Armored mesh bags: collecting mycelium of mycorrhizal fungi in a tropical rainforest

Mycorrhizal fungi play a vital role in soil processes and form a large part of belowground biodiversity in tropic ecosystems. Unlike temperate forests, many tree species in tropical stands are dominated by arbuscular mycorrhizal fungi (AMF), which form high amount of extraradical mycelium and even rhizomorphs, penetrating both soil and leaf litter. The quantification of mycelium biomass in natural systems often being conducted with the use of sand filled in-growth mesh bags made of nylon mesh [1]. The use of plastic polymer for mesh bags in temperate systems is justified by low bioavailability of the material that can be hardly decomposed by soil bacteria and fungi or penetrated by roots or soil invertebrates. In a number of tropical forests, the activity of invertebrates is more aggressive, mostly due to the termites foraging, resulting in the disruption of the integrity of the mesh bags and consequent samples loss.Here we introduce the use of dual-walled in-growth mesh bags made of nylon and stainless steel. Its relevance was attested in Dong Nai (Cat Tien) National Park in Southern Vietnam. The harvested biomass of extraradical EMF mycelium and taxonomic composition of EMF and AMF fungi was compared in regular and dual-walled in-growth mesh bags across two tropical rainforests, dominated by both EMF and AMF associated tree species. The biomass was accessed with PLFA analysis for fungal biomarkers 18:2ω6,9, 18:1ω9 [2] and 18:0 [3]. The taxonomic composition of extraradical mycelium from harvested in-growth mesh bags was studied with new-generation sequencing, including specific primer pair WANDA/AML2 for the SSU ribosomal RNA gene of AMF fungi and ITS fragment (primer pair ITS3/ITS4) for ectomycorrhizal fungi.Double-walled mesh bags remained completely intact, while about 40% of regular mesh bags were damaged by termite activity after 180 days of exposure. The biomass of extraradical mycelium of ectomycorrhizal fungi was comparable between forests dominated by EMF and AMF associated trees and reached 114.5 and 122.1 µg of «fungal» carbon g-1 of substrate respectively. The total amount of the three measured PLFA biomarkers did not differ between both variants of in-growth mesh bags and surrounding soil, while the median values were slightly higher for mesh bags compared to soil (1.9 and 1.6 µg g-1 of substrate respectively). Application of ITS fragment was 100% positive among harvested mycelium samples from both forests, while AMF fungi were detected in 62% of samples from the forest dominated by AMF-associated trees and 70% samples from the forest dominated by EMF-associated trees.This study was supported by Alexander von Humboldt Foundation (project 3.4-1071297-RUS-IP).References:[1] Wallander H, Nilsson LO, Hagerberg D, Bååth E (2001) Estimation of the biomass and seasonal growth of external mycelium of ectomycorrhizal fungi in the field. New Phytol 151:753–760. https://doi.org/10.1046/j.0028-646x.2001.00199.x[2] Ruess L, Chamberlain PM (2010) The fat that matters: Soil food web analysis using fatty acids and their carbon stable isotope signature. Soil Biol Biochem 42:1898–1910. https://doi.org/10.1016/j.soilbio.2010.07.020[3] Chen J, Ferris H, Scow KM, Graham KJ (2001) Fatty acid composition and dynamics of selected fungal-feeding nematodes and fungi. Comp Biochem Physiol B Biochem Mol Biol 130:135–144. https://doi.org/10.1016/S1096-4959(01)00414-