Studies to date on push-pull effects on the fall armyworm (FAW) embedded in the mechanisms according to the current state of knowledge of the system.

Volatiles (and potentially other traits) of the intercrop repel the herbivorous insect and additionally attract its parasitoids, while volatiles (and potentially other traits) of the border crop attract herbivores away from the main crop (Pickett et al., 2014; Khan et al., 2018; Eigenbrode et al., 2016). The pull effect of the border crop Napier grass, Pennisetum purpureum was observed in earlier systems, as it attracted stemborers (Khan et al., 1997b), but could not be confirmed for the FAW with the border crop Brachiaria cv Mulato II (Sobhy et al., 2022). For more detail on all studies, see supplementary table S1.

Heatmap comparing the log10-transformed peak area of the non-zero hits of substances present in at least 2/3 of the samples of at least one of the Desmodium species in the field, showing relative peak areas detected in field- or pot-grown (bioassay conditions) D. intortum and D. incanum, with pot-grown maize for comparison.

Peak intensity can only be meaningfully compared within the same substance. The x-axis displays the species and their growth conditions (field or pot) and the y-axis shows the target substances in alphabetical order. The color indicates mean of the log-10 transformed peak area of all samples with the substance present. The grey color indicates that a substance occurs in more than one, but less than 2/3 of the samples from the field. “Maize Infested” refers to maize plants that were exposed to FAW eggs, as moths were allowed to oviposit on the plants two nights prior to volatile sampling. Sample sizes: Ambient Control Field = 4, D. incanum Field = 14, D. incanum Pot = 3, D. intortum Field = 11, D. intortum Pot = 5, Maize infested Pot = 4, Maize Pot = 4. Figure 2—figure supplement 1. Per-sample heatmap

A: Principal component analysis plot based on the normalized peak areas of the 25 target substances. Sample sizes: Ambient Control Field = 4, D. incanum Field = 14, D. incanum Pot = 3, D. intortum Field = 11, D. intortum Pot = 5, Maize infested Pot = 4, Maize Pot = 4 B: Loading plot of the projection of the variables on the first two dimensions. The x-axis displays PC1 (Dimension 1) and the y-axis shows PC2 (Dimension 2). The color indicates the cos2, whereby dark and long arrows indicate a better representation of a loading on these first two dimensions.

Mass fragments of the unknown features detected by EI-MS in field D. intortum and / or D. incanum

Origin of the reference standards of all identified target substances present in field D. intortum and / or D. incanum. TMTT = (3E,7E)-4,8,12-Trimethyltrideca-1,3,7,11-tetraene, DMNT = (E)-4,8-Dimethylnona-1,3,7-triene

A: Setup of the control with two maize plants inside the cage. B: Setup of the treatments with direct contact with one maize and one Desmodium plant inside the cage. C: Setup of the treatments with indirect contact with two maize plants placed inside the cage and one Desmodium plant placed in proximity to one of the maize plants, but unreachable for the moths. D: The boxplots display the relative number of eggs (light grey boxes) or egg batches (dark grey boxes) per position (x axis) and treatment (superordinate boxes). The lower and upper hinges correspond to the 25th and 75th percentiles, while the whiskers extend to the largest and smallest non-outlier values, respectively. Outliers are values outside a window of 1.5 x the interquartile range and are plotted individually. Desmodium is abbreviated with ‘Des’ and treatment is abbreviated with ‘treat’. See Table 3 for a model-based statistical analysis and Figure 4—figure Supplement 1 for a breakdown of eggs per repetition and batch. Sample sizes (n, replicate units of a given treatment): Control = 21, D. incanum direct = 19, D. intortum direct = 20, D. incanum indirect = 19, D. intortum indirect = 20. Figure 4—figure supplement 1. Total egg count per repetition and per batch during oviposition bioassays Figure 4—figure supplement 2. Schemes and photographs of the experimental setup of the choice oviposition bioassays Figure 4—figure supplement 3. Scheme of the cage setup in greenhouse 1B Figure 4—figure supplement 4. Scheme of the cage setup in greenhouse 3B Figure 4—figure supplement 5. Photographs of the egg collection process

