Effect of Osmotic Stress on the Growth, Development and Pathogenicity of Setosphaeria turcica

Abstract

Osmotic stress is a severe condition frequently encountered by microorganisms; however, there is limited knowledge on the influence of hyperosmotic stress on the growth, development and pathogenicity of phytopathogenic fungi. Here, three osmotic conditions (0.4 M NaCl, 0.4 M KCl, and 0.6 M sorbitol supplemented in potato dextrose agar medium) were used to identify the effect of osmotic stress on the growth, development and pathogenicity of Setosphaeria turcica which is a plant pathogenic fungus and causes northern corn leaf blight disease in maize, sorghum, and related grasses. In osmotic stress, the growth rate of mycelium was decreased, and the number of vesicular structures and flocculent secretion outside the hypha cell wall were significantly increased. The qRT-PCR results showed that the osmotic stress quickly activated the HOG-MAPK pathway, up-regulated the expression of the downstream genes, and these genes were most highly expressed within 30 min of exposure to osmotic stress. Furthermore, the germination rate and the yield of conidia were significantly higher under osmotic stress than in the control. A pathogenicity analysis confirmed that pathogenicity of the conidia which were cultured under osmotic stress was significantly enhanced. By analyzing the knock-out mutants of an osmotic stress responsed gene StFPS1, an aquaglyceroporin downstream of the HOG-MAPK pathway, we found that StFPS1 was involved in the formation of appressorium and penetration peg, which affected the penetration ability of S. turcica. In summary, our work explained the correlation between osmotic stress and growth, development, and pathogenicity in S. turcica.

Keywords: HOG-MAPK; Setosphaeria turcica; StFPS1; osmotic; pathogenicity.

FIGURE 1

Observation of the Setosphaeria turcica phenotype when cultured under osmotic conditions. (A) Colony morphology and (B) the growth rate of S. turcica in potato dextrose agar (PDA) and 0.4 M NaCl, 0.4 M KCl, and 0.6 M sorbitol culture media. The results are shown as mean colony diameter from three independent experiments. (C) Morphology and (D) the growth rate of aerial hyphae under the above four culture mediums; (E) morphology of basal hyphae under the above four culture conditions. These results are shown as mean length of hyphae from three independent experiments. Data are presented as the mean ± SD of three independent experiments. **P < 0.01.

FIGURE 2

Transmission electron microscopy (TEM) of hyphae of S. turcica cultured on PDA and 0.4 M NaCl. (A) Representative TEM images (magnification, ×2,000, ×12,000, and ×30,000, respectively) of hyphae. (B) The mean number of vesicles in each cell. (C) The mean diameter of the cell wall. Data are presented as the mean ± SD of each cell. **P < 0.01.

FIGURE 3

qRT-PCR analysis of the relative expression of HOG-MAPK related genes at different treatment times in 0.4 M NaCl treatment. Data are presented as the mean ± SD of three independent experiments.

FIGURE 4

Observation of conidia germination after 20 days of culture under the three osmotic conditions (0.4 M NaCl, 0.4 M KCl, and 0.6 M sorbitol) and control (PDA). (A) The number of conidia produced by the hyphae under the four media. (B) The conidial suspension was incubated on cellophane at 25°C in the dark and observed at 3, 6, 9, and 11 h after inoculation under a microscope and (C) the quantitative statistics from three independent petri dishes. ap: appressorium, Data are presented as the mean ± SD of three independent experiments.**P < 0.01.

FIGURE 5

Observation of appressorium development after 20 days of culture under the three osmotic conditions (0.4 M NaCl, 0.4 M KCl, and 0.6 M sorbitol) and control (PDA). The conidial suspension was incubated on cellophane at 25°C in the dark and observed at 12, 24, 36, 48, 60, and 72 h after inoculation under a microscope. h: invading hyphae.

FIGURE 6

Pathogenicity of S. turcica after 20 days of culture under the 0.4 M KCl and control (PDA). (A) Conidial suspensions were inoculated on maize B73 seedling whorl at the 4–6 leaf stage, and results were scored at 12 days post-inoculation and (B) the mean size of the lesion. **P < 0.01, bar = 1 cm.

FIGURE 7

Identification of the StFPS1 knockout mutant (ΔStFPS1) and functional analysis of ΔStFPS1. (A) S. turcica wild-type (WT) and ΔStFPS1, identified by amplifying a fragment of the hygromycin B phosphotransferase gene (HPH) using specific primers (HPH-L/HPH-R; left) and fragment including the StFPS1 upstream flanking fragment and HPH using specific primers (StFPS1-UpL/HPH-UpR; right). (B) Observation of the appressorium development of WT and ΔStFPS1 at 12, 24, 48, 72, and 96 h after inoculation under a microscope. (C) Mycelial suspensions were inoculated on maize B73 seedling whorl at the 4–6 leaf stage, and results were scored at 10 days post-inoculation, bar = 1 cm. (D) Comparison of the cellophane penetrating ability of WT and ΔStFPS1 after the strains were incubated on cellophane at 25°C in the dark for 5 days and then removed the cellophane and continued to incubate for 2 days for observation. (E) High performance liquid chromatography analysis of HT-toxins of WT and ΔStFPS1.