Relative C. biennis biomass and P content were significantly correlated (P ≤ 0.05) and were both greater in M+ than in M– plants (Fig. 1) independent of disturbance (Table 1). However, in the case of S. jacobaea, mycorrhizal inoculation and disturbance had no effect on relative biomass production or relative P acquisition (Table 1, Fig. 1). The results of harvest 1 (not shown) were not different from those of harvest 2.

The biomass and plant P content of both perennials, C. biennis and S. jacobaea, were increased by mycorrhizal inoculation (Table 2, Fig. 2), and P content was positively correlated with biomass (P ≤ 0.05). Disturbance significantly increased P content (non-disturbed pots 0.85 ± 0.17 mg, disturbed pots 1.35 ± 0.24 mg) and biomass (non-disturbed pots 0.30 ± 0.05 g, disturbed pots 0.48 ± 0.08 g) of S. jacobaea but had no effect on these parameters for C. biennis (Table 2). Plants grown in monocrop or multicrop did not affect plant biomass or P content of either S. jacobaea or C. biennis (Table 2). The results of harvest 1 (not shown) were not different from those of harvest 2.

No roots of M− plants contained AM fungal structures, while AM fungi were present in roots of all M+ plants (see Table S4). Although microscopic assessment of AM fungal abundance in roots was performed only in M− plants, a control sample of M+ plants intended for determination of AM fungal colonization determined plant roots were highly colonized by AM fungi (Table S3).

Plant growth in monocrop or multicrop did not significantly affect the abundance of any AM fungal taxa in roots of S. jacobaea or C. biennis (Table 3). Disturbance tended to decrease the abundances of R. irregularis and F. mosseae (C. claroideum was not present in roots of any plant of harvest 2) in the roots of C. biennis (Table 3, Fig. 3). However, disturbance had no effect on AM fungal abundances in roots of S. jacobaea (Table 3).

The abundances of individual AM fungal taxa were not significantly different between roots of weedy annual plant C. canadensis or its paired perennial plant S. jacobaea (Table 4, Fig. 4). The abundances of R. irregularis did not differ between weedy annual plant E. annuus roots and paired perennial C. biennis. However, E. annuus was colonized by lower abundances of F. mosseae, compared to paired C. biennis (Table 4, Fig. 4). Whereas disturbance equally increased the abundance of F. mosseae of both species, E. annuus and C. biennis,grown in competition (non-disturbed pots 30 ± 6 × 10⁵ CN mg⁻¹ roots, disturbed pots 45 ± 6 × 10⁵ CN mg⁻¹ roots) (Table 4). The results of harvest 1 (not shown) were not different from those of harvest 2.

C. canadensis is an important weedy annual, expanding throughout introduced, as well as native, areas. The rapid global expansion of C. canadensis has been attributed to a combination of factors. For example, effective reproduction and effective utilization of newly created habitats are likely mechanisms promoting C. canadensis expansion (e.g.,³¹). Despite having an annual life strategy, association with AM fungi has also been hypothesized as a mechanism for successful expansion of C. canadensis^32–34. In agreement with other research^35–38, C. canadensis formed associations with AM fungi in our current study. However, the lack of any negative effect of C. canadensis presence on performance of S. jacobaea in our study does not support the possibility that AM fungi could contribute to C. canadensis spread; the coexistence of these two plants was independent of AM symbiosis in our experiment. While AM symbiosis was beneficial for P-uptake and biomass production of S. jacobaea, the symbiosis did not increase the plant performance of S. jacobaea when grown with C. canadensis, presumably because AM fungi were beneficial for both plant species in our study. A previous study, contrary to our results, reported negative responses of perennial Anthemis cotula on neighbouring C. canadensis were mediated by AM fungi³⁶. Therefore, it should be cautioned that mycorrhizal effects between neighboring plants of different species is likely context-dependent, as our findings showed no influence from mycorrhiza in the competition of C. canadensis with the perennial plant S. jacobaea.

