Maize (Zea mays) is one of the main crops for human consumption and animal fodder in the world (Fig. S1a), and accounts for more than one-third of China’s cereal production (FAO 2016). We chose sweet maize single hybrid Kennian 1 in this study because it is a major cultivar in China’s commercial production, and our previous study has shown that this cultivar is colonized by B. bassiana (Sui et al. 2020).
Beauveria bassiana [BbHOSD1 (A3)] was isolated from a dead grub (Holotrichia oblita) at the Institute of Plant Protection, Jilin Academy of Agricultural Sciences in 2010 (Fig. S1b). The strain was deposited in the China General Microbiological Culture Collection Center (CGMCCC No. 19373). The fungus was cultured and grown on potato dextrose agar (PDA, Hopebio Spectrum Instruments Co., Ltd., Shanghai, China) for 15-20 days at 26 ± 0.7°C in the dark, and conidia were harvested by scraping with a sterile spatula, and then kept at 4°C in dark storage before use.
Asian corn borer (Ostrinia furnacalis, Fig. S1c) is one of the most serious insect pests of corn in China and causes ca. 30% yield losses (Wang et al. 2014). The eggs of O. furnacalis were obtained from maize stands in the field, and an O. furnacalis colony was established in the laboratory (air temperature 26.4 ± 1°C, 70-75% relative humidity, L16:D8) using artificial diet. Adults were used to oviposition trial, and larvae were utilized in survivability studies.
The B. bassiana inoculation experiment was conducted ca. May 2017. Maize plants were treated by one of two treatments: (1) maize inoculated with sterilized water containing 0.05% Tween-80 (Dingguo, Beijing, China) solution (Uninfected control), (2) maize inoculated with B. bassiana suspension containing 0.05% Tween-80 solution (Infected). To establish B. bassiana as an endophyte in Z. mays, we used two inoculation methods (Sui et al. 2020): seed immersing and soil drench inoculation. Seeds of maize were surface-sterilized (dipped in 70% ethanol 5 min and then immersed in 2% NaOCl for 3 min). Half of the sterilized maize seeds were immersed in B. bassiana conidial suspension (1 × 108 conidia ml-1 containing 0.05% Tween-80) for 12 hours, and then were sown 6 cm below the surface of 23 g autoclaved peat soil (Humin substrate, Fenghong Co., Jilin, China, 121°C for 2 h, 0.1MPa) in a plastic pot (35 cm in diameter and 45 cm in height). For the soil drench inoculations, 200 ml of a B. bassiana conidial suspension (1 × 108 conidia ml-1 containing 0.05% Tween-80) was applied on day 7, 12, 17, and 22 after sowing respectively to ensure colonization. For the control treatments, other half of the sterilized seeds were immersed of a sterile 0.05% Tween-80 solution and the soil was drenched with 200 ml of sterile 0.05% Tween-80 solution. Each treatment had 20 pots with three maize seeds per pot. The plants were grown in the greenhouse (air temperature 27.5 ± 0.8°C, and relative humidity 63.5 ± 14.2%) with 14L:10D light cycle.
Maize leaf colonization by B. bassiana was assessed by plating sterilized leaf segments on PDA 20 day after seedling emergence (Tefera and Vidal 2009). A portion of the fourth entirely fully developed leaflet from each plant was sampled, surface-sterilized by 100% ethanol, and then placed on sterile tissue paper in a laminar flow cabinet (Sui et al. 2019). Nine 1 cm2 leaf pieces of each leaf per plant were placed onto PDA, and incubated for 20 days at 26°C in the dark. Identification of B. bassiana outgrowth from the leaves was based on colony and conidial morphology (Fernandes et al. 2006, Fig. S1d), all plants from each treatment were tested. Colonization rates were calculated as follows: colonization rate (%) = (the number of B. bassiana colonized plants/total number of plants) × 100. In this study, we observed natural B. bassiana endophytism at a rate of 3.7% in uninfected maize, and the endophytism rate was 43.3 % in B. bassiana-inoculated treatments (Fig. S2). Thus, the colonized plants in the infected treatments were utilized for further experiments, and colonized plants in the uninfected treatment were removed. In total, each treatment had 20 pots with one plants per pot, with 10 pots for oviposition selection trials of O. furnacalis, and the other 10 pots for the collection of volatile compounds.
