Mycorrhiza (2006) 17:51–65 DOI 10.1007/s00572-006-0083-4 ORIGINAL PAPER Methods to control ectomycorrhizal colonization: effectiveness of chemical and physical barriers François P. Teste & Justine Karst & Melanie D. Jones & Suzanne W. Simard & Daniel M. Durall Received: 19 January 2006 /Accepted: 25 August 2006 / Published online: 15 November 2006 # Springer-Verlag 2006 Abstract We conducted greenhouse experiments using ecological question of interest, Topas\ or the employment Douglas-fir (Pseudotsuga menziesii var. glauca) seedlings of mesh with pore sizes <1 μm are suitable for restricting where chemical methods (fungicides) were used to prevent mycorrhization in the field. ectomycorrhizal colonization of single seedlings or physical methods (mesh barriers) were used to prevent formation of Keywords Ectomycorrhizal colonization . mycorrhizal connections between neighboring seedlings. Common mycorrhizal networks . Hyphal restriction . These methods were chosen for their ease of application in Fungicides . Mesh barriers the field. We applied the fungicides, Topas\ (nonspecific) and Senator\ (ascomycete specific), separately and in combination at different concentrations and application Introduction frequencies to seedlings grown in unsterilized forest soils. Additionally, we assessed the ability of hyphae to penetrate In mycorrhizal research, evaluation of mycorrhizal effects μ mesh barriers of various pore sizes (0.2, 1, 20, and 500 m) to on plant performance often requires comparisons between form mycorrhizas on roots of neighboring seedlings. Ecto- mycorrhizal and non-mycorrhizal plants. Creating effective, mycorrhizal colonization was reduced by approximately 55% yet feasible methods to control mycorrhizal colonization in \ −1 with the application of Topas at 0.5 g l . Meshes with pore the field has become of utmost importance as there has μ sizes of 0.2 and 1 m were effective in preventing the been a recent demand to increase the ecological relevance formation of mycorrhizas via hyphal growth across the of mycorrhizal research (Read 2002). This requires moving mesh barriers. Hence, meshes in this range of pore sizes away from laboratory-based work to experiments con- could also be used to prevent the formation of common ducted in natural environments. mycorrhizal networks in the field. Depending on the Currently, most studies have obtained non-mycorrhizal plants by employing one of three methods: substrate François P. Teste and Justine Karst contributed equally to this work. sterilization (via autoclaving, steam sterilization, or gamma irradiation), the creation of mutant plants unable to form * : F. P. Teste ( ) S. W. Simard mycorrhizas, or the use of fungicides applied to soil around Department of Forest Sciences, University of British Columbia, Vancouver, B.C. V6T 1Z4, Canada plant roots. Sterilizing soil can result in substantial changes e-mail: [email protected] in its chemical and physical properties (Lensi et al. 1991; Chambers and Attiwill 1994; Sheremata et al. 1997; Shaw J. Karst et al. 1999); moreover, its application in the field is futile Department of Botany, University of British Columbia, Vancouver, B.C. V6T 1Z4, Canada because contamination is certain. The development of : defective plants that lack the ability to form mycorrhizas M. D. Jones D. M. Durall has been limited to a few plant species associating with Biology and Physical Geography Unit, arbuscular mycorrhizal fungi (AMF) (Marsh and Schultze University of British Columbia Okanagan, 3333 University Way, 2001). More research is also required to determine whether Kelowna, B.C. V1V 1V7, Canada the functioning of mutants is otherwise identical to 52 Mycorrhiza (2006) 17:51–65 nonmutant plants (Kahiluoto et al. 2000). Of the fungicides, Booth 2004; Kranabetter 2005) or provide root-free benomyl has been effectively used to reduce arbuscular compartments where mycorrhizal hyphae can explore and mycorrhizal colonization of plants in the field by as much grow. To allow movement of soil solution while restricting as 80% (Hartnett and Wilson 1999; Wilson et al. 2001; penetration of roots and hyphae, mesh with pores 1 μmor Callaway et al. 2004; Dhillion and Gardsjord 2004). smaller has been used (Robinson and Fitter 1999; Johnson Benomyl, no longer licensed for use in some countries et al. 2001; Zabinski et al. 2002; Cardoso et al. 2004), even and relatively ineffective against basidiomycetes, is how- though it is unknown whether it prevents formation of ever, not an option to control ectomycorrhizal (EM) fungi. mycorrhizal connections between plants. Furthermore, Fungicides have generally not been employed in EM given that hyphal width varies (from 1.5 to 9 μm), a mesh systems (but see Page-Dumroese et al. 1996; Manninen et with pore sizes