Study organisms

Trichoderma harzianum T22 (recently re-classified as Trichoderma afroharzianum [51]; for consistency with previous studies further referred to as T. harzianum) is the active ingredient in several biopesticides and biofertilizers such as Trianum-P (Koppert Biological Systems, The Netherlands) from which it was isolated [52, 53]. Beauveria bassiana ARSEF 3097 was acquired from the Agricultural Research Service Collection of Entomopathogenic Fungal Cultures (ARSEF; New York, USA), and represents the active ingredient in several commercially available bioinsecticides such as Naturalis^® (Intrachem, Italy). The strain can colonize diverse plant species endophytically following artificial inoculation, including sweet pepper, besides its direct entomopathogenic capability [25, 54]. The fungal strains were preserved at -80°C in 35% glycerol on potato dextrose agar (PDA) plugs, until used in the experiments. For all experiments, sweet pepper (Capsicum annuum L.) cv ‘IDS RZ F1’ (Rijk Zwaan, The Netherlands) was used. Plants were grown in a 3:1 mixture of potting mix (Universal potting mix; Agrofino, Belgium) and perlite under controlled conditions in a climate cabinet (MD1400, Snijders Labs, The Netherlands) at 23 ± 1˚C, 65 ± 2% RH and a 16L:8D photoperiod, with white LED lights to provide a photosynthetic flux density of 790 μmol photons m^-2 s^-1.

Nezara viridula was reared in insect cages (47.5 × 47.5 × 47.5 cm) (BugDorm, MegaView Science Co. Ltd., Taiwan) under controlled conditions (ECL02, Snijders Labs, The Netherlands) at 25 ± 1°C, 70 ± 2% RH and a 16L:8D photoperiod. The population was established from individuals obtained from the University of Palermo [10], to which yearly newly field-caught specimens from Flanders (Belgium) were introduced. Stink bugs were fed with seasonal organic vegetables (such as tomatoes, cabbage and beans) and organic seeds (sunflower, soybean and peanut). A wet cotton roll served as a water source, while sweet pepper plants along with paper towels were provided as oviposition substrate. Food and water were replaced every two to three days, and newly laid eggs were systematically collected by carefully removing the egg masses from the paper towels, cage mesh or sweet pepper leaves. Both the eggs and N. viridula nymphs that emerged from the collected eggs were kept under the same conditions as described above, while adults were utilized for the continuation of the rearing and the experiments.

Trissolcus basalis individuals were kept in 50 mL polypropylene vials with a cotton plug (VWR International, USA) under the same conditions as described for N. viridula. Every two days a few drops of honey water (80:20 v/v) were provided on the cotton plug as a food source. To maintain the parasitoid colony, N. viridula egg masses were individually exposed to two mated T. basalis females. Parasitized egg masses were then placed in new vials until the emergence of the wasps. Twenty-four hours prior to the experiments, female T. basalis that were 5–7 days old were individually transferred into small 6 mL glass vials sealed with a cotton plug, and incubated as described above (supplemented with a drop of honey water on a piece of parafilm). Subsequently, parasitoids were transferred to the bioassay room and allowed to acclimate for approximately 1 h before the tests. To avoid bias from parasitoids reared on different egg masses, individuals from different egg masses were randomized over the different treatments.

Fungal inoculation and treatments

The preparation of fungal spore suspensions and subsequent plant inoculation were performed as outlined previously [6, 25]. In short, stored agar plugs from the fungi were plated on PDA (Oxoid Holdings Ltd., United Kingdom) (T. harzianum T22) or Sabouraud dextrose agar medium supplemented with 0.25% yeast extract (SDAY) (Oxoid Holdings Ltd., United Kingdom) (B. bassiana ARSEF 3097), and incubated at 25°C for seven days. Next, sterile physiological saline solution (0.8% NaCl) was added to the plates, and the spores were carefully scraped off to prepare the fungal spore suspensions. To eliminate mycelial fragments, the suspensions were filtered through a microcloth (Mira Cloth, Merck, USA) and washed three times with physiological saline solution. After determining the concentration of conidia using a Bürker hemocytometer, the spore suspensions were diluted to a final concentration of 1 × 10⁷ conidia mL^-1. Before conducting the experiments, conidial viability was checked by plating an aliquot of 100 μL of 1 × 10³ conidia mL^-1 on three agar plates and counting the numbers of germinated and ungerminated conidia under the microscope after incubation at 25°C for 24 h. Spores were considered germinated when the germ tube was at least two times longer than the spore diameter. The germination tests demonstrated > 90% viability rate for all conidial suspensions used. Plant inoculation was performed when the plants had reached the first true leaf stage (BBCH stage 101). Therefore, after rinsing the seedling roots with tap water, the roots were submerged for 18 h in 10 mL of either the conidial spore suspension (Bb, B. bassiana; Th, T. harzianum) or physiological saline solution to obtain non-inoculated, control plants (Co). Subsequently, the seedlings were transplanted in 11 cm diameter plastic pots filled with the same potting mixture as mentioned above. Plants were then put in controlled conditions as mentioned before, ensuring that plants subjected to different treatments could not make any contact with each other.

