Impact of endochitinase-transformed white spruce on soil fungal biomass and ectendomycorrhizal symbiosis

Abstract

The impact of transgenic white spruce [Picea glauca (Moench) Voss] containing the endochitinase gene (ech42) on soil fungal biomass and on the ectendomycorrhizal fungi Wilcoxina spp. was tested using a greenhouse trial. The measured level of endochitinase in roots of transgenic white spruce was up to 10 times higher than that in roots of nontransformed white spruce. The level of endochitinase in root exudates of three of four ech42-transformed lines was significantly greater than that in controls. Analysis soil ergosterol showed that the amount of fungal biomass in soil samples from control white spruce was slightly larger than that in soil samples from ech42-transformed white spruce. Nevertheless, the difference was not statistically significant. The rates of mycorrhizal colonization of transformed lines and controls were similar. Sequencing the internal transcribed spacer rRNA region revealed that the root tips were colonized by the ectendomycorrhizal fungi Wilcoxina spp. and the dark septate endophyte Phialocephala fortinii. Colonization of root tips by Wilcoxina spp. was monitored by real-time PCR to quantify the fungus present during the development of ectendomycorrhizal symbiosis in ech42-transformed and control lines. The numbers of Wilcoxina molecules in the transformed lines and the controls were not significantly different (P > 0.05, as determined by analysis of covariance), indicating that in spite of higher levels of endochitinase expression, mycorrhization was not inhibited. Our results indicate that the higher levels of chitinolytic activity in root exudates and root tissues from ech42-transformed lines did not alter the soil fungal biomass or the development of ectendomycorrhizal symbiosis involving Wilcoxina spp.

FIG. 1.

Levels of endochitinase activity (A) in root tissues and (B) in root exudates for the four ech42-transformed lines and the control white spruce trees. The values for bars labeled with the same letter are not significantly different as determined by Waller-Duncan's multiple-range test (P ≤ 0.05). The error bars indicate standard errors of the means. CT, control.

FIG. 2.

(A) Levels of fungal biomass in pots based on measurement of ergosterol for the controls and the four transformed lines. (B) Levels of mycorrhizal colonization for the four ech42-transformed lines and the control white spruce trees. The values for bars labeled with the same letter are not significantly different as determined by Waller-Duncan's multiple-range test (P ≤ 0.05). The error bars indicate standard errors of the means. CT, control.

FIG. 3.

(A and B) Stereomicroscope views of root tips colonized (A) by Wilcoxina spp. (MG1) and (B) by both Wilcoxina spp. and P. fortinii (MG2). (C and D) Variation in the numbers of Wilcoxina sp. and P. fortinii ITS molecules in root tips belonging to MG1 and MG2, respectively, as determined by amplification by real-time PCR with primer pairs specific for the two fungal taxa.

FIG. 4.

Results of ANCOVA performed with the real-time PCR data. The circles indicate observed values, and the fitted lines indicate the predicted values based on the ANCOVA model.

FIG. 5.

Transverse section of a root tip containing fungi belonging to MG1 collected from ech42-transformed line 1. The arrow indicates a Hartig net, and the double arrowheads indicate intracellular hyphae. C, cortical cell; p, tannin-filled cell; S, stele; n, nucleus.