Plant diseases caused by pathogenic microorganisms represent a serious threat to plant productivity, food security, and natural ecosystems. An effective framework for early warning and rapid response is a crucial element to mitigate or prevent the impacts of biological invasions of plant pathogens. For these reasons, detection tools play an important role in monitoring plant health, surveillance, and quantitative pathogen risk assessment, thus improving best practices to mitigate and prevent microbial threats. The need to reduce the time of diagnosis has prompted plant pathologists to move towards more sensitive and rapid methods such as molecular techniques. Considering prevention to be the best strategy to protect plants from diseases, this review focuses on fast and reliable molecular methods to detect the presence of woody plant pathogens at early stage of disease development before symptoms occur in the host. A harmonized pool of novel technical, methodological, and conceptual solutions is needed to prevent entry and establishment of new diseases in a country and mitigate the impact of both invasive and indigenous organisms to agricultural and forest ecosystem biodiversity and productivity.

Fusarium circinatum is the causal agent of pitch canker disease on numerous Pinus spp. This aggressive fungus may infect pine seed cryptically and, therefore, can easily be spread long distances by the seed trade. F. circinatum has recently been listed as a quarantine organism in numerous countries throughout the world, which prompted the development of a specific and sensitive tool for the detection of this pathogen in conifer seed. A new detection protocol for F. circinatum based on a biological enrichment step followed by a real-time polymerase chain reaction (PCR) assay was developed. Several enrichment protocols were compared and a 72-h incubation of the seed with potato dextrose broth was the most efficient technique to increase F. circinatum biomass before DNA extraction. The relative accuracy, specificity, and sensitivity of the real-time PCR assay was evaluated in comparison with a previously published conventional PCR test on 420 seed DNA extracts. The real-time PCR described here proved to be highly specific and significantly more sensitive than the conventional PCR, and enabled the detection of F. circinatum in samples artificially contaminated with less than 1/1,000 infected seed, as well as in naturally infected samples. Last, in order to routinely check the quality of the seed DNA extracts, a primer–probe combination that targets a highly conserved region within the 18S ribosomal DNA in plants or fungi was successfully developed. This assay allows for quick and reliable detection of F. circinatum in seed, which can help to prevent long-distance spread of the pathogen via contaminated seed lots.

Accelerating international trade and climate change make pathogen spread an increasing concern. Hymenoscyphus fraxineus, the causal agent of ash dieback, is a fungal pathogen that has been moving across continents and hosts from Asian to European ash. Most European common ash trees (Fraxinus excelsior) are highly susceptible to H. fraxineus, although a minority (~5%) have partial resistance to dieback. Here, we assemble and annotate a H. fraxineus draft genome, which approaches chromosome scale. Pathogen genetic diversity across Europe and in Japan, reveals a strong bottleneck in Europe, though a signal of adaptive diversity remains in key host interaction genes. We find that the European population was founded by two divergent haploid individuals. Divergence between these haplotypes represents the ancestral polymorphism within a large source population. Subsequent introduction from this source would greatly increase adaptive potential of the pathogen. Thus, further introgression of H. fraxineus into Europe represents a potential threat and Europe-wide biological security measures are needed to manage this disease.

Dothistroma pini, D. septosporum, and Lecanosticta acicola are fungal pathogens that cause severe foliage diseases in conifers. All three pathogens are listed as quarantine organisms in numerous countries throughout the world and, thus, are subject to specific monitoring. Detection and identification of these pathogens still often relies on cumbersome and unsatisfactory methods that are based upon the morphological characterization of fungal fruiting bodies and conidia. In this study, we present the development of several new molecular tools that enable a rapid and specific in planta detection of each of these pathogens. Several DNA extraction procedures starting from infected needles were compared and four commercial DNA extraction kits provided DNA of satisfactory quality for amplification by polymerase chain reaction (PCR). In addition, we developed several sets of conventional PCR primers, dual-labeled probes (DLPs), and duplex-scorpion probes (DSPs), all of which targeted each pathogen. Their ability to detect the pathogens in a series of naturally infected needle samples was compared. The quadruplex DLP real-time assay proved to be more sensitive than the DSP assay and conventional PCR but the two real-time probe formats yielded identical results in the naturally infected samples. Both real-time assays proved to be significantly superior to the technique of humid chamber incubation, which often failed to produce spores for the accurate identification of the pathogens.