Oviposition selection of O. furnacalis for uninfected and infected maize
Oviposition preferences of O. furnacalis for host plants inoculated by B. bassiana were examined by two-choice tests (De Moraes et al. 2001, Rizvi et al. 2016). Ten mated O. furnacalis females were released into a pyramidal screen cages (120 cm × 60 cm × 120 cm), which contained two plants (one plant as an uninfected control and the other as an infected treatment) at the 7-8 plant leaf stage when O. furnacalis often oviposit their eggs in the field. Females were released into these cages at 19:00, considering the nocturnal activity of O. furnacalis. After 72 h, these females were removed from these experimental cages. The number of egg mass and eggs they have laid were counted. These oviposition cages were placed in the greenhouse with at an air temperature of 26.5 ± 0.8°C and relative humidity of 63.5 ± 14.2%. The position of each pot in each cage was randomly determined, and the distance between pots was 35 cm. The cages were separated from each other by at least 1.2 m. Plants and insects were used once, and 10 replicates (cages) were performed.
Collection and measurement of maize volatile
When conducting the oviposition experiment, we collected samples of volatile compounds from treated maize leaves using solid phase microextraction (SPME filed sampler 100-μm polydimethylsiloxane, Supelco [Sigma-Aldrich] Bellefonte, PA, USA), and analyzed and identified these compounds using gas chromatography linked to mass spectroscopy (Agilent 5975, Agilent Technologies, Madrid, Spain). Volatiles were collected using SPME techniques at ambient temperature (26.4 ± 0.6 °C). Three entire young leaves per plant were sampled because O. furnacalis preferred to lay eggs on these leaves (Zhu et al. Observation), and then were placed into a Teflon sampling bag with polyperfluoroethylene propylene (E-Switch, Du Pont Co, USA). We analyzed 23 samples in total (18 plants and 5 ambient controls). A detailed description of volatile collection and chemical analysis is available in Appendix S1. Relative amounts of each chemical compound from these tested plants in each replicate were calculated by the proportion of peak area of each chemical compound to total peak area of all chemical compounds, and then used for further analyses.
Electronantennogram responses of O. furnacalis to chemical compounds
The electronantennographic (EAG) responses of gravid O. furnacalis females were recorded using the EAG instrument with a data acquisition interface board (Type IDAC-02) and a universal single-ended probe (Type PRS-1) and related software (PC-EAG version 2.4) from Syntech (Hilversum, Netherlands). The solutions of tested chemical stimuli (with liquid paraffin as the solvent) at different concentrations (0.001, 0.01, and 0.1μg/μl), and the antennae of live gravid moths were used for the EAG recordings. Sixteen chemical compounds were used to examine the EAG responses of gravid O. furnacalis, and yet 1-penten-3-one, 3-carene, 1-penten-3-ol, azulene, and 2-ethyl furan were not included since commercial standards were not available (for a detailed list of compounds see Table S1). EAG values were recorded by using a standard method (Zhu et al., 2016). Solutions were applied (10 μl) to a filter paper strip (5 mm × 60 mm), and the solvent was allowed to evaporate for 30 s before the strip was placed inside a glass Pasteur pipette. Ten microliters of liquid paraffin was used as the control. Test stimulations were carried out by applying puffs of air for 2 s through a Pasteur pipette containing the filter paper as the stimulus. Puffs of the test stimuli were applied at 30 s intervals in randomized order of each chemical. Puffs with liquid paraffin were applied at the beginning and at the end of each tested compounds for monitoring relatively-stable baseline during the antennal preparation. These chemical compounds were tested from low to high dosage. Mated females (15-20 individuals) were tested for each compound and concentration. EAG responses were normalized with respect to the solvent control (Sun et al. 2014). To calculate the relative EAG values, the mean response to the sample minus the mean response to the control was divided by the mean response to the control.
