The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield

Abstract

Disease resistance strategies are powerful approaches to sustainable agriculture because they reduce chemical input into the environment. Recently, Piriformospora indica, a plant-root-colonizing basidiomycete fungus, has been discovered in the Indian Thar desert and was shown to provide strong growth-promoting activity during its symbiosis with a broad spectrum of plants. Here, we report on the potential of P. indica to induce resistance to fungal diseases and tolerance to salt stress in the monocotyledonous plant barley. The beneficial effect on the defense status is detected in distal leaves, demonstrating a systemic induction of resistance by a root-endophytic fungus. The systemically altered "defense readiness" is associated with an elevated antioxidative capacity due to an activation of the glutathione-ascorbate cycle and results in an overall increase in grain yield. Because P. indica can be easily propagated in the absence of a host plant, we conclude that the fungus could be exploited to increase disease resistance and yield in crop plants.

Fig. 1.

Colonization pattern of P. indica in barley roots. (a) Fungal hyphae enter roots via root hairs from 10-day-old plants. The fungus forms pear-shaped chlamydospores within root hairs and proceeds into rhizodermis cells. (b) The fungus grows into the root cortex tissue. (c) Longitudinal section. The fungus was not detected in the central part of the roots beyond the endodermis. Fungal structures were visualized by 0.01% acid fuchsin-lactic acid (28) (red in a and c), or they were stained for mitochondrial respiratory activity by the succinate dehydrogenase assay (29) (black in b).

Fig. 2.

Impact of P. indica on salt-stress tolerance and root infections by F. culmorum. (a) Shoot fresh weight of P. indica and control (noninfested) plants was determined in 5-week-old plants that had been grown for the final 2 weeks in the presence of 100 or 300 mM NaCl, in hydroponic culture. Data points are representative of three independent experiments. Error bars, SD. (b) Plant phenotypes demonstrating the protective potential of P. indica toward F. culmorum.(c) Mean fold change of root and shoot weights relative to noninfested (no P. indica or F. culmorum) 4-week-old plants. CP, P. indica-infested; CF, Fusarium-infected; PF, P. indica-infested, Fusarium-infected. Error bars, SD. Columns labeled with the same letter represent not-significantly-different means, according to multiple unpaired Student t tests (P < 0.05), after ANOVA.

Fig. 3.

Ascorbate, dehydroascorbate (DHA) content, and DHA reductase (DHAR) activity in P. indica-infested roots. Ascorbate content (a), DHA content (b), and DHAR activity (c) were measured in roots of 1-, 2-, and 3-week-old P. indica-infested (shaded columns) and control (free of P. indica, open columns) barley plants. Values are means of three or four samples. Similar results have been obtained with three independent sets of experiments. Error bars, SD. Within each frame, ^* and ^** indicate statistically significant differences between roots of infested and noninfested plants (unpaired Student t test; ^*, P < 0.05; ^**, P < 0.01).

Fig. 4.

Systemic disease resistance conferred by P. indica. (a) Severity of powdery mildew infection (disease index) was calculated as colonies produced by B. graminis on the youngest leaf of 3-week-old barley plants (cultivar Ingrid), with roots either not infested (Control) or infested (P. indica). (b) Cellular responses to powdery mildew attack were evaluated by counting cells showing an active defense response, a hypersensitive response of the whole cell (Hypersensitive reaction), a local defense stopping a penetration attempt (Nonpenetrated cell), or a successful penetration (Haustoria), visible as the successful formation of a fungal haustorium in the cell. Error bars, SD. ^* and ^** indicate statistically significant differences between leaves of infested and noninfested plants (unpaired Student t test; ^*, P < 0.05; ^**, P < 0.01).

Fig. 5.

Expression of potential marker genes for known resistance pathways. Shown are Northern blots of 10 μg of total RNA from leaves of 3-week-old barley plants with roots infested (+) or not infested (-) with P. indica. Plants were harvested either directly or at 12 or 24 h after inoculation with powdery mildew (B. graminis f.sp. hordei, race A6). Blots were hybridized with radio-labeled JIP-23, BCI-1, and PR-5 probes. After removal of the probe, they were subsequently hybridized with a 28S rRNA probe to confirm equal transfer of RNA onto the membrane.