larger than 1 μm may restrict penetration of al. 1998). Ectomycorrhizal fungal communities are more some mycorrhizal fungal species, but not others. Conse- taxonomically diverse than arbuscular mycorrhizal fungal quently, the mesh pore size could alter the EM fungal communities, thus, requiring a broad spectrum fungicide to community composition. Ectomycorrhizal fungi vary in adequately decrease EM colonization. Of the three methods their ability to absorb and transport nutrients and water currently employed to control mycorrhization, the use of (Simard and Durall 2004); therefore, any alteration of the fungicides appears to be the most feasible for field research community may affect transport within the CMN. in EM systems. The objective of this study was to examine the Two fungicides, Topas\ and Senator\,havebeen effectiveness of chemical and physical methods at control- suggested by greenhouse managers for control of EM ling formation of ectomycorrhizas on Douglas-fir seedlings. hyphal growth. Propiconazole, the active ingredient in We tested the effectiveness of the fungicides, Topas\ and Topas\ (25% a.i.), interferes with ergosterol biosynthesis, Senator\, at various concentrations and application fre- which is critical to the formation of fungal cell membranes quencies. We predicted that both fungicides would reduce (Kendrick 2000). The lack of normal sterol production EM colonization, however, we expected that colonization slows or stops the growth of the fungus, effectively of ascomycete fungi would be particularly reduced with the preventing further infection and/or invasion of host tissues application of Senator\. Thus, the composition of the EM (Kendrick 2000). Propiconazole incorporated into agar fungal community would be altered compared to untreated media at 1 ppm or higher inhibited growth of many EM controls. In addition, we tested the effectiveness of nylon fungal strains (Zambonelli and Iotti 2001; Laatikainen and mesh with various pore sizes at preventing hyphal Heinonen-Tanski 2002). Colonization of Pinus sylvestris penetration and its effects on EM community composition roots by EM fungi decreased by approximately 20%, with of neighboring seedlings. We predicted that percent some morphotypes affected more than others, when colonization and similarity of EM communities between propiconazole was applied for two consecutive years in seedlings on opposite sides of the mesh barrier would the field at a rate of 250 g l−1 every 2 weeks (Manninen et decrease with decreasing mesh pore size. al. 1998). Thiophanate-methyl, the active ingredient in Senator\ (70% a.i.), interferes with the functioning of microtubules, so that treated cells cannot divide. Thiopha- nate-methyl targets the cells of ascomycetes (Kendrick Materials and methods 2000), but to our knowledge has not been used to control EM fungi. Field soil collection Studies of common mycorrhizal networks (CMNs) in plant communities form a unique subset of studies on On August 27–28 of 2003, we collected 600 l of soil from mycorrhizal effectiveness (Simard and Durall 2004). They the Black Pines variable retention cut (also known as a require comparisons between plants that are linked with green-tree retention cut where some trees are not harvested) those that are not linked by a CMN (Simard et al. 1997; and adjacent forest approximately 50 km northwest of Booth 2004). In these studies, control plants may be Kamloops, British Columbia (120°26′ W, 50°42′ N). The mycorrhizal, but hyphal linkages between plants must be Black Pines variable retention cut occurs in the dry cool absent. While non-mycorrhizal or non-linked controls are subzone of the Interior Douglas-fir (IDFdk) biogeoclimatic easily established in the laboratory using substrate sterili- zone (Meidinger and Pojar 1991). It has an elevation of zation techniques, this is more problematic in the field 1,180 m above seal level (asl) and loamy Gray Luvisolic where seedlings are grown in native soils. Mesh barriers soil (Krzic et al. 2004). The plant community is dominated constructed of either steel or nylon have been used to by residual Douglas-fir [Pseudotsuga menziesii var. glauca prevent formation of ectomycorrhizal connections between (Beissn.) Franco] and subalpine fir [Abies lasiocarpa plants (e.g., Francis and Read 1984; Schüepp et al. 1992; (Hook.) Nutt.] trees and advanced regeneration (saplings), Mycorrhiza (2006) 17:51–65 53 with shrub and herbaceous layers dominated by soopolallie two fungicides (both from Engage Agro, Guelph, Ontario, [Sherpherdia canadensis (L.) Nutt.] and pinegrass (Cala- Canada). The three rates of application were: 0.5, 1, or magrostis rubescens Buckley), respectively. 1.5 ml l−1 of Senator\; and 0.5, 1, or 1.5 g l−1 of Topas\. We collected forest floor