Abstract
The pathogenic fungus Ophiostoma novo-ulmi spreads within the secondary xylem vessels of infected elm trees, causing the formation of vessel plugs due to tyloses and gels, which ultimately result in Dutch elm disease. Foliage discoloration, wilting and falling from the tree are typical external leaf symptoms of the disease followed by the subsequent death of sensitive trees. Cellulolytic enzymes produced by the fungus are responsible for the degradation of medium molecular weight macromolecules of cellulose, resulting in the occurrence of secondary cell wall ruptures and cracks in the vessels but rarely in the fibers (Ďurkovič et al., 2014). The goal of this procedure is to evaluate the extent of cellulose degradation by a highly aggressive strain of O. novo-ulmi ssp. americana × novo-ulmi. Size-exclusion chromatography (SEC) compares molecular weight distributions of cellulose between the infected and the non-infected elm trees, and reveals changes in the macromolecular traits of cellulose, including molecular weights, degree of polymerization, and polydispersity index. 13C magic angle spinning nuclear magnetic resonance (13C MAS NMR) spectra help to identify and also to quantify the loss of both crystalline and non-crystalline cellulose regions due to degradation. The procedure described herein can also be easily used for other woody plants infected with various cellulose-degrading fungi.
Keywords: Ophiostoma novo-ulmi, Ulmus spp., Crystalline cellulose, Size-exclusion chromatography, Nuclear magnetic resonance
Materials and Reagents
Equipment
Software
Procedure
Notes
We have found that syringyl to guaiacyl (S/G) ratio in lignin affected the cellulose degradability by O. novo-ulmi in the infected elm trees (Ďurkovič et al., 2014). Other recent studies also revealed that an S/G ratio has a significant influence on the cross-linking between lignin and other cell wall components, thus modifying the microscopic structure and topochemistry of the cell wall, the cell wall degradability during chemical and hot-water pretreatments, and the successive hydrolysis of cellulose to glucose (Li et al., 2010; Studer et al., 2011; Papa et al., 2012). Therefore, we suggest using standard analytical methods such as alkaline nitrobenzene or cupric oxidations, NMR, pyrolysis–gas chromatography–mass spectrometry (Py–GC–MS) or others to determine lignin monomer composition as quantified by the S/G ratio. Thereby, both the lignin monomer composition and the cellulose degradation data can provide a more complete view of the biodegradation process caused by cellulose-degrading fungi.
Acknowledgments
The authors thank Dr. Miloň Dvořák, Dr. Jana Krajňáková, Dr. Miroslava Mamoňová, Dr. Ingrid Čaňová, Dr. Jaroslav Ohanka and Mr. Miroslav Rusnák for their technical assistance. This work was funded by the Slovak scientific grant agency VEGA (1/0149/15). This protocol has been adapted from our previous work (Ďurkovič et al., 2014).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.