Erigeron annuus is another example of a weedy annual plant successfully increasing in abundance throughout Europe. The expansion of E. annuus has been credited to increased seed survival, rapid seedling and rosette development, and/or allelopathic activity³⁹. Because E. annuus readily forms mycorrhizal symbiosis^40–42, mechanisms allowing successful expansion of E. annuus could also include AM symbiosis. However, based on our results, we cannot confirm AM fungi as a mechanism used by this annual plant when in competition with the perennial C. biennis. This is partly supported by Rinauldo et al.⁴³ and Veiga et al.⁴⁴ who found weeds grown in community to be negatively influenced by AM fungi. In fact, in our current study, AM fungi increased the relative biomass and P content of perennial C. biennis when paired with E. annuus. As we hypothesized, the greater mycorrhizal responsiveness of perennial plants compared to annual plants⁴⁵ play a role in the higher responsiveness of perennial C. biennis on AM fungal inoculation, compared to annual E. annuus. Based on our results, we suggest the more responsive perennial C. biennis is more competitive in soils with a natural occurrence of AM fungi, when paired with E. annuus.

The mycorrhizal benefit of perennial C. biennis was nevertheless independent of the initial soil disturbance even though the disturbance decreased the abundance of AM fungi in their roots. Therefore, although C. biennis and E. annuus were associated with similar AM fungal taxa with the potential for resource sharing through intact CMNs in non-disturbed soil, this was not likely a major mechanism driving the positive effects of AM fungi or promoting greater competitive performance of C. biennis over the annual E. annuus. Alternatively, it is likely the disturbed CMNs recovered rapidly following disturbance. Rapid recovery of CMNs after disturbance would lead to similar results between disturbed and intact CMNs.

Both plant tissue P content and biomass production of perennial S. jacobaea increased following disturbance. However, the relative performance of S. jacobaea, when grown with co-occurring weedy annual plant C. canadensis, was not affected by disturbance, as the performance of C. canadensis increased to the same extent as S. jacobaea. Soil disturbance likely increased soil aeration and microbial activity, temporarily increasing P-availability and subsequent P supply to both co-occurring plants⁴⁶. Furthermore, mycorrhizal plants did not differ from nonmycorrhizal plants, in either undisturbed or disturbed soils, indicating host plant reliance on AM fungi for P-uptake was apparently not substantial under these conditions.

Although increases in nutrient availability as a result soil disturbance has been previously reported^46,47, in our study initial soil disturbance did not affect performance of nonmycorrhizal C. biennis; increased P availability caused by the disturbance was not reflected as increased P uptake by the plant. Because P availability for both AM inoculated plants and non-inoculated plants in both disturbed and undisturbed treatments was constant, increased P supply would be beneficial for all plants if P-availability was limited, and any increase in P uptake would not be considered luxurious. In fact, in our study, P did not appear to be limiting, as presence of weedy annual plant E. annuus did not affect the P content in C. biennis shoots. However, although the overall system was not P limiting, P could be limiting for individual plants when in competition with a neighboring plant more efficient at P-uptake, which likely occurred when perennial C. biennis acquired less P compared to corresponding perennial S. jacobaea when grown under the same conditions. Reduced P-uptake by C. biennis may be because another factor that we did not measure was more limiting for this species.

Disturbance did not influence the performance of perennial C. biennis or the co-occurring weedy annual plant E. annuus. Contrary to our hypothesis, based on the ruderal nature of weedy annual plants, the single soil disturbance did not play a role in the performance of weedy annual plant E. annuus when grown in competition with perennial plant C. biennis, or C. canadensis when grown in competition with perennial S. jacobaea. In a previous study, a single soil disturbance did not influence performance of Inula conyzae, a native plant species when grown in competition with Echinops sphaerocephalus, a non-native invasive plant species common in Eastern Europe²⁹, and the authors propose the lack of response to disturbance was likely due to a rapid recovery of CMNs following disturbance. We propose a similar rapid recovery of the CMNs occurred in our current study, and therefore, plants with intact CMNs were not at an advantage over plants with initially disturbed mycelium.

In our study, P was not limiting, and a single soil disturbance did not affect AM fungi or alter AM benefits for either the perennial or the weedy annual plants. This indicates CMNs following a single soil disturbance will likely recover quickly and, therefore, not support the performance and subsequent expansion of weedy plants species as we initially hypothesized. However, we caution that mycorrhizal fungi may be considerably more important under other soil disturbances, especially long-term disturbances such as drought, salinity, soil compaction, or nutritional stress^48,49.