Mixed model applied for oviposition bioassays On Maize plant: Comparison of the laid eggs on the maize plants for all treatments with the following formula: RelEggNoMaize ∼ ContrvsTreat + IndvsDir + IncvsInt + Treatment + (1| Greenhouse) + (1| Group:CageNo) + (1| Start.Date) + (1| Rep) On Desmodium plant: Comparison of the laid eggs on the Desmodium plants for all treatments with the following formula: RelEggNoDesmodium ∼ ContrvsTreat + IndvsDir + IncvsInt + Treatment + (1| Greenhouse) + (1| Group:CageNo) + (1| Start.Date)+ (1| Rep) Descriptions of the terms: RelEggNoMaize & RelEggNoDesmodium = The dependent variable was determined by number of eggs laid on maize or Desmodium relative to the total number of eggs laid in one repetition. ContrvsTreat = All Desmodium treatments are compared with the control treatment IndvsDir = The treatments with direct and indirect contact with Desmodium plants are compared. IncvsInt = D. incanum and D. intortum are compared against each other. Treatment = All treatments are compared against each other to detect effects which are not related to terms used before. Greenhouse = Treatments were carried out equally in two different greenhouses. Group:CageNo = Twenty-five cages were repeatedly used, whereby each cage could be unambiguously identified with the group and cage number. Start.date = Treatments were carried out at five different start dates. Rep = All repetitions of the control were inserted twice with inversion of the values of the left and right maize plants. Any effects of the single repetitions are displayed in this term.

A: Line plot displaying the number of segment changes per minute until the experiment ended after 5 min. The line width indicates the number of overlaying lines and the colors indicate the moth activity level: ‘Settled after 2 min’ = No segment changes after 2 min. ‘Settled after 3 min’ = No segment changes after 3 min. ‘Little Activity’ = Moths showed max. 2 segment changes in the last 3 min of the experiment. ‘High Activity’ = Moths showed higher activity with at least 6 segment changes in the last 3 min of the experiment. The mean of all repetitions is represented by the black dashed line. B: Setup of the dual-choice assay for a Desmodium treatment comparing a maize plant (left) vs. a maize and a Desmodium plant (right). The moths are released through an opening in the center (at 0 cm) and observed for 5 min. C: Violin plot displaying the duration of stays in each segment in treatments with D. incanum and D. intortum (comparison of maize and maize + Desmodium) with the inclusion of the individual data points per repetition (black) and their mean (red): The x-axis displays the location in the dual-choice assay and the y-axis the total duration that each moth stayed in each segment. Below the x-axis, the last position of the moths that were considered settled (settled after <3 min or little activity) is displayed. See Table 4 for the statistical analysis and Figure 5—figure Supplement 1 for the set-up and segmentation of the dual-choice assay setup. Sample sizes (n, replicate units of a given treatment: Control = 18, D. incanum = 18, D. intortum = 19.) Figure 5—figure supplement 1. Photograph of the experimental setup of the dual-choice assay

Mixed model applied for dual-choice assay: Side of Maize: Comparison of the duration of stay in the compartment of the maize plants for all treatments with the following formula: (y_logitMaize ∼ (MaizevsDes + Treatment) + PosMaize + (1|Date)+(1|Rep) Side of Desmodium: Comparison of the duration of stay in the compartment of the Desmodium treatment for all treatments with the following formula: (y_logitDesmodium ∼ (MaizevsDes + Treatment) + PosMaize + (1|Date)+(1|Rep) Descriptions of the terms: y_logitMaize and y_logitDesmodium = The dependent variable was determined by the logit-transformed accumulated duration of the stays in minutes in the two compartments (10 cm −50 cm) closer to the maize plant or a maize plant combined with a Desmodium plant. ContrvsTreat = The D. incanum and D. intortum treatments are both compared with the control treatment. Treatment = The two Desmodium species are compared with each other. PosMaize = The position of the maize plant and the maize + Desmodium treatment were equally placed on the left or right side of the dual-choice assay. Rep = All repetitions of the control were inserted twice with inversion of the values of the left and right maize plants.

