Aboveground herbivory does not affect mycorrhiza-dependent nitrogen acquisition from soil but inhibits mycorrhizal network-mediated nitrogen interplant transfer in maize

Abstract

Arbuscular mycorrhizal fungi (AMF) are considered biofertilizers for sustainable agriculture due to their ability to facilitate plant uptake of important mineral elements, such as nitrogen (N). However, plant mycorrhiza-dependent N uptake and interplant transfer may be highly context-dependent, and whether it is affected by aboveground herbivory remains largely unknown. Here, we used ¹⁵N labeling and tracking to examine the effect of aboveground insect herbivory by Spodoptera frugiperda on mycorrhiza-dependent N uptake in maize (Zea mays L.). To minimize consumption differences and ¹⁵N loss due to insect chewing, insect herbivory was simulated by mechanical wounding and oral secretion of S. frugiperda larvae. Inoculation with Rhizophagus irregularis (Rir) significantly improved maize growth, and N/P uptake. The ¹⁵N labeling experiment showed that maize plants absorbed N from soils via the extraradical mycelium of mycorrhizal fungi and from neighboring plants transferred by common mycorrhizal networks (CMNs). Simulated aboveground leaf herbivory did not affect mycorrhiza-mediated N acquisition from soil. However, CMN-mediated N transfer from neighboring plants was blocked by leaf simulated herbivory. Our findings suggest that aboveground herbivory inhibits CMN-mediated N transfer between plants but does not affect N acquisition from soil solutions via extraradical mycorrhizal mycelium.

Keywords: Zea mays; common mycorrhizal networks; insect herbivory; mycorrhiza; nitrogen.

Figure 1

Mycorrhizal symbiosis promotes the growth and nutrient uptake of maize plants. Maize plants were inoculated with Rhizophagus irregularis (Rir) and cultivated in self-designed boxes (A). Five weeks post-Rir inoculation, maize plant roots were harvested for the measurement of mycorrhizal root colonization rate (B) and mycorrhizal observation by wheat germ agglutinin (WGA) fluorescence staining (C) (bar scale: 25 μm). Plant height (D), leaf area (E), and dry weight (F) were measured, followed by the analysis of phosphorus content (G), ZmPht1;6 (a mycorrhizal symbiosis marker gene in maize) expression (H), nitrogen content (I) and N transporter gene expressions (J–L). For analysis of root colonization, n = 6; plant height and leaf area, n = 20; dry weight, n = 10; nitrogen content, n = 5; phosphorus content, n = 8; expression of ZmPht1;6 and N transporter genes, n = 3. Data are means ± SE. Asterisks at the top of the bars indicate statistical significance (Student’s t-test, *P < 0.05; ***P < 0.001; ns, no significance). NM, non-mycorrhizal maize plants; AM, arbuscular mycorrhizal maize plants.

Figure 2

Mycorrhiza-mediated nitrogen uptake from soil by maize plants. Maize plants were inoculated with the mycorrhizal fungus Rhizophagus irregularis (Rir) and cultivated in the boxes with a PVC core in each box (A). Five weeks post Rir inoculation, the PVC core was injected with (¹⁵NH₄)₂SO₄ solution, and was then either rotated daily (for the purpose of damaging the mycelium) or non-rotated (intact for maintaining mycelial contact to labeling ¹⁵N). Plants were harvested to measure the root colonization rate (B) and the accumulation of shoot ¹⁵N (C) and root ¹⁵N (D) ten days after labeling N injection. For the analysis of root colonization, n = 6; ¹⁵N accumulation, n = 4. Data are the means ± SE of biological replicates. Asterisks indicate significant differences (Student’s t-test, *P < 0.05; ***P < 0.001; ns, no significance).

Figure 3

Mycorrhiza-mediated nitrogen uptake from soil by maize plants is independent of aboveground herbivory. Maize plants were inoculated with Rir and were cultivated in self-designed boxes, with a PVC core in each box as illustrated in experiment III. Five weeks post Rir inoculation, the PVC core was injected with (¹⁵NH₄)₂SO₄ solution, and was then either rotated daily (for the purpose of severing the mycelium) or nontreated (Intact) for 10 days, during which W+OS treatment was introduced (A). Plants were then harvested to measure the root colonization rate (B), shoot ¹⁵N (C), root ¹⁵N (D), and ZmAMT3;1 (a mycorrhizal inducible nitrogen transporter gene) expression (E). The ¹⁵N in Intact-’W+OS’ minus the ¹⁵N in Rotated-’W+OS’ (to counteract the effect of root acquisition of ¹⁵N) was present as a ‘Control’, and ¹⁵N in Intact+’W+OS’ minus the ¹⁵N in Rotated+’W+OS’ (to counteract the effect of herbivory on root acquisition of ¹⁵N) was present as ‘W+OS’ in (C) and (D). NM, non-mycorrhizal roots; AM, arbuscular mycorrhizal roots. For the analysis of root colonization, n = 6; ¹⁵N accumulation, n = 4; ZmAMT3;1 expression, n = 3. Data are the means ± SE of biological replicates. Asterisks indicate significant differences (Student’s t-test, *P < 0.05; ns, no significance).

Figure 4

Common mycorrhizal network-mediated N transfer between maize plants was affected by herbivory. Maize plants were inoculated with Rir and cultivated in self-designed mesocosms as demonstrated in experiment IV (A), RC, root compartment; CMNs, common mycorrhizal networks; ERM, extraradical mycorrhizal mycelia. The CMNs established between the two maize plants were either damaged daily or intact. Five weeks after Rir inoculation the mycorrhizal colonization rate in the roots was measured (B, E). Thereafter, 10 days after ¹⁵N labeling and W+OS treatment, ¹⁵N was detected in unlabeled plants (C, D) and ¹⁵N-labeled plants (F, G). Data are the means ± SE of the three (or six) biological replicates. Asterisks indicate significant differences (Student’s t-test, *P < 0.05; ***P < 0.001; ns, no significance).

Figure 5

Diagram of pathways for N uptake in maize plants. (A) Pathway for maize plant N uptake under no stress. (B) Diagram of the pathway for maize plant N uptake under herbivory. Myc-N, N uptake by mycorrhizal pathway from soil; root-N, N uptake by root from soil; transfer-N, N obtained from the CMN-connected neighboring plant; , N transferred from a CMN-connected neighboring plant was blocked.