Variability in Alternaria alternata spore characteristics under different culture conditions: implications for automatic detection using air flow cytometry

Vol.: (0123456789) Aerobiologia https://doi.org/10.1007/s10453-024-09831-z ORIGINAL PAPER Variability inAlternaria alternata spore characteristics underdifferent culture conditions: implications forautomatic detection using air flow cytometry IoannaPyrri· NicolasBruffaerts· MarkoRadovic· ElizabetD’hooge· LjiljanaJanjusevic· BrankoSikoparija Received: 3 April 2024 / Accepted: 28 June 2024 © The Author(s), under exclusive licence to Springer Nature B.V. 2024 revealed overall differences in spore morphology pattern. Standardizing culture conditions and accounting for variability in spore properties are essential for improving the reliability of bioaerosol monitoring systems. Further research is needed to refine detection methods for A. alternata and other airborne fungal spores. Keywords Fungi· In vitro culture· Spore morphology· Raman spectrum 1 Introduction Alternaria Nees is a ubiquitous, omnipresent genus comprising approximately 300 species exhibiting diverse functional modes ranging from saprobes, to endophytes, to pathogens (Woudenberg et al., 2013). Species of Alternaria are known as serious plant pathogens causing blights, leaf spots, and rots on over 380 host species, postharvest pathogens, causative agents of phaeohyphomycosis in immuno-compromised patients or airborne allergens (Samson et al., 2004). Among Alternaria spp., A. alternata (Fr.) Keissl. (1912) is one of the most common and most studied fungi of the aeromycota (Woudenberg etal., 2015). Prolonged exposure to A. alternata can increase the risk of allergic asthma development and exacerbations (Rivas etal., 2022) and the risk is not limited to the outdoor environment only as confirmed by identification of notable quantity of Alt a 1 antigen in dust sampled from furniture and floors Abstract Airborne fungal spores of the genus Alternaria pose challenges for accurate airborne spore identification by automatic bioaerosol monitors, because of their significant implications for public health and agriculture due to their role as airborne allergen and plant pathogen. These systems require high-quality reference data for training algorithms by machine learning. Alternaria alternata was cultured on three different media, including exposure to UV light to favor sporulation. Spore morphology was evaluated both macroscopically and microscopically, and chemical analysis was conducted using micro-Raman spectroscopy to assess spore composition. Significant differences were observed in colony morphology and spore characteristics among culture media. While typical spores predominated, atypical forms were also identified, which may represent a potential bias for identification. Comparative analysis with air samples by the Hirst method also I.Pyrri(*) Department ofBiology, Section ofEcology andSystematics, National andKapodistrian University ofAthens, Athens, Greece e-mail: ipyrr[email protected]r N.Bruffaerts· E.D’hooge Mycology andAerobiology, Sciensano, Brussels, Belgium M.Radovic· L.Janjusevic· B.Sikoparija BioSense Institute, Research Institute forInformation Technologies inBiosystems, University ofNovi Sad, NoviSad, Serbia Aerobiologia Vol:. (1234567890) and their significant positive correlation to appearance of asthma symptoms in inhabitants (Salo etal., 2006). Because of the significant negative effects of Alternaria on public health, a rapid, accurate identification and quantification of Alternaria spores present in the air would be of great value. In recent years, automated monitors have been developed for real-time measurements of pollen grains and fungal spores suspended in the outdoor air. These devices automatically identify bioaerosols of interest based on their morphology and chemical properties (Huffman etal., 2020) by using advanced machine learning methods that usually depend on the reference data upon which they will be trained (Buters et al., 2024). Tummon etal. (2024) recently emphasized the importance of standardizing these automatic bioaerosol monitoring methods by using high-quality training datasets to maximize the performance of measurements. These data can be collected either by exposing the instrument to aerosolized bioparticles of known taxonomical identity or by the manual selection of environmental observations. For fungal spores, only the latter approach has been tested operationally, confirming that it either does not provide specific enough data for identifying separate taxa (Simović etal., 2023) or it requires a tedious cleaning procedure (Erb etal., 2024). Therefore, for a proper application in aerobiological monitoring, it seems more reasonable to propose a reference data creation protocol based on in vitro growth and sporulation of fungi. However, the variability in sporulation performance and in the characteristics of fungal spores grown on different culture media are serious limitations in the way to standardize such method. This approach involves supplying reference spore material to an automated monitor in order to accurately identify each particle type. To achieve this, dominant airborne fungi such as Alternaria are cultured to harvest their spores and supply them to the samplers. This investigation aims to investigate a cultivation-based approach to produce A. alternata spores suitable for tests in morphology-based automatized aerobiological monitoring. 