A: Setup of the no-choice assay for a Desmodium treatment. The moths are released through an opening in the right side (at 0 cm) and their movement observed for 5 min. The plant odor was released from the outlet at 100 cm, while the moths were allowed to move freely up to 115 cm. All repetitions that showed activity in the last two minutes were excluded, as they are considered to have made no clear decision. B: Violin plot with the inclusion of the individual data points per repetition displaying the distance flown in the first, initial movement and the final location that was considered the finally chosen position. The x-axis displays the treatment and the y-axis the total distance that a moth moved or flew. See Table 5 for the statistical analysis. The sample sizes corresponded to 19 - 20 for all samples, whereby all repetitions with activity in the last 2 min of the experiment were removed. Remaining sample sizes (n, replicate units of a given treatment): Control = 17, Maize = 17, Maize + D. incanum = 16, Maize + D. intortum = 17.

Mixed model applied for no-choice bioassays Initial Distance: Comparison of the distance flown in the first movement bigger than 5 cm with the following formula: InitialDistance ∼ ContrvsTreat + MaizevsDes + Treatment + (1|Date) +(1|RepNo) Landing Distance: Comparison of the landing distance of the moths that showed no movement for the last two minutes of the experiment with the following formula: LandingDistance ∼ ContrvsTreat + MaizevsDes + Treatment + (1|Date) + (1|RepNo) Descriptions of the terms: InitialDistance = Distance flown of a moth in the first movement bigger than 5 cm from the release point LandingDistance = Final landing distance of a moth after the last movement. All repetitions with movements in the last 2 min of the experiment were considered as not decisive and excluded from the statitics. ContrvsTreat = All treatments including plants are compared to a control treatment with only an empty bag. MaizevsDes = All Desmodium treatments (maize + Desmodium plant) are compared with the maize (alone) treatment Date = Date the experiment was conducted RepNo = Order in which the repetitions were measured. Treatment = All treatments are compared against each other to detect effects which are not related to terms used before.

Stacked barplot displaying the moth activity between 6 pm and 1 am.

Several pairs of moths were observed mating for several hours up until 1 pm. Explanation color code: Mating = Two moths mate, Plant = Moth sits on the plant and therefore might oviposit, Active = Moth actively move or flies, Inactive = Moth does not move and sits on the frame or the net, Unchanged = Moth does not move from the starting point, No Information = Moth could not found and therefore no information was obtained about its activity.

Heatmap comparing the log10-transformed peak area of all samples.

The x-axis displays the species and their growth conditions (field or pot) and the y-axis shows the target substances in alphabetical order. The color indicates mean of the log-10 transformed peak area, whereby transparency indicates that no peak was found.

A: Variance of total egg count per repetition. The x-axis shows the sum of all eggs found in all positions within one repetition. The grey points display the total egg count per repetition and the black diamonds show the mean value over all data points per treatment. B: Boxplot including individual data points of the egg batch size (egg number per batch) per location (x-axis) and per treatment (superordinate boxes). Desmodium is abbreviated with ‘Des’ and treatments is abbreviated with ‘treat’. Sample sizes: Control = 21, D. incanum direct = 19, D. intortum direct = 20, D. incanum indirect = 19, D. intortum indirect = 20.

Upper Line: Schematic drawing of the placement of the plants in the cages according to the treatments. The rectangle represents the cage, the plant in the beige pot represents maize and the plant in the red pot Desmodium. Lower Line: Photos of the experimental setup of three treatments: control (left), D. incanum direct (center), and D. intortum indirect (right).

