Airborne pollen are largely studied to obtain information about the atmospheric content of natural allergens. Aerobiological monitoring networks have been established to provide reliable data that facilitate the timely initiation of preventive actions aimed at minimizing allergic symptoms. Airborne pollen are usually identified and counted using an optical microscope, but as such procedures are extremely time-consuming, more expedient options are being explored. We have assessed the potential of Fourier transform infrared (FT-IR) spectroscopy as an alternative method for the rapid and reliable identification of allergenic pollen using six well-known allergenic pollen taxa and obtaining the respective FT-IR spectra. In doing this, a first IR spectral library has been created. The spectra of unknown pollen were compared to those of the reference library, and two pollen taxa of a mixed sample were identified.

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Metabarcoding is a promising DNA-based method for identifying airborne pollen from environmental samples with advantages over microscopic methods. This method requires several preparatory steps of the samples, with the extraction protocol being of fundamental importance to obtain an optimal DNA yield. Currently, there is no consensus in sample preparation and DNA extraction, especially for gravimetric pollen samplers. Therefore, the aim of this study was to develop protocols to process environmental samples for pollen DNA extraction and further metabarcoding analysis, and to assess the efficacy of these protocols for the taxonomic assignment of airborne pollen, collected by gravimetric (Tauber trap) and volumetric samplers (Burkard spore trap). Protocols were tested across an increasing complexity of samples, from single-species pure pollen to environmental samples. A short fragment (about 150 base pair) of chloroplast DNA was amplified by universal primers for plants (trnL). After PCR amplification, amplicons were Sanger-sequenced and taxonomic assignment was accomplished by comparison to a custom-made reference database including chloroplast DNA sequences of 46 plant families, including most of the anemophilous taxa occurring in the study area (Trentino, Italy, Eastern Italian Alps). Using as a benchmark the classical morphological pollen analysis, it emerged that DNA metabarcoding is applicable efficiently across a complexity of samples, provided that sample preparation, DNA extraction and amplification protocols are specifically optimized.

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