Abstract
Pest introductions via trade in tree seed may result from a lack of adequate survey and validation protocols. Developing better diagnostic protocols to identify potentially harmful pests and pathogens in forest tree seed is of critical importance. High-throughput sequencing-based barcoding and metabarcoding provide effective tools for screening potentially harmful organisms in various plant materials, including seeds. However, the sample size needed to detect the total microorganism diversity of a community is a major challenge in microbiome studies. In this work, we examined how increasing sample size (ranging between 100 and 1000 seeds) influences diversity of fungal communities detected by high throughput sequencing in Pinus sylvestris seeds. Our results showed that as sample size increased, fungal alpha diversity also increased. Beta-diversity estimators detected significant differences between the mycobiota from different samples. However, taxonomic and functional diversity were not correlated with sample size. In addition, we found that increasing the number of PCR replicates resulted in a higher abundance of plant pathogens. We concluded that for the purpose of screening for potentially harmful pathogens using HTS, greater efforts should be made to increase the sample size and replicates when testing tree seed.
Highlights
Pest introductions via trade in tree seed may result from a lack of adequate survey and validation protocols
Phytosanitary requirements for international trade are established by the International Plant Protection Convention (IPPC) through International Standards for Phytosanitary Measures (ISPMs) in order to allow safe plant trade, help prevent the introduction and spread of invasive alien species and advise appropriate measures for control when pests become established
The aim of this work was to determine if the total fungal community detected in Pinus sylvestris varies in samples with different numbers of seeds analysed, hypothesizing that the full coverage is obtained when more seeds are tested
Summary
Pest introductions via trade in tree seed may result from a lack of adequate survey and validation protocols. The new rules were implemented with several acts, including lists of regulated pests, high-risk plants and plant products and priority pests This new regulation introduces more effective measures than previously in place for the protection of the European territory, discrepancies among member states in the application of the regulations could result in a weakening of the EU biosecurity status[5]. Another issue not considered in the new legislation is the possibility that alien pests and pathogens enter states on either non- or unknown hosts and asymptomatic hosts[6,7]. Without seed inspection, seed trade could lead to new pest introductions
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