To perform the experiments, four weeks after inoculation, fungus-inoculated and non-inoculated plants (ca. six weeks old) were individually exposed to two gravid N. viridula females or were left untouched. Female stink bugs were allowed to feed only (F) or feed and oviposit, hereafter simply referred to as oviposit (O). Stinkbugs are known to feed on plant tissues while laying eggs [55]. Hence, this resulted in three treatments: (1) feeding, (2) oviposition, and (3) no infestation. For the first two treatments, plants were exposed to stink bugs in cages (30 × 30 × 30 cm) under controlled conditions (23 ± 1˚C, 65 ± 2% RH and a 16L:8D photoperiod), while the uninfested plants were incubated in cages under the same conditions. In both the feeding and oviposition treatments stink bug feeding was confirmed by visual observation, with females consistently observed inserting their stylets into leaf tissue. Plants were used in the experiments 24 h after exposure to feeding or within 24 h after discovery of an egg mass (inspected daily). On average, egg masses contained approximately 80 eggs.

Two-choice olfactometer bioassays

The olfactory responses of T. basalis were assessed in a two-choice Y-tube olfactometer [56] made from a polycarbonate body (stem 9 cm; arms 8 cm at a 130° angle; internal diameter 1.5 cm) sandwiched between two glass plates. Charcoal-filtered air at a flow rate of 0.8 L min^-1 was pumped into each of the olfactometer arms by passing a glass bottle (diameter 12 cm; height 52 cm) containing a test plant as odour source. Plants were carefully placed in the glass bottles after wrapping the pot and soil with aluminium foil to limit belowground VOCs from moving into the headspace and impact parasitoid behaviour. The Y-tube olfactometer was positioned at an incline of 20° and homogeneously illuminated from above by four 24 W T5 TL-fluorescent tubes (16 × 549 mm, 1350 lumen, 5500 K) to stimulate movement of the insects towards the bifurcation. The table was closed off by white curtains, to exclude any visual bias from the surroundings [56]. A single female wasp was introduced at the entrance of the Y-tube and allowed to walk freely in the olfactometer. Next, the olfactory behaviour of the introduced wasps was recorded for ten minutes using a camera (Logitech C920), and the recorded videos were analysed by CowLog 3 software [57]. Wasp responses were recorded for 10 min and assessed in terms of residence time, i.e., the time spent by the wasps in each arm after crossing a virtual line, defined at 5 mm distal to the bifurcation of the Y-tube olfactometer. In the Y-tube olfactometer, females of T. basalis have the tendency to explore first the entire area and then spend more time in the arm with the most attractive odour. Thus, olfactory responses for this species are better predicted by analysing residence time data rather than first-choice (first arm chosen) data as often done for other parasitoids [55, 58, 59]. Each bioassay was replicated using five pairs of plants, and ten female wasps per pairwise combination were tested (in total 50 wasps). Each wasp was only tested once. The position of the plants was switched after testing five wasps in order to identify any unforeseen bias in the setup. In total, six pairwise combinations were tested, as presented in Table 1. At the end of the assay, all polycarbonate olfactometer parts were rinsed with fragrance-free laboratory detergent and deionized water, followed by air-drying. The glass components were rinsed with deionized water and acetone, after which they were incubated at 175°C overnight. All bioassays were conducted at 25 ± 2°C, 65 ± 5% RH, and were performed between 09:00 and 18:00. Experiments were set up randomized over several days.

