Impact of Endochitinase-Transformed White Spruce on Soil Fungal Biomass and Ectendomycorrhizal Symbiosis

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.White spruce [Picea glauca (Moench) Voss] is a tree species with an extensive distribution in boreal and subboreal forests and with significant ecological roles (37, 38). It is also an important commercial species for production of pulpwood and construction-grade lumber. However, in nurseries and plantations, white spruce is sensitive to multiple fungal diseases (23, 29, 42, 62, 76). Climate change scenarios suggest that diseases could result in increased mortality in conifer forests (22, 48). Genetic engineering offers a potential means to mitigate biotic and abiotic stresses.During the last 2 decades, chitinase genes isolated from plants, fungi, or bacteria have been studied and used to transform crops or trees in order to increase their resistance to plant-pathogenic fungi. One potential goal is improving white spruce tolerance to fungal infection through insertion of a chitinase gene. Chitin is a biopolymer of β-(1-4)-linked molecules of N-acetylglucosamine (NAG), a derivative of glucose, and is the primary constituent of the fungal cell wall and arthropod exoskeleton (3, 51). Chitinases are plant defense pathogenesis-related proteins (6, 11) that break down the chitin chain either by cleavage of internal glycoside bonds (endochitinases), by hydrolysis of the nonreducing end of the chitin chain (exochitinases), or by hydrolysis of NAG oligomers and trimers into NAG monomers (chitobiases). Endo- and exochitinase genes have been well characterized using sugar beet (Beta vulgaris) (44) and the filamentous fungal genus Trichoderma (14, 24, 69). Chitinolytic genes have been inserted into the genomes of cultivated plants and trees in an attempt to boost plant chitinase activity. Among the different genes involved in the production of chitinolytic enzymes, the ech42 endochitinase gene from Trichoderma harzianum has been inserted into plant genomes to enhance their resistance against phytopathogenic fungi. In McIntosh apple cultivars transformed with the ech42 gene there was limited attack by the apple scab fungus Venturia inaequalis (5). Transgenic black spruce (Picea mariana) expressing the ech42 gene was more resistant to the root rot pathogen Cylindrocladium floridanum (45).However, field deployment of crops and trees genetically transformed to improve nonspecific resistance against phytopathogenic fungi has raised concerns about the impact on nontarget fungi, including potentially beneficial symbionts. This is particularly worrisome when nonspecific constitutive promoters control expression of the resistance gene and the gene is expressed in all tissues from roots to leaves. As a consequence, the natural colonization of such transformed plants by endophytic or mycorrhizal fungi can be altered.Mycorrhizal fungi play a key role in plant nutrition (55) by mobilizing and transferring nutrients to the host through an intimate and highly organized association with plant roots (52, 63). Furthermore, their involvement in soil nutrient recycling (56) makes mycorrhizal symbiosis a major ecological process that is important for the health of soil and forest ecosystems. Crops, fruits, and forest trees exhibit mycorrhizal colonization by arbuscular mycorrhizae, ectomycorrhizae, and ectendomycorrhizae (EEM). While numerous studies have addressed the impact of transgenic plants on arbuscular mycorrhizae (10, 26, 64, 68, 72, 73) and ectomycorrhizae (32, 43, 50, 60), no previous study focused on EEM.Ectendomycorrhizal fungi can be distinguished from ectomycorrhizae by the presence of a thin or fragmented mantle and intracellular penetration into root cortical cells. All EEM fungi identified so far belong to the Ascomycetes, and these fungi are represented by several genera of Helotiales and Pezizales (77). EEM fungi are prevalent in conifer and deciduous tree nurseries (27, 39, 40, 70) and are also very common on seedling root tips at disturbed sites (15, 16, 19). The prevalence of EEM fungi on seedling roots, from which the genus Wilcoxina is frequently recovered (16, 67), suggests that they can play a significant role in establishment and growth of seedlings (77) and provide protection against root diseases (31, 61). Consequently, the potentially negative effects of chitinase-transformed trees on ectendomycorrhizal fungi could be detrimental to plant health.The present study addressed the potential impact of ech42-transformed white spruce on soil fungal biomass and ectendomycorrhizal symbiosis. It was hypothesized that (i) the soil fungal biomass in a transgenic white spruce rhizosphere is less than the soil fungal biomass in a control tree rhizosphere and (ii) the development of Wilcoxina spp. on root tips of transgenic white spruce is less important than the development of Wilcoxina spp. on root tips of control trees. To test these hypotheses, 5-year-old white spruce trees transformed with the 35S promoter-ech42 construct were analyzed by performing a greenhouse trial. The amount of soil fungal biomass was estimated using measurements of ergosterol in soil. A real-time PCR method was developed to detect changes in the quantity of ectendomycorrhizal hyphae involved in colonization of transgenic white spruce root tips.