Greenhouse 1B: Schematic graph of the cage setup in the greenhouse 1B.

This greenhouse provided space for 15 cages in total, whereby three replicates of each treatment could be repeated per cycle. The plants display the placement of the plants in the replicates of the second cycle from 23. – 26.05.2023. To minimize volatile interference of neighboring setups, the treatments were placed in groups next to each other (represented by letters) with a distance of at least one cage length in between, which corresponded to 60 – 100 cm. Due to space limitations, the cages of group H were placed slightly closer, with an approximate distance of 50 cm. The positions of the treatments were swapped between each cycle so that each treatment was conducted at each position once. In the treatments where Desmodium plants were placed outside the cages, the Desmodium plants were normally placed between the cages and additionally on the outside of the outermost cage of a group (as can be seen on the cages of G). However, as all treatments were photographed, in retrospect several misplaced plants were noticed (see cage K2). Therefore, it must be considered that only conclusions about the influence of Desmodium volatiles in proximity versus those at a distance greater than 0.5 m can be made and any volatiles reaching longer distances might have affected neighboring replications. Light grey squares = tables, dark grey squares = cages, yellow circle = maize plants, green circle = Desmodium plants; The red circle indicated the situation where a plant was misplaced and therefore was closer to the neighboring repetition.

Greenhouse 3B: Schematic graph of the cage setup in the greenhouse 3B.

This greenhouse consisted of 10 cages in total, whereby two replicates of each treatment could be repeated per cycle. The plants display the placement of the plants in the cycle of 23. – 26.05.2023. To minimize volatile interference of neighboring setups, the treatments were placed in groups next to each other (represented by letters) with the distance of at least one cage length in between, which corresponded to 60 – 100 cm. Due to space limitations on two tables, the cages of group E were placed slightly closer with an approximate distance of 50 cm. The positions of the treatments were swapped between each cycle, with all treatments being conducted in 4/5 positions. In the treatments where Desmodium plants were placed outside the cages, the Desmodium plants were normally positioned either between the cages or on the outer sides. However, as all treatments were photographed, in retrospect several misplaced plants were noticed (see Figure 4—figure Supplement 3 Figure of Greenhouse 1B, cage K2). Therefore, it must be considered that only conclusions about the influence of Desmodium volatiles in proximity versus those at a distance greater than 0.5 m can be made and any volatiles reaching longer distances might have affected neighboring replications. Light grey squares = tables, dark grey squares = cages, yellow circle = maize plants; green circle = Desmodium plants.

Fall armyworm eggs are normally laid staggered in clusters and with hair-like scales on the surface, which complicates accurate egg counting (see picture A).

Therefore, eggs were collected with sticky tape to separate the layers and spread all eggs out in one dimension (B). The egg batches were taped to white paper, taking care not to squash eggs, and photographed using a UV imaging system (Syngene, Cambridge, England) against UV light (312 nm) coming from underneath the paper (C). A script for semiautomatic counts in ImageJ (Version 1.54f, National Institutes of Health, USA) was developed and used for counting the exact number of eggs per picture (D).

Experimental setup of the dual-choice assay with a D. incanum treatment on the left and maize on the right.

The photograph was taken in the light, while the experiments were conducted in the dark, using only red light. Yellow marks: Air exhaust at the centre of the dual-choice assay covered with a fine-meshed net. Red marks: Two red light bulbs placed symmetrical approx. 20 cm above the upper rim of the dual-choice assay. White marks: Dual-choice assay body (30 x 30 x 100 cm) and conceptual separation of the length in five segments of 20 cm. Green marks: Air transfer from the top of the plant bags via polytetrafluoroethylene (PTFE) tubing to the dual-choice assay. Blue marks: Air inlet for charcoal-filtered air provided by volatile collection kits via PTFE tubing.