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Table 1. Overview of the pairwise combinations tested in Y-tube olfactometer assays.

https://doi.org/10.1371/journal.pone.0304220.t001

Dynamic headspace sampling and analysis of volatile organic compounds

For each treatment, volatiles were collected by dynamic headspace sampling to assess the VOC composition of the aboveground plant parts according to the procedures described in Wilberts et al. [25] with a few modifications. Volatiles were sampled from a different set of plants (n = 9–10) of the same age and treated in a manner identical to those that were tested in the Y-tube olfactometer bioassays. Plants were placed individually in a glass dome with a height of 20 cm and a diameter of 23 cm. The dome was sealed using aluminium plates around the stem without constricting the plants. To avoid external VOCs from entering the system, charcoal-filtered air was pumped in at a rate of 300 mL min^-1, while simultaneously air was drawn out at a rate of 200 mL min^-1 through a stainless steel tube (89 mm length; 6.4 mm outer diameter; 5 mm inner diameter) filled with 200 mg Tenax TA adsorbent (20/35 mesh; 165 CAMSCO, Houston, TX, USA), so that positive pressure was maintained within the dome. Collections were carried out under controlled laboratory conditions (23 ± 2°C; 65 ± 5% RH) for a period of 3 h (lights on), after acclimatization of the plants to the room and the glass domes for 30 min. Between subsequent volatile collections, the glass domes were rinsed with water and acetone and incubated at 175°C for 2 h. After VOC collection, the above-ground parts of the plants were cut and weighed for normalization of the VOC data (see below). Background VOCs from empty glass domes were collected at regular time intervals.

Desorption of volatiles from the Tenax TA, as well as separation and detection of volatiles, was carried out using a Thermal Desorber TD100-xr (Markes International Ltd., UK) connected to a 7890B gas chromatograph (GC) coupled to quadrupole-time-of-flight mass spectrometer (Q-ToF) (both from Agilent Technologies, USA). Volatiles released from the adsorbent at 250°C for 10 min under a helium flow of 30 mL min^-1 were simultaneously re-collected in an electronically-cooled sorbent trap (Markes International Ltd., UK) at 0°C. Once the desorption and re-collection process was completed, volatile compounds were released from the cold trap by ballistic heating at 40°C s^-1 to 280°C, which was then kept for 5 min, while volatiles were transferred to a 30 m length × 0.25 mm inner diameter × 1 μm film thickness DB-5MS analytical column (Phenomenex, USA), placed inside the oven of a GC (Agilent Technologies, USA) for further separation. The GC oven temperature was initially held at 40°C for 2 min and was raised at 10°C min^-1 to 100°C, where it was held for 1 min. The temperature was then raised at 5°C min^-1 to 140°C and was then immediately raised at 10°C min^-1 to a final temperature of 280°C, where it was kept for 1 min under a constant helium flow of 1.2 mL min^-1. Column effluents were ionized by electron impact ionization at 70 eV and detected with an accurate mass Q-ToF MS (Agilent Technologies, USA), acquiring mass spectra from 35–400 m/z at an acquisition rate of 5 spectra s^-1. The transfer line and ion source of the Q-ToF MS were set at 280 and 230°C, respectively.

Chromatograms recorded for the presence of plant volatile compounds using MassHunter deconvolution software (Agilent Technologies, USA) were converted to Xcalibur data through a two-step raw data conversion program available in MetAlign software [60]. Automated baseline correction, peak selection (S/N > 3), and alignments of all extracted mass signals of the raw data were processed following an untargeted metabolomic workflow using MetAlign software, producing detailed information on the abundance (peak height) of the mass signals representing the available metabolites [60]. Subsequently, the extracted mass features were reconstructed into potential compounds using the MSClust software through data reduction employing unsupervised clustering and extraction of putative metabolite mass spectra [61]. Chromatograms were visually inspected to ensure that true peaks were selected and that no peaks were missing. Tentative identification of volatile metabolites was based on a comparison of the reconstructed mass spectra with those in the NIST 2014 and Wageningen Mass Spectral Database of Natural Products MS libraries, as well as experimentally obtained linear retention indices (LRIs).

