DNA-based detection of grapevine trunk-disease pathogens from environmental spore samples

In California vineyards, spore dispersal of fungi that cause grapevine trunk diseases Botryosphaeria dieback and Eutypa dieback occurs with winter rains. Spores infect through pruning wounds made to the woody structure of the vine in winter. Better timing of preventative practices that minimize infection may benefit from routine spore-trapping, which could pinpoint site-specific time frames of spore dispersal. To speed pathogen detection from environmental spore samples, we identified species-specific PCR primers and protocols. Then we compared the traditional culture-based method versus our new DNA-based method.

PCR primers for Botryosphaeria-dieback pathogen Neofusicoccum parvum and Eutypa-dieback pathogen Eutypa lata were confirmed species-specific, through extensive testing of related species (in families Botryosphaeriaceae and Diatrypaceae, respectively), other trunk-disease pathogens, and saprophytic fungi that sporulate in vineyards.

Consistent detection of N. parvum was achieved from spore suspensions used fresh or stored at -20°C, whereas consistent detection of E. lata was achieved only with a new spore-lysis method, using zirconia/silica beads in a FastPrep homogenizer (MP Biomedicals; Solon, Ohio, USA), and only from spore suspensions used fresh. Freezing E. lata spores at -20°C made detection inconsistent.•From environmental samples, spores of E. lata were detected only via PCR, whereas spores of N. parvum were detected both via PCR and in culture.

Premise: Within a broader study on leaf fossilization in freshwater environments, a long-term study on the development and microbiome composition of biofilms on the foliage of aquatic plants has been initiated to understand how microbes and biofilms contribute to leaf decay and preservation.

Here, water lily leaves are employed as a study model to investigate the relationship between bacterial microbiomes, biodegradation, and fossilization. We compare four DNA extraction kits to reduce biases in interpretation and to identify the most suitable kit for the extraction of DNA from bacteria associated with biofilms on decaying water lily leaves for 16S rRNA amplicon analysis.

Methods: We extracted surface-associated DNA from Nymphaea leaves in early stages of decay at two water depth levels using four commercially available kits to identify the most suitable protocol for bacterial extraction, applying a mock microbial community standard to enable a reliable comparison of the kits.

Results: Kit 4, the FastDNA Spin Kit for Soil, resulted in high DNA concentrations with better quality and yielded the most accurate depiction of the mock community. Comparison of the leaves at two water depths showed no significant differences in community composition.

Discussion: The success of Kit 4 may be attributed to its use of bead beating with a homogenizer, which was more efficient in the lysis of Gram-positive bacteria than the manual vortexing protocols used by the other kits. Our results show that microbial composition on leaves during early decay remains comparable and may change only in Bio Basic Bead Homogenizers later stages of decomposition.

This chapter covers the various methods of mechanical cell disruption and tissue homogenization that are currently commercially available for processing small samples s < 1 mL) to larger multikilogram production quantities. These mechanical methods of lysing do not introduce chemicals or enzymes to the system. However, the energies required when using these “harsh,” high mechanical energy methods can be enough to damage the very components being sought.

The destruction of cell membranes and walls is effected by subjecting the cells (a) to shearing by liquid flow, (b) to exploding by pressure differences between inside and outside of cell, (c) to collision forces by impact of beads or paddles, or (d) a combination of these forces.Practical suggestions to optimize each method, where to acquire such equipment, and links to reference sources are included. Several novel technologies are presented.