Oviposition bioassays of O. furnacalis with chemical compounds
For O. furnacalis adult oviposition bioassays in the presence of individual volatile compounds, a transparent polythylene cage (15 cm × 8 cm × 8 cm) was used, modified from De Moraes et al. (2001) and Huang et al. (2009). The oviposition cage consisted of an oviposition container and a lid (17 cm × 10 cm). Two holes (3 cm in diameter) were cut in the lid, and two glass cuvette tubes (3 cm in diameter and 8 cm in height) containing filter paper with 10μl of either a test chemical or liquid paraffin (the solvent) covered the two holes. One hole received the tested compounds or distilled water, and the other the solvent (the control). The distance between the two holes was 5 cm. A piece of wax paper was adhered to the inside of the container because females will lay eggs on wax paper. The opening of the glass tubes were covered with wax paper that been perforated ten times with a needle to allow volatiles to pass through into the oviposition cage. Experiments were performed using mated females (temperature 26.0 ± 0.9°C, RH 71.3 ± 12.4％, photoperiod 14L:10D). Six compounds, 3-hexen-1-ol, 2-ethyl-1-hexanol, α-pinene, β-caryophyllene, naphthalene, and caproaldehyde at a concentration of 0.1 ug/ul for each chemical compound were used to examine oviposition bioassays of gravid O. furnacalis due to higher EAG responses of O. furnacalis for them (Table S1). Eggs laid on the wax paper were counted after 72 h. Mated females (18-26 individuals) were tested for each chemical compound. Oviposition stimulation index (OSI) was calculated using the following formula to determine whether the compounds repelled or attracted females to oviposit (Huang et al. 2009):
In this equation, T is the number of eggs on wax paper in the presence of the tested compound, and C is the number on wax paper of the control. The OSI ranges from -100 to 100, and when OSI = 0, this indicates that oviposition on the tested compound was equal to that of the control, when OSI < 0, this indicates that the tested compound acted as repellent, when OSI > 0, the chemical tested was an attractant.
Performance measurement of O. furnacalis
To evaluate the performance of O. furnacalis after they oviposit their eggs on uninfected and infected maize, we conducted a no-choice rearing experiment that mimicked the situation in which the larvae have no possibility of switching to another plant after hatching. The second-instar O. furnacalis larvae were placed into a container (35 cm × 20 cm × 15 cm in size) for rearing with fresh maize leaf and stalk and had participated in the oviposition selection experiment. The larvae were reared in full-sib groups of forty individuals in one container for one replicate, with ten replicates for each treatment, and were allowed to develop until pupation. Every two days, the treated maize food in these containers was replaced by fresh food, and the number of larvae, pupae, adults, and individual infected by B. bassiana were recorded.
Measurements of maize characteristics
Plant morphological variables including height of each maize plant, leaf length and leaf width for the third to fifth leaf in the middle stratum of each maize plant were measured (Duan et al. 2021). All ten maize plants per treatment were measured. The average of the three leaves of each plant was used for further analysis. Total nitrogen and total carbon of the maize was measured. Ten plants (including aboveground leaf and stalk) per treatment were collected, and were dried in an oven at 80°C for 48 h. The dried maize plants were then grounded in Willey mill equipped with a 1 mm mesh screen before chemical analyses. Five samples of 2 mg per plant were measured using an element analyzer (vario EL cube, ELEMENTAR). The average of five samples per plant and each replicate was further analyzed.
For the oviposition and performance data (number of egg masses, number of eggs, and number of surviving larvae, pupal, adults, and infected cadavers), we used generalized linear mixed models with a Poisson distribution and a log link function to examine the difference in oviposition selection and offspring fitness between uninfected and infected maize plants by B. bassiana. For B. bassiana inoculation, the amount of plant volatile emission data (inoculation rate, relative amount of each compound), and characteristics of maize plants, we used a generalized linear mixed models with Gaussian distribution and identity link function to test the inoculation rate of B. bassiana, and the effects of B. bassiana on the emission of plant volatiles. We used the nlme package for these above analyses. To examine the differences in EAG responses among the four concentrations for each compound, and the relationships between insect performance and chemical properties of maize we used linear mixed models (LMMs) with the lm-function of the vegan package. For post hoc analysis, we used Tukey’s post hoc tests with the mulcomp package. For the oviposition bioassay data (number of eggs), chi-squared goodness of fit test was used to examine oviposition preferences of O. furnacalis for distilled water, liquid paraffin, and chemical compounds. All analyses were carried out in R (version 3.6.0 × 64, 2019, The R Foundation for Statistical Computing Platform).