Fitness-related traits of Trissolcus basalis

In order to assess effects of fungal inoculation on fitness-related traits of T. basalis, we measured the proportion of parasitized eggs within a single egg mass, laid on fungus treated and non-inoculated plants. Additionally, the size (i.e. right hind tibia length) of female wasps that successfully emerged from these egg masses was measured. In insects, and especially in hymenopteran parasitoids, adult body size is known to be positively associated with fitness and is commonly used to assess parasitoid performance [62]. Within 24 h after N. viridula oviposition, one T. basalis female was introduced in a fine mesh bag enclosing the leaf containing the egg mass. After 24 h, the female parasitoid was removed, and the parasitized egg masses were left on the leaf until right before parasitoid emergence. Then, they were carefully removed from the leaf and were placed into 50 mL plastic vials until the wasps’ emergence. The proportion of the egg mass that was parasitized was recorded, and freshly emerged wasps were killed in 96% ethanol and kept in phosphate-buffered saline (PBS) until further use. Measurements were conducted using a stereoscope (Zeiss SteREO Discovery.V12) with AxioVision SE64 Rel. 4.9.1 software for image acquisition and analysis. A total of ten randomly selected female wasps from five egg masses were included in the experiment for each of the three treatments.

Statistical analysis

Behavioural data were analysed by linear mixed models (LMMs) with fungal treatment as a fixed factor and each plant pair (with the parasitoid individual nested within the plant pair) as a random factor, to account for pseudoreplication [56]. For each pairwise combination, a separate LMM was used. Significance of the fixed term in the model was determined using likelihood ratio tests (LRTs), whereas model fit was assessed with residual plots [63]. A similar LMM test followed by Tukey’s multiple comparison test was also used to analyse the effect of fungal inoculation on the size of wasps. Prior to statistical analysis of the volatile emissions data, the peak height of each compound was normalized by dividing by the biomass (aboveground fresh weight (g)) of the plant used during VOC collection to account for plant-to-plant variation within treatment groups. To assess whether overall VOC emissions differed between treatments, a permutational multivariate analysis of variance (perMANOVA) was performed on the log-transformed values, based on 1000 permutations, using the adonis2 function of the vegan package [64]. Further, a heatmap of calculated Z-scores was constructed using the heatmap.2 function from the gplots package [65] to visualize differences in the VOC profiles among treatments [65]. Z-scores were calculated for the average of the log-transformed data per treatment, by subtracting the average value of all treatments for the respective compound from the average value of that treatment and dividing by the respective standard deviation. Subsequently, the data were analysed in the same pairwise comparisons as those made in the behavioural assays to explain potential differences in wasp behaviour. Therefore, VOC emissions were visualized by non-metric multidimensional scaling (NMDS) using the Bray-Curtis dissimilarity measure, in the vegan package in R [64]. Additionally, a perMANOVA was performed as described above to assess differences in overall VOC profiles among treatments. Furthermore, for each tested pair of treatments, differences in the peak heights of individual volatile compounds were analysed, after checking assumptions of normality, using a Student’s t-test or Wilcoxon rank-sum test (for compounds with a normal or non-normal distribution, respectively) on log-transformed values. To correct for multiple testing, the raw P values were adjusted using the Benjamini-Hochberg correction, to control the false discovery rate at 5% [66]. A significance level of α = 0.05 was used to determine significant differences, and results were visualized using the ggplot2 package. All analyses and visualization of the data were performed in R version 3.6.3 [67].

Two-choice olfactometer bioassays

In two-choice Y-tube olfactometer bioassays, females of T. basalis significantly preferred sweet pepper volatiles induced by N. viridula oviposition over volatiles from non-infested control plants (Co versus Co_O: P = 0.009) (Fig 1). More specifically, the wasps spent 60.5% more time in the olfactometer arm containing the oviposition-induced plant volatiles. Yet wasps did not show any significant response when given a choice between non-infested control plants and plants subjected to N. viridula feeding only (Co versus Co_F: P = 0.868). Likewise, wasps did not discriminate between volatiles from non-inoculated and fungus-inoculated plants that were subjected to feeding, regardless of the identity of the fungal strain (Co_F versus Bb_F: P = 0.158; Co_F versus Th_F: P = 0.440). By contrast, T. harzianum significantly increased the wasps’ preference to volatiles induced by egg deposition compared to the volatiles emitted by non-inoculated plants that were subjected to egg deposition, with wasps spending 38.4% more time in the olfactometer arm from the fungus-inoculated plant (Co_O versus Th_O: P = 0.019). When eggs were deposited on plants that were inoculated with B. bassiana, wasps preferred the odour from non-inoculated plants (Co_O versus Bb_O: P = 0.009). In this case, their residence time in the olfactometer arm containing plant volatiles from B. bassiana-inoculated plants was 36.2% higher compared to the control arm (Fig 1).

