Weather Variables Associated with Spore Dispersal of Lecanosticta acicola Causing Pine Needle Blight in Northern Spain

In the last decade, the impact of needle blight fungal pathogens on the health status of forests in northern Spain has marked a turning point in forest production systems based on Pinus radiata species. Dothistroma needle blight caused by Dothistroma septosporum and D. pini, and brown spot needle blight caused by Lecanosticta acicola, coexist in these ecosystems. There is a clear dominance of L. acicola with respect to the other two pathogens and evidence of sexual reproduction in the area. Understanding L. acicola spore dispersal dynamics within climatic determinants is necessary to establish more efficient management strategies to increase the sustainability of forest ecosystems. In this study, spore counts of 15 spore traps placed in Pinus ecosystems were recorded in 2019 and spore abundance dependency on weather data was analysed using generalised additive models. During the collection period, the model that best fit the number of trapped spores included the daily maximum temperature and daily cumulative precipitation, which was associated to higher spore counts. The presence of conidia was detected from January and maximum peaks of spore dispersal were generally observed from September to November.

Dothistroma septosporum Not Detected in Pinus sylvestris Seed Trees from Investigated Stands in Southern Poland

In recent years, the decline of pine stands in Europe, including Poland, has been caused by the emerging needle pathogen Dothistroma septosporum. Although this fungus appears to preferentially infect Pinus pini, P. pinaster or P. radiata in Southern Europe, it has been reported in stands of P. nigra, P. mugo and P. sylvestris from Southern Poland. Our preliminary tests of symptomatic needles of diseased pines, including black pine (P. nigra), showed the presence of both D. septosporum and D. pini—the latter as the first report in Poland. No other endophytic pathogen, i.e., Lecanosticta acicola or Cenangium ferruginosum, were found. More extensive molecular surveying based on β-tub2 amplification of DNA in needle samples from 72 seed trees of P. sylvestris in nine different Forest Districts of Southern Poland did not find the presence of D. septosporum. Our study revealed that the seed trees from which we collected propagation material were free from the pathogen, and its endophytic behavior was not confirmed in our testing. Consequently, these investigated trees of P. sylvestris should be suitable for seed collection and propagation, following the requirements of “good” phytosanitary quality as “pathogen-free” pine seeds used for reforestation.

First report of Mycosphaerella dearnessii Rostrup, teleomorph of Lecanosticta acicola (Thüm.) Syd., in Europe

Conifers severely affected by brown needle blight disease caused by the ascomycete Lecanosticta acicola (Thüm.) Syd. show defoliation, reduced growth and death. Although L. acicola is known in Europe since 1942, its teleomorph, Mycosphaerella dearnessii Rostrup, has not yet been found. In this brief, we address the occurrence of Mycosphaerella dearnessii in Pinus radiata D. Don cast dead needles collected in the north of Spain (Basque country). To our knowledge, this is the first report of Mycosphaerella dearnessii Rostrup, teleomorph of Lecanosticta acicola (Thüm.) Syd., in Europe.

Rapid Detection of Pine Pathogens Lecanosticta acicola, Dothistroma pini and D. septosporum on Needles by Probe-Based LAMP Assays

Needle blights are serious needle fungal diseases affecting pines both in natural and productive forests. Among needle blight agents, the ascomycetes Lecanosticta acicola, Dothistroma pini and D. septosporum are of particular concern. These pathogens need specific, fast and accurate diagnostics since they are regulated species in many countries and may require differential management measures. Due to the similarities in fungal morphology and the symptoms they elicit, these species are hard to distinguish using morphological characteristics. The symptoms can also be confused with those caused by insects or abiotic agents. DNA-based detection is therefore recommended. However, the specific PCR assays that have been produced to date for the differential diagnosis of these pathogens can be applied only in a well-furnished laboratory and the procedure takes a relatively long execution time. Surveillance and forest protection would benefit from a faster diagnostic method, such as a loop-mediated isothermal amplification (LAMP) assay, which requires less sophisticated equipment and can also be deployed directly on-site using portable devices. LAMP assays for the rapid and early detection of L. acicola, D. pini and D. septosporum were developed in this work. Species-specific LAMP primers and fluorescent assimilating probes were designed for each assay, targeting the beta tubulin (β-tub2) gene for the two Dothistroma species and the elongation factor (EF-1α) region for L. acicola. Each reaction detected its respective pathogen rapidly and with high specificity and sensitivity in DNA extracts from both pure fungal cultures and directly from infected pine needles. These qualities and the compatibility with inexpensive portable instrumentation position these LAMP assays as an effective method for routine phytosanitary control of plant material in real time, and they could profitably assist the management of L. acicola, D. pini and D. septosporum.

Development of a Rapid Loop-Mediated Isothermal Amplification Assay for the Detection of Dothistroma septosporum

A Loop-Mediated Isothermal Amplification (LAMP) assay was developed for the detection of the pine pathogen Dothistroma septosporum (G. Dorog.) M. Morelet. The specificity of the LAMP assay was tested using a selection of pine needle fungi, including Dothistroma pini Hulbary, and Lecanosticta acicola (Thüm.) Syd.; only D. septosporum DNA was amplified by the test. In terms of sensitivity, the assay was able to detect as little as 1 pg of total D. septosporum DNA. This assay enables DNA extracted from diseased host needles to be rapidly tested for the presence of D. septosporum using relatively simple to operate equipment away from a fully equipped molecular biology laboratory.