The Foliar Microbiome Suggests that Fungal and Bacterial Agents May be Involved in the Beech Leaf Disease Pathosystem

Beech leaf disease (BLD) is a recently discovered disease that is causing severe damage to American beech (Fagus grandifolia) in northeastern North America. The recently described nematode Litylenchus crenatae subsp. mccannii was detected in BLD-affected foliage and may be associated with the disease. However, speculation on the direct role of the nematode in infection still remains. In this study, we profiled the microbial communities associated with asymptomatic, symptomatic, and naïve (control) American beech foliage by using a high-throughput sequence-based metabarcoding analysis of fungi, bacteria, phytoplasmas, and nematodes. We then used both a differential abundance analysis and indicator species analysis as well as several diversity metrics to try and discover microbes associated only with symptomatic foliage. To do so, we amplified the organism-specific phylogenetic informative regions of the 16S, 18S, and internal transcribed spacer (ITS)1 regions using Illumina MiSeq. Our results detected the amplicon sequence variant (ASV) associated with the nematode L. crenatae subsp. mccannii but in all symptom types. However, four ASVs associated with the bacterial genera Wolbachia, Erwinia, Paenibacillus, and Pseudomonas and one ASV associated with the fungal genus Paraphaeosphaeria were detected only in symptomatic samples. In addition, we identified significant differences based on symptom type in both the a- and b-diversity indices for the bacterial and fungal communities. These results suggest that L. crenatae subsp. mccannii may not be fully responsible for BLD but, rather, that other microbes may be contributing to the syndrome, including the putative nematode endosymbiont Wolbachia sp.