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Fig 1. Olfactory behaviour of Trissolcus basalis females in a Y-tube olfactometer when given the choice between odour sources coming from differently treated sweet pepper plants.

Plants were uninfested or subjected to Nezara viridula feeding or oviposition, and inoculated with Beauveria bassiana ARSEF 3097 (blue) or Trichoderma harzianum T22 (red) or mock-inoculated with physiological saline solution (green). Bars represent the mean (mean ± SE) time spent by the female wasps (n = 50) in each olfactometer arm over an observation period of 600 s. P values in bold indicate significant differences in the residence times (P ≤ 0.05), when compared to a 50:50 distribution (linear mixed model).

https://doi.org/10.1371/journal.pone.0304220.g001

Volatile composition

Volatile analysis revealed a total of 75 compounds in the headspace of sweet pepper plants subjected to the different treatments (S1 Table and S1 Fig). These compounds belonged to the class of terpenoids (52), nitrogen-containing compounds (3), fatty acid derivatives (7), and benzenoids or phenylpropanoids (13) (S1 Table). PerMANOVA did not indicate any statistical differences in VOC composition between the different treatments when considering all data together (F_6,59 = 1.462, P = 0.055). When focusing on the pairs of treatments examined in the olfactometer bioassays, the NMDS ordination plots revealed no clear separation in the VOC composition among the compared treatments (Fig 2). Likewise, perMANOVA did not reveal any significant differences in the VOC profiles among the treatments compared (Co versus Co_O: F_1,16 = 1.101, P = 0.307, Fig 2A; Co versus Co_F: F_1,16 = 1.521, P = 0.159, Fig 2B; Co_F versus Bb_F: F_1,16 = 1.264, P = 0.264, Fig 2C; Co_F versus Th_F: F_1,16 = 0.936, P = 0.414, Fig 2D; Co_O versus Bb_O: F_1,16 = 0.650, P = 0.691, Fig 2E; Co_O versus Th_O: F_1,16 = 0.567, P = 0.750, Fig 2F). When looking at the individual detected VOCs, no significant differences were found after correcting for multiple hypothesis testing (S2 Table).

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Fig 2. Non-metric multidimensional scaling (NMDS) ordination plots based on Bray-Curtis dissimilarities of plant VOCs emitted by differently treated sweet pepper plants.

Plants inoculated with Beauveria bassiana ARSEF 3097 (blue) or Trichoderma harzianum T22 (red), or mock-inoculated with physiological saline solution (green), were uninfested (square) or subjected to Nezara viridula feeding (triangle) or oviposition (circle). Each dot represents an individual sample (n = 9–10 per treatment). The star represents the group centroid. The circled numbers correspond to the pairwise comparisons in the Y-tube olfactometer assays (Table 1): (a) Co versus Co_O, (b) Co versus Co_F, (c) Co_F versus Bb_F, (d) Co_F versus Th_F, (e) Co_O versus Bb_O, and (f) Co_O versus Th_O.

https://doi.org/10.1371/journal.pone.0304220.g002

Fitness-related traits of Trissolcus basalis

Fungal inoculation did not affect the size of T. basalis parasitoid females that emerged from the egg masses deposited on inoculated sweet pepper plants (χ² = 1.40, df = 2, P = 0.495; Fig 3). Overall, hind tibia lengths of wasps were similar across the treatments (B. bassiana-inoculated plants = 377.17 ± 4.41 μm; T. harzianum-inoculated plants = 389.98 ± 3.22 μm; non-inoculated control plants = 381.38 ± 2.99 μm). Likewise, the proportion of the egg mass that was parasitized did not differ between the treatments (χ ² = 0.25, df = 2, P = 0.882; S2 Fig) (B. bassiana-inoculated plants = 93 ± 5.7%; T. harzianum-inoculated plants = 97 ± 1.6%; non-inoculated control plants = 94 ± 4.8%).

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Fig 3. Performance of Trissolcus basalis parasitoid individuals (determined by hind tibia length (μm)), emerged from Nezara viridula egg masses deposited on plants inoculated with Beauveria bassiana ARSEF 3097 (blue) or Trichoderma harzianum T22 (red), or mock-inoculated with physiological saline solution (green).

The lower, middle and upper lines of the boxplots correspond to the first quartile, median and third quartile, respectively, while the diamond represents the average.

https://doi.org/10.1371/journal.pone.0304220.g003