Winrhizo

Nine plant development parameters were measured on five six-leaves stage plants per plot (25 plants per treatment). The monitoring of root development was based on root dry biomass and root system architecture via the quantification of total root length, surface, and number, as well as average root diameter (WinRHIZO; Regent Instruments Inc., Québec City, Canada). The monitoring of shoot development was based on shoot dry biomass and stem diameter at the root collar, and on foliar morphology via the quantification of the length and average width of the fifth leaf from the shoot bottom (WinFOLIA; Regent Instruments Inc.). Data obtained with formulation F2 have been reported by [20 (link),21 (link)].

We conducted root architecture (root length) analysis on entire root systems of plants in the sterile treatment. Root architecture in the sterile treatment has been found to correlate with mycorrhizal response (Koziol & Bever, 2015 (link); Seifert et al., 2009 (link)); smaller root systems tend to be more responsive to mycorrhizal fungi, whereas larger ones tend to be less responsive. Root systems were quantified using WinRhizo (Regent Instruments Inc.).
We also inspected root colonization in a subset of study plants to confirm that our sterile controls did not have AMF colonization and that the AMF treatments showed AMF colonization. Root colonization was assayed using Trypan Blue staining and subsequent microscope inspection (Giovanetti & Mosse, 1980 ). Stained roots were mounted on slides and were analyzed using 25 vertical transects for colonization as well as for presence of arbuscules, vesicles, coils, and hyphae (Table S4).

The roots from six harvested plants from each condition and genotype were immersed in water and scanned using a flatbed scanner followed by analysis using WinRHIZO software (Regent Instruments, Canada) to determine total root length (TRL). Shoot lengths were determined at harvest by measuring above ground shoot length with a ruler. The aerial parts of the plants were separated and dried in an oven (75°C) for 48 h and weighed using a weighing scale. The chlorophyll content was measured as a spad value (SPAD-502Plus, United States). The remaining six plants of each treatment were harvested, and roots were immediately frozen in liquid nitrogen and stored in a − 80°C freezer until they were extracted metabolomics analyses.

Root system and root hair measurements were obtained at 4, 11, 17, 20, 23, and 28 DAT. Roots were scanned (Epson 10000XL) and root hairs were measured using a stereomicroscope (Leica, MZ12) inside the EcoFAB-N chambers (Fig. 1C–F). Depending on the developmental stage, root hairs were measured in a pre-decided region from the tip of the primary root, specifically 1–4 mm (4 DAT), 2–8 mm (11 DAT), 2–10 mm (17 DAT), and 4–15 mm (23 and 28 DAT). Later the images were analysed by ImageJ (version 2.0.0) and total root length, primary root length and root hair length were analysed through time. Lateral root length was calculated by subtracting the primary root length from the total root length.
Invasive root phenotyping was performed at 11, 20, and 28 DAT. Root fresh and dry weight were determined on a subsection of the available plants, listed in the respective figure legends. At 28 DAT, five roots from each group were scanned (Epson 10000XL) and binarized with WinRHIZO (Regent Instruments Inc., Canada), for determination of root length and comparison to non-invasive imaging.

Cucumber root systems were scanned at 300 dpi using a special scanner (Expression 4990, Epson, Long Beach, CA, United States). Root-related growth parameters (Table 1) were determined after analysis of scanned images with a computer image-analysis software (Win RHIZO, Regent Instruments, Inc., Canada) and ImageJ software (V1.50b) (Abràmoff et al., 2004 ).

Fifteen representative plants per plot were tagged to measure the total leaf area every 10 days, starting when the first treatment entered into silking. Individual leaf area was calculated as the product of leaf length and width multiplied by 0.75. Subsequently, GLA was estimated visually until the canopy of all the plants fully turned yellow (Lisanti et al., 2013 (link)). Three maize plants per plot were sampled to measure the dynamics of LRLD every 20 days, from the beginning of the first treatment that entered into silking until the last treatment reached maturity. The same root sampling method was used as previously described (Supplementary Figure 2). Functional live roots can be distinguished by staining red using 2,3,5-triphenyltetrazolium chloride (TTC). The detail procedure referred to Stūrīte et al. (2005) (link) is as follows: first, fresh roots were quickly incubated in breakers containing 0.6% (w/v) TTC, 0.06 M phosphate buffer, and 0.05% (v/v) Tween 20, at 24°C for 20 h; then, the roots were scanned with an Epson Perfection scanner, and the live root lengths were analyzed with Win RHIZO (Regent Instruments, Inc., Canada) pixel color classification method. LRLD was calculated by dividing the live root length by the sampling core volume for each of the soil layers.