2 Materials andmethods 2.1 Alternaria alternata strain, growth and sporulation conditions The A. alternata strain IHEM 18586 from the Belgian collection for medical and veterinary fungi and yeast (BCCM-IHEM) was cultured in three broad spectrum media for fungi, namely Potato Dextrose Agar (PDA) ([https:// bccm. belspo. catal ogs be// ihemmediadetai ls? Cultu reMed iaID= 905]), Malt extract Agar (MA) ([https:// bccm. belspo. be/ catal ogs/ ihemmediadetai ls? Cultu reMed iaID= 902]) and V8 agar (V8) (5% V8 juice (Campbell Soup Co.), 1g MgSO4•7H2O, 1g KH2PO4, 20g Pastagar, 1mL oligoelements for 1L at pH 5.5). The lyophilized stock product was previously cultured in a slant tube with diluted Sabouraud medium (S10) ([https:// bccm. belspo. be/ catal ogs/ ihemmediadetai ls? Cultu reMed iaID= 908]) in order to reactivate the growth capacity of the strain. In total, 76 Petri dishes were inoculated, i.e. 36 on PDA, 24 on MA and 16 on V8. Inoculation was performed by striating the whole dish surface with a piece of S10 medium that is loaded with sporulating material in order to avoid forming canyons of sterile mycelium between colonies. The dishes were incubated at 25°C in the dark for 7–10days. In addition, one Petri dish of each medium was selected for exposure under UV light (366nm) for 3days in order to further stimulate sporulation, as compared to non-exposed control dishes. 2.2 Morphological analysis The growth rate and sporulation of colonies were evaluated after two weeks, both macroscopically and microscopically by using a stereoscope. Lactic acid-based mounts were prepared from samples taken from the center, the middle and the edge of the sporulating colonies that were visible in the Petri dish. In total, 1000 individual spores were randomly counted and grouped into two categories, the typical multicellular pear-shaped conidia with beak and the non-typical unicellular ones that are globose to subglobose to ellipsoidal. In addition, samples obtained by the Hirst method, by using a 7-day volumetric spore sampler (Burkard Manufacturing Co Ltd or Lanzoni s.r.l.), were selected to perform a side-by-side comparison with the Aerobiologia Vol.: (0123456789) cultured samples. A day with very high concentration of Alternaria spores was selected for each of the three following geographic areas: Brussels (Belgium) on 11/08/2023, Kanjiža (Serbia) on 13/08/2023 and Athens (Greece) on 7/7/2023. The whole sampling surface was observed and all Alternaria spores were counted and characterized. Microscopic observations were made using a Nikon Eclipse E600 microscope (Nikon Corporation, Tokyo, Japon) at 400X magnification and photographed by the camera with the NIS-Elements software (version 3.0). Slides prepared from UV-exposed cultures as well as the slides selected from the Hirst method were observed with a Zeiss AxioImager A1 Differential Interference Contrast microscope at 400X magnification and photographed. 2.3 Chemical analysis Spores from each growth condition were harvested with a suitable pump into a dry modified Eppendorf tube (Bruffaerts et al., in preparation) for subsequent analysis. Micro-Raman spectroscopy investigation of the chemical peculiarities of individual A. alternata spores was performed on confocal Horiba Xplora plus system equipped with 532nm laser excitation source, 100 × objective, Peltier cooled CCD detector and piezo-controlled sample stage. Substrates for Raman measurements were prepared using e-beam evaporation technique on Si wafer. Initially, thin film of Ti was deposited as an adhesion layer, followed by deposition of 200nm thick gold layer. The Raman spectra of single spores were collected in the 500–2000 cm−1 range, using 1800g mm−1 grating and with 10% of 532nm laser power (approx. 8mW). The depth mapping profile of A. alternata spores was established using an automated Z controller to examine the distribution of Raman signature features at various locations from the surface to the middle of the cell. The reference or zero point was determined by adjusting the optical image until the edge of the spore was clearly visible and then a number of measurements were recorded with 100nm Z step size. 3 Results anddiscussion 3.1 Macromorphology of sporulating colonies The macromorphological features of the A. alternata colonies growing on the selected broad spectrum synthetic nutrient media were recorded in detail. The cultured Alternaria strain grew rapidly and matured within 5–7days. Colony diameter reached a size of 6–9 cm following incubation at 25 °C for approximately 7 days. The colonies on PDA were densely floccose, dark olive brown with several areas covered by grayish secondary mycelium (Fig.1). The colonies grown on MA after 7–10days of incubation were dark olive brown to black, granulate with few areas of gray secondary mycelium (Fig.1). Colonies grown on V8 after 7–10days of incubation were olivaceous brown, sparsely granulose and with very few areas of hyaline secondary mycelium (Fig.1). Mycelium was present in MA cultures but almost absent in V8 cultures. In general, sporulation was the most abundant on MA. The colonies exposed to UV light did not develop gray aerial mycelium (Fig.1). When the different cultures were observed under a stereomicroscope, it was evident that the colonies on V8 consisted of simple, short conidiophores producing branched chains of porospores, whereas on PDA and MA the vegetative phase was mainly showing. The macromorphology of colonies incubated under UV light was similar to the ones grown without UV irradiation with the exception that on PDA and MA there were no gray areas of aerial mycelium (Fig.1). 3.2 Micromorphology of sporulating colonies The micromorphology of A. alternata was also studied on all three culture media. Hyphae were septate and olivaceous. Conidiophores were short, brown in color, occasionally having a zig-zag appearance toward the apex. Typical A. alternata conidia were pale brown to dark brown, obclavate to obpyriform, small or moderate in size, with several transverse and few oblique and/or longitudinal septa, constricted at the septa, with a short beak at the tip or beakless, smooth to verruculose. They were produced in simple (PDA) to complex branched chains (MA, V8) depending on the culture medium. Secondary conidiophores were formed apically or laterally with one or a few conidiogenous loci. Abundant sporulation was Aerobiologia Vol:. (1234567890) observed on MA colonies followed by similar sporulation on PDA and V8 but with a striking difference on the amount of vegetative phase that was prevalent on PDA. Ginoya et al. (2015) who tested A. alternata growth characteristics on PDA, Host leaf extract agar, Host fruit extract agar, Oatmeal agar, Richards’ agar, Czapek’s Dox agar and Rose Bengal agar, noticed abundant sporulation on PDA and variations of conidia size and beak length among the media. They also reported significant variation among isolates from diverse geographic regions grown on the same medium thus concluding that there is an inherent variation. In addition, irregular, atypical spore shapes were present (Fig. 2). There were globose to subglobose aseptate brown spores as well as hyaline to pale Fig. 1 Colony morphology of Alternaria alternata (IHEM 18586) 1–3. MA, PDA, V8 after 7–10days of incubation, 4–6. MA, PDA, V8 after 7days of incubation, 7–9. MA, PDA, V8 after 10days of incubation the last 3 under UV-light Aerobiologia Vol.: (0123456789) ellipsoidal 0–1 septate spores. The culture media used differed in the amount of atypical spores that were present, with PDA containing the highest amount of atypical spores, both with and without UV exposure (Table1). Colonies on PDA and V8 and exposed to UV light exhibited higher rates of atypical spores. On PDA (Fig.2), the vegetative phase dominated but there were more obclavate conidia with a short beak Fig. 2 Alternaria alternata (IHEM 18586) spores on MA (1–2), on PDA (3–4) and on V8 (5–6) incubated under UV light. Atypical conidia in circles Aerobiologia Vol:. (1234567890) and with oblique and longitudinal septa. Porospores with an obovoid to barreloid shape, similar to spores of the former genus Ulocladium, were also present as in all PDA colonies (Fig.2). Selected samples obtained by the Hirst method in dates with anticipated high Alternaria concentration and originating from Brussels, Kanjiža and Athens were also studied in detail. Alternaria spores trapped on the slides were observed very carefully in order to evaluate the diversity in morphology and the presence of atypical spores like those in the cultures on synthetic media. The entire sampling area on the slide was thoroughly scanned. In the examined slides, there were spores corresponding to typical A. alternata conidia but also to spore-morphology of other species present in the air, with longer spores and elongated beaks (Fig.3). Spores without beak, with less or no longitudinal septa, broken or desiccated were also observed and they were all considered as Alternaria sensu lato to include those that cannot be differentiated from allied genera. Immature spores single or two celled similar to those found in our cultures they were not detected in the examined slides from Kanjiža, Brussels and Athens (Table2). In a quality control exercise (Galán etal., 2021) where an environmental sample from Szczecin (Poland) was examined Table 1 Number of spores counted in ten random optical fields in mounts prepared from 1 mm2 piece of colony transferred to lactic acid. Mounts were prepared in triplicates from the center, middle and edge of the colony. The average spore values and the atypical/typical ratio are depicted Synthetic medium Typical Atypical Ratio (%) Center Middle Edge Average SD Center Middle Edge Average SD MA 2035 1816 2153 2001 171 146 99 114 119.67 24 7.2 MA UV 2135 2116 1932 2061 112 127 110 98 111.67 15 5.9 PDA 1169 973 954 1032 119 183 137 133 151.00 28 15.6 PDA UV 806 753 673 744 67 165 142 125 144.00 20 20.5 V8 1181 985 422 862 394 70 55 58 61.00 8 5.9 V8 UV 1123 1041 387 850 403 83 75 66 74.67 9 7.4 Fig. 3 Alternaria spores on environmental air sampling slides from Brussels (