We conducted a greenhouse experiment to study pairwise interactions between mycorrhizal weedy annual and perennial plants (weedy annual—perennial: Conyza canadensis—Senecio jacobaea; Erigeron annuus—Crepis biennis). Erigeron annuus (L.) Pers. and Conyza canadensis (L.) Cronq., are common annual weedy plant species of seminatural plant communities in Central Europe (Czechia). The corresponding perennial species, Crepis biennis L.; and Senecio jacobaea L., are from the same plant family (Asteraceae) as the weedy annual plant species and occur abundantly in E. annuus or C. canadensis inhabited plant communities. We imposed two mycorrhizal treatments: inoculated with mycorrhiza (M+) or not (M-), combined with a disturbance treatment: soil initially mechanically disturbed or not (leading to intact or disturbed CMNs in M+ pots). Biomass production of perennial plants in the presence of weedy annual plants were assessed for perennial plants in monocrops (perennial paired with perennial) or multicrops (perennial paired with weedy annual). Competition between the perennial and weedy annual plant was evaluated at two experimental harvest times [64 days (harvest 1) or 91 days (harvest 2)] after plant transplantation. For the experimental design see Fig. 5. Each treatment combination contained five replicate pots with a total of 160 pots set up at the beginning of the experiment (see Fig. 5). The positions of the pots in the greenhouse were randomized.

Similar to Řezáčová et al.²⁹, plants were grown in 2-L pots (11 × 11 × 20 cm, w × d × h) filled with a substrate comprised of 10:45:45 mixture of 10% γ-irradiated (> 25 kGy) Central European field soil from Litoměřice, Czechia (N50°31′54.53′′ E14°06′7.10′′), 45% autoclaved zeolite MPZ 1–25 from Zeopol (https://www.zeolity.cz, grain size 1–2.5 mm), and 45% autoclaved quartz sand (grain size < 3 mm). Physicochemical properties of the substrate are detailed in Řezáčová et al.⁵¹ or Table S1.

Mycorrhizal pots (M+) were inoculated with 36 g of AM fungal inoculum consisting of potting substrate containing root fragments of leek (Allium porrum L.), used as the host plant to develop pot cultures of AM fungal isolates, all widespread generalists of Central European origin. Inoculum in our current study contained the following AM fugal taxa: Claroideoglomus claroideum (N. C. Schenck& G. S. Sm.) C. Walker & Schuessler (2010) BEG 155, Funneliformis mosseae (T.H. Nicolson & Gerd.) C. Walker & Schuessler (2010) BEG 161 and Rhizophagus irregularis (N.C. Schenck & G.S. Sm.) C. Walker & Schuessler (2010) BEG 158 (https://www.i-beg.eu). These monospecific inocula were mixed as a volume ratio 1:1:1 (v:v:v) to create a synthetic community of widespread generalist AM fungi, resulting in 492 spores of. C. claroideum, 588 spores of F. mosseae and 612 spores of R. irregularis added to each pot. Nonmycorrhizal (M–) pots received 36 g non-mycorrhizal (mock) inoculum, which was added 4–5 cm below soil surface of each pot. The mock inoculum contained substrate and leek root fragments from cultures grown under the same conditions, for the same amount of time, as the M+ pot cultures (above), but without AM fungi, as assessed microscopically.

To promote the growth of CMNs in the M+ plots, we used Festuca pratensis Huds. as a nurse plant, which was also used in the M− pots to control for nutrient depletion (Fig. 5). Seeds of F. pratensis were directly sown into each pot in the middle of January. After 48 days, substrate (and CMNs in M+ pots) in half of both M+ and M− pots were mechanically disrupted as follows: a long metallic spatula was inserted to the full depth of the pot, and wide circular movements progressing from one corner of the pot to the other, disturbing the entire volume of the pot (Fig. 5). Microscopic assessment of soil samples (according to Newman⁵²) of 6 M+ determined hyphal length densities and these densities were similar to a previous experiment⁵³.

The seeds of all the experimental plants were field collected in the Czech Republic. Our study complies with relevant institutional, national, and international guidelines and legislation on handling plant material.

Seeds were germinated for two weeks at room temperature on wet filter paper contained in Petri dishes. One of the empty corners of the pot was planted with one perennial species, whereas the opposite corner was planted with perennial or weedy annual plant species (see Fig. 5). Note that sowing was conducted immediately after soil disturbance simulations. Plants in M+ treatments were planted into non-disturbed soil with intact CMNs, or into initially disturbed soil where CMNs needed to reestablish from spores and hyphal fragments. Thirty-six days following the initial disturbance, shoots of all nurse plants were harvested, dried, and weighed. Nurse plants did not re-grow after harvest and there were no significant differences between nurse plant biomass grown in multicrop or monocrop pots.

