The Abundance of Endofungal Bacterium Rhizobium radiobacter (syn. Agrobacterium tumefaciens) Increases in Its Fungal Host Piriformospora indica during the Tripartite Sebacinalean Symbiosis with Higher Plants

Abstract

Rhizobium radiobacter (syn. Agrobacterium tumefaciens, syn. "Agrobacterium fabrum") is an endofungal bacterium of the fungal mutualist Piriformospora (syn. Serendipita) indica (Basidiomycota), which together form a tripartite Sebacinalean symbiosis with a broad range of plants. R. radiobacter strain F4 (RrF4), isolated from P. indica DSM 11827, induces growth promotion and systemic resistance in cereal crops, including barley and wheat, suggesting that R. radiobacter contributes to a successful symbiosis. Here, we studied the impact of endobacteria on the morphology and the beneficial activity of P. indica during interactions with plants. Low numbers of endobacteria were detected in the axenically grown P. indica (long term lab-cultured, lcPiri) whereas mycelia colonizing the plant root contained increased numbers of bacteria. Higher numbers of endobacteria were also found in axenic cultures of P. indica that was freshly re-isolated (riPiri) from plant roots, though numbers dropped during repeated axenic re-cultivation. Prolonged treatments of P. indica cultures with various antibiotics could not completely eliminate the bacterium, though the number of detectable endobacteria decreased significantly, resulting in partial-cured P. indica (pcPiri). pcPiri showed reduced growth in axenic cultures and poor sporulation. Consistent with this, pcPiri also showed reduced plant growth promotion and reduced systemic resistance against powdery mildew infection as compared with riPiri and lcPiri. These results are consistent with the assumption that the endobacterium R. radiobacter improves P. indica's fitness and thus contributes to the success of the tripartite Sebacinalean symbiosis.

Keywords: P. Indica; endobacteria; endofungal bacteria; endophytes; plant growth promotion bacteria; root colonization; tripartite symbiosis.

FIGURE 1

Detection of endobacteria in Piriformospora indica by Fluorescence In Situ Hybridization (FISH). The fungus was grown in liquid complete medium (CM) medium for 3 weeks, and fixed for FISH analysis using the universal bacterial probe EUB-338-FITC (Table 1). FISH staining (green signal) detects bacteria in hypha (A), chlamydospores (B), and crushed mycelium (C). (D) FISH detection of pure RrF4 cells (positive control). White arrows point to bacteria. Microscopic analyses were done at 1,000-fold magnification; Bars indicated 10 μm.

FIGURE 2

Quantification of endobacteria in P. indica. (A) Three-day-old barley seedlings were dip-inoculated with chlamydospore solutions and cultured on ½ Murashige-Skoog (MS) in sterile jars. For comparison, chlamydospores (lcPiri) were cultured in liquid CM medium in a flask in the absence of root tissue. Endobacteria were quantified in barley roots (at 7 and 14 dpi) and in 14-day-old liquid cultures. (B) Chlamydospores of lcPiri were cultured in liquid CM medium for 3 days. Subsequently, root extracts and root pieces, respectively, from fresh barley roots were added to the cultures. Mycelium from each culture was harvested after 7 days and assessed for endobacteria. Mean values based on three independent biological replicates are given. Different letters on the top of the bars indicate statistically significant differences tested by one-way analysis of variance performed with the Tukey test (P < 0.05).

FIGURE 3

Re-isolation of P. indica from barley roots and quantification of endobacteria. Seedlings inoculated with lcPiri were cultured on ½ MS in sterile jar for 2 weeks. Thereafter roots were surface-sterilized, cut into small pieces, and cultured on solid or in liquid CM medium, respectively. (A) P. indica colony developing around a surface-sterilized root piece on CM agar medium. (B) P. indica colony developing around a surface-sterilized root piece in liquid CM medium. (C) Relative amount of endofungal bacteria in cultures of riPiri-1 and sub-culture riPiri-2 was quantified with ITS targets of RrF4 related to the Tef gene of P. indica. Mean values and standard errors based on three independent biological replicates. Different letters indicate statistically significant differences tested by one-way analysis of variance performed with the Tukey test (P < 0.05).

FIGURE 4

Piriformospora indica cultures grown from single protoplasts in the presence of antibiotics. (A) Fungal colonies regenerated from single protoplasts of lcPiri cultures grown for 5 days on CM medium containing 300 μg/mL spectinomycin and 300 μg/mL ciprofloxacin. (B) Colonies grown on CM medium without antibiotics. (C) Single colony picked up from A and propagated on medium with the same antibiotics combination for 10 days. (D) Single colony picked up from B and propagated without antibiotics for 10 days. (E): Microscopy of P. indica mycelium from image C (cultured in the presence of antibiotics). (F) Microscopy of the P. indica culture from image D (cultured in the absence of antibiotics). Chlamydospores are clearly visible in this culture. (G,H) Nuclei were stained with DAPI and show blue fluorescence under the fluorescence microscope. Mycelium from an antibiotics-treated (G) and a control culture (H).

FIGURE 5

GFP-tagged RrF4 sticking around P. indica. Chlamydospores of P. indica were germinated in liquid CM medium for 3 days, before a GFP-RrF4 suspension was added to the culture. (A) GFP-tagged RrF4 stacking around the hyphae. The square box showed GFP-tagged RrF4 around a hyphal tip. (B) GFP-tagged RrF4 at the surface of chlamydospores. Bars indicated 10 μm.

FIGURE 6

Colonization of barley roots by riPiri, lcPiri, and pcPiri. Three-day-old seedlings were dip-inoculated with respective mycelia. Seedlings were grown in soil in a growth chamber and harvested after 1 week for quantification and WGA-staining. (A) Relative amount of R. radiobacter bacteria based on the genome ratio of RrF4 vs. P. indica on barley roots. (B) Quantification of the amount of P. indica mycelium on barley roots. (C) Pear-shaped chlamydospores and mycelium in a root inoculated with riPiri. (D) Mycelium in a root inoculated with pcPiri. Mean values and standard errors of three independent biological replicates are given. Different letters on the top of the bars indicate statistically significant differences tested by one-way analysis of variance performed with the Tukey test (p < 0.05). Bars indicated 10 μm.

FIGURE 7

Biological activity conferred by pcPiri, lcPiri and riPiri on barley. Three-week-old plants inoculated with riPiri, lcPiri, pcPiri, and non-inoculated control plants were used for biomass assessments and a pathogen assay. (A) Shoot fresh weights (FW); (B) Root FW. (C) Number of Bgh pustules on detached leaves. (D) Three-week-old barley plants treated with P. indica cultures. Bars indicate standard errors based on three independent biological replicates. Letters on the top of the bars indicate statistically significant differences tested by one-way analysis of variance performed with the Tukey test (p < 0.05).

FIGURE 8

Biological activity conferred by pcPiri, lcPiri, and riPiri on Arabidopsis. Seedlings were dip-inoculated with P. indica cultures and incubated on petri dishes. (A) Shoot FW; (B) Root FW; (C) Relative amount of P. indica in Arabidopsis roots (colonization density). (D) Root structure (primary and secondary root formation) of 3-week-old Arabidopsis seedlings. Bars indicate standard errors based on three independent biological replicates. Letters on the top of the bars indicate statistically significant differences tested by one-way analysis of variance performed with the Tukey test (p < 0.05).