The Free-Living Stage Growth Conditions of the Endophytic Fungus Serendipita indica May Regulate Its Potential as Plant Growth Promoting Microbe

Abstract

Serendipita indica (former Piriformospora indica) is a non-obligate endophytic fungus and generally a plant growth and defence promoter with high potential to be used in agriculture. However, S. indica may switch from biotrophy to saprotrophy losing its plant growth promoting traits. Our aim was to understand if the free-living stage growth conditions (namely C availability) regulate S. indica's phenotype, and its potential as plant-growth-promoting-microbe (PGPM). We grew S. indica in its free-living stage under increasing C availabilities (2-20 g L^-1 of glucose or sucrose). We first characterised the effect of C availability during free-living stage growth on fungal phenotype: colonies growth and physiology (plasma membrane proton pumps, stable isotopic signatures, and potential extracellular decomposing enzymes). The effect of the C availability during the free-living stage of the PGPM was evaluated on wheat. We observed that C availability during the free-living stage regulated S. indica's growth, ultrastructure and physiology, resulting in two distinct colony phenotypes: compact and explorer. The compact phenotype developed at low C, used peptone as the major C and N source, and displayed higher decomposing potential for C providing substrates; while the explorer phenotype developed at high C, used glucose and sucrose as major C sources and casein and yeast extract as major N sources, and displayed higher decomposing potential for N and P providing substrates. The C availability, or the C/N ratio, during the free-living stage left a legacy to the symbiosis stage, regulating S. indica's potential to promote plant growth: wheat growth promotion by the explorer phenotype was ± 40% higher than that by the compact phenotype. Our study highlights the importance of considering microbial ecology in designing PGPM/biofertilizers. Further studies are needed to test the phenotypes under more extreme conditions, and to understand if the in vitro acquired characteristics persist under field conditions.

Keywords: free-living stage; fungal phenotype; morphology; physiology; plant-growth-promoting-microbes; symbiosis stage.

FIGURE 1

Effect of increasing C availabilities (glucose or sucrose) on Serendipita indica’s colony biomass (free-living stage). *** shows significant effects (p < 0.01) and “ns” shown non-significant (p > 0.05). Different letters show significant differences between C availabilities (p < 0.05). Symbols are the mean of 10 colonies per replicate (n = 3) ± SD.

FIGURE 2

Effect of two contrasting C availabilities on Serendipita indica’s ultrastructure (free-living stage): 2 g L^–1 of glucose (a–c) and 20 g L^–1 of glucose (d–f). V, vacuoles; d, dolipores; P, parenthosomes; cw, cell wall; ESP, extracellular polymeric substances.

FIGURE 3

Effect of two contrasting C availabilities (2 and 20 g L^–1 of glucose) on Serendipita indica’s (free-living stage) proton (H⁺) transport of plasma membrane ATPase (P-H⁺-ATPase): vanadate inhibition curve of the treatment of 2 g L^–1 glucose (A); vanadate inhibition curve of the treatment of 20 g L^–1 (B); maximum fluorescence (F_max – C), and initial velocity (V0) of P-H⁺-ATPase H⁺ transport (D). CTR, control; + Van, preincubation with 0.2 M vanadate. *** shows significant difference by Student’s t test (p < 0.01). Bars are the mean of 10 colonies per replicate (n = 3) ± SD.

FIGURE 4

Effect of increasing C availabilities (glucose or sucrose) on Serendipita indica’s (free-living stage) stable isotopic signatures of C (δ¹³C – A) and N (δ¹⁵N – B). Arrows on the right side of the graphs indicate δ¹³C and δ¹⁵N of several medium components that can be C or N sources for S. indica. Arrows are the mean of three measurements per medium component (n = 3). *** shows significant effects (p < 0.01) and “ns” shown non-significant (p > 0.05). Different letters show significant differences between C availabilities (p < 0.05). Symbols are the mean of 10 colonies per replicate (n = 3) ± SD.

FIGURE 5

Legacy of increasing C availabilities (glucose) during the fungal free-living stage on biomass of Serendipita indica-inoculated wheat plants (symbiosis stage). Dashed line represents the biomass produced by non-inoculated plants. *** shows significant effects (p < 0.01) and “ns” shown non-significant (p > 0.05). Different letters show significant differences between C availabilities (p < 0.05). Symbols are the mean of 10 colonies per replicate (n = 3) ± SD.

FIGURE 6

C availability in the growth medium triggers nutrient imbalances which regulate Serendipita indica’s phenotype during the free-living and symbiosis stages (when later colonising the plant host). Low C availability (below 5 g L^–1 of glucose or sucrose) leads to C deprivation which triggers the C scavenging mode displayed by the compact phenotype. High C availability (above 5 g L^–1 of glucose or sucrose) leads to N and P limitation which triggers the N and P scavenging mode displayed by the explorer phenotype. When S. indica colonises the plant host (symbiosis stage), the explorer phenotype provides greater benefits to plant growth than the compact phenotype.