Annual and perennial plants were grown for an additional 28 days (harvest 1) or 55 days (harvest 2) (March–May) 2018 in a greenhouse at the Institute of Microbiology, Prague with average day and night temperatures of 24 °C and 20 °C, respectively. Day length was extended to 12 h using supplemental lighting. Temperature and daylength were selected to represent typical field conditions of Central Europe (March–May). Following nurse plant planting, each pot received 65 ml of Long Ashton mineral nutrient solution every week⁵⁴ with the P concentration reduced to 20% of the original recipe^45,55, to avoid constraint of AM fungal or plant growth due to P limitation.

The experiment was harvested twice to assess M+ annual and perennial plant production following initial disturbance. A harvest, plant shoots were cut at the hypocotyl–root interface and subsequently dried for 3 days at 65 °C to determine shoot dry weight. Substrate was thoroughly washed from roots, roots were weighed and cut into 1.5 cm fragments, and fragments were divided into three portions. One portion of the root fragments was immersed in 50% ethanol to determine AM root colonization. The second was stored in a freezer at −20 °C for molecular analyzes. The final portion was weighed, dried and reweighed, to determine root water content and root dry biomass.

Plant dry weight and tissue P content were assessed to evaluate benefits provided by AM fungi to each plant. P concentration in aboveground plant tissues was assessed as described in Řezáčová et al.⁵⁶. Orthophosphate concentration of the extracts was measured using the malachite green method⁵⁷. Shoot P content (hereafter referred as plant P content) was calculated from shoot nutrient concentration using total shoot dry biomass.

The composition of AM fungal communities in the roots was assessed using quantitative real-time PCR (qPCR) markers (mt5, moss, and clar) targeting sequence-specific motifs of Rhizophagus, Funneliformis, and Claroideoglomus, respectively, in the mitochondrial (Rhizophagus) or nuclear (Funneliformis and Claroideoglomus) large ribosomal subunit RNA genes as described previously⁵⁸.

Presence of AM fungal root structures was assessed microscopically in all M– pots, using one composite root sample for each M– pot (following McGonigle et al.⁵⁹) after staining the roots with trypan blue (⁶⁰; with minor modifications as in Řezáčová et al.⁵¹).

Analyses of variance (ANOVA) with P < 0.05 as the significance cutoff level were calculated in R 4.2.0 statistical environment (R Core Team, 2022, http://www.R-project.org/⁶¹) after data was assessed for conformity with ANOVA assumptions (i.e., normality and homogeneity of variances). The analyses were conducted independently for harvest 1 and 2. Data are presented as mean values and standard errors for each treatment combination.

To assess if AM fungal inoculation and soil disturbance altered the plant performance of perennial or co-occurring paired weedy annual plants, we conducted two-way ANOVAs with the following factors: mycorrhizal inoculation and soil disturbance on perennial plant share in multicrops. Measures of P and biomass estimate the perennial plant share as the fraction of total plant biomass or plant P content that is the contributed to the perennial plant in each multicrop^29,57.

To assess if plant growth in monocrop or multicrop, and disturbance, affect mycorrhizal shoot P content and biomass of the perennial plants, we used three-way ANOVAs with the following factors: mycorrhizal inoculation, soil disturbance and plant community composition (monocrop or multicrop). Plant community composition (monocrop or multicrop) was assessed to determine biomass and plant P content of each perennial plant species growing in monocrops or growing with the respective paired weedy annual plant.

To assess if the weedy annual plants and soil disturbance affect AM fungal abundances in the paired perennial plants, we performed two-way ANOVAs with the following factors: plant community composition (monocrop or multicrop) and soil disturbance on AM fungal taxon abundances of the perennial plant species growing in monocrops and multicrops. Additionally, to assess if the AM fungal taxon abundances differed between co-occurring perennial and weedy annual plants, and if this was affected by disturbance, we conducted two-way ANOVAs with the factors: plant species and soil disturbance on abundance of AM fungal taxa of each perennial plant species and paired weedy annual plants grown in multicrops.