Perfection v600 photo

After 3 days of treatment, plates were scanned with CCD flatbed scanners (EPSON Perfection V600 Photo, Seiko Epson, Nagano, Japan), and images used to quantify root parameters with FIJI, by using the tool ‘segmented line’ (Schindelin et al., 2012; Figure S2) as described in Ristova and Busch (2017). In particular, we quantified: primary root length on day 10 (P), growth rate of P after treatment (P2), branching zone or the length of P between the first and last visible lateral root (R), average lateral root length (LRL), and visible lateral root number (LR.No). Length was measured in millimeters. Other traits were calculated: lateral root density of P, LRD_P (LR.No/P), lateral root density of R, LRD_R (LR.No/R), total lateral root length, TLRL (LR.No*LRL), total root length, TRL (TLRL + P), and length ratio, LRR (TLRL/P). For detailed protocol, see Ristova and Busch (2017).

Plates with mycelia were scanned with an Epson scanner (Perfection V600 Photo, Epson, Germany), and the files imported into ImageJ (Schindelin et al., 2012 (link)). Mycelial coverage on each plate was delineated using a free-hand selection tool and measured with the built-in “Measure” function.
AMF colonization was determined by the “magnified intersections method” described in detail by McGonigle et al. (1990) (link). In brief, roots were cut in about 1 cm pieces and we counted the fungal structures of 150 intersections per sample after staining with Trypan Blue.
For determination of AMF colonization marker 11-carboxyblumenol, three leaf disks per AMF inoculated of the first and second stem leaf were harvested 6 and 8 weeks after AMF inoculation. 11-Carboxyblumenol levels were determined as markers of arbuscule colonization and quantified following the protocol of Wang et al. (2018a) (link).

To allow equal spacing of Petri dishes on the scanner bed, a black construction paper mask was created by cutting ∼ 2.5-cm circles about 1 cm apart from each other. The mask was accommodated on a flatbed scanner (Perfection V600 Photo, Epson, Suwa, Japan) and the Petri dishes were placed on top of the carved circles. Ninety-six-well plates were placed directly on the scanner bed. Scans were acquired at 600 dpi and by covering the scanner with a cardboard box. All images were saved in jpg format and processed in FireAlpaca v. 2.11.17 on a Windows 11 Home v. 23H2 computer, to replace the original background with a black background (Additional file 6: Fig. S4). The final resolution of the images after editing was maintained at 600 dpi. Image analysis was performed with Tomato Analyzer (TA) v. 4.0 on a Windows 8.1 with Bing computer. Tomato Analyzer calibration was done using a color checker (ColorChecker Passport Photo 2, Calibrite LLC, Wilmington, DE, USA) and setting illuminant D65, observer 10° of the CIELab color space. The Color Test function of TA (TACT) was used to obtain L*, a*, b*, hue, and Chroma of all samples. Parameters a* and b* were used to compute the a*/b* ratio.

We collected a total of 75 (50 male and 25 female) beetles from a large metapopulation near Butt Mountain, Virginia, in June 2019. We housed subjects individually in small, plastic containers (5 cm × 2.5 cm × 5 cm) under natural light conditions (14.5:9.5 hr light:dark cycle ± 17 min) and room temperature (22 ± 3°C) at the Mountain Lake Biological Station (Salt Pond Mountain). Containers included a small amount of mulch over a layer of plaster of Paris to help maintain a humid environment and mimic natural substrate. We added a small piece of Ganoderma tsugae (a polypore fungus) fruiting body as a food source and provisioned water ad libitum. Beetles remained in isolation in their containers for 12 days before trials began. All individuals were assigned a unique ID by adding colored dots to their elytra using nontoxic Testors^® Enamel paint. We recorded elytra length to the nearest 0.01 mm from images taken with a flatbed scanner (Epson Perfection V600 Photo) in ImageJ (Abramoff et al., 2004; Formica et al., 2012). Age and reproductive status are impossible to determine from wild‐caught B. cornutus, so we did not control for the age or experience of our individuals.