, Richmond, VA, USA) were used to collected VOCs according to dynamic headspace collection method reported by Guo et al. (2022). After collection, the trapped volatiles were eluted using chromatography-grade n-hexane (99.9%) and then were analyzed using an Agilent 6890 gas chromatograph (GC) coupled to an Agilent 5975 Mass Spectrometer (MS). The procedures for the GC-MS analysis were the same as described in Du et al. (2016) with the exceptions that the GC was equipped with a DB-5MS column (60 m × 0.25 mm × 0.15 µm, Agilent, USA) rather than a HP-5MS column (30 m × 0.25 mm × 0.25 μm) and the injector temperature was 250℃ other than 210℃. Following injection, the column temperature was maintained at 37℃ for 6 min, followed by an increase in temperature of 2℃/min to 70℃ for 5 min, and then an increase of 5℃/min up to 200℃, at last maintained at 200℃ for 5 min. Compounds were tentatively identified by comparing mass spectra with NIST Standard Reference Database 98 (Agilent Technologies, Palo Alto, CA, USA). Compounds were quantified by their total ion abundance relative to that of the internal standard (n-nonyl acetate).
Five specific VOCs from PDA, including methyl 2-methyl butyrate, styrene, methyl caproate, butyl caprylate, and n-tetradecane, were chosen for electroantennogram (EAG) measurements. Four concentrations (10-1, 10-2, 10-3 and 10-4 (v/V)) of five individual compounds were prepared. All chemicals (purities ≥ 95%) were purchased from commercial companies, which methyl 2-methyl butyrate and styrene were from J & K Chemical Ltd. (Shanghai, China), methyl caproate, butyl caprylate, and n-tetradecane from TCI Development Co., Ltd. (Shanghai, China). The test solutions were made by firstly dissolving 100 μL of compounds into 900 μL of laboratory-grade mineral oil, and then serially diluted with mineral oil to the desired concentrations. The test solutions were stored at -20℃ for further analyses.
EAG recordings were performed on 3- to 4-day-old mated YPM females that the moths at this stage were eggs-loading and sensitive to signals used for oviposition location (Belmain et al. 2002). The method of EAG recordings was the same as that described by Du et al. (2016). Stimulus was delivered and tested in increasing doses on the antennae of mated YPM females with mineral oil and n-hexanol being used as control and standard stimuli, respectively. EAG test was run for a variable number of replicates per day, and each compound at each concentration was tested on 15 antennae. In each test, the control and standard stimuli were applied subsequently after four successive stimulations. Normalization was achieved by dividing the peak EAG amplitude of the test puff with the average EAG amplitude of the two nearest standard stimulations after subtracting the amplitude recorded in response to the mineral oil.
Y-tube olfactometer experiments
The preference of mated YPM females to apples with or without five specific VOCs (methyl 2-methylbutyrate, styrene, methyl caproate, butyl caprylate, and n-tetradecane) from PDA was tested using Y-tube olfactometer. For Y-tube olfactometer assays, apples with and without five specific VOCs from PDA (the amount was referred to the concentrations in fungi-infected apple fruits (Table S1)) were separately placed into the chambers of the Y-tube. The test procedure was similar to that in our previous study (Guo et al. 2022). Each individual moth was used only once, and totally 80 mated female moths were tested for each treatment. The selection rate in the Y-tube olfactometer experiment was defined as the number of females that made a selection for apples with exogenous compounds divided by the total number of females that made a selection between apples with and without exogenous compounds.
Data obtained from oviposition selection experiments, behavioral assay in four-arm olfactometers, EAG tests, and the absolute content of host plant VOCs were subjected to analysis of variance (ANOVA) using Tukey-HSD test. The data of Y-tube olfactometer experiments were analyzed using non-parametric Chi-square analysis (Females with no choice were excluded from statistical analyses). Quantification of volatiles measured as the absolute content of each compound was analyzed using principal component analysis (PCA) using the software program SIMCA P+ 11.0 (Umetrics AB, Umeå, Sweden). All statistics except the PCA analysis were performed using the SPSS16.0 statistical software. Graphs were generated in the program of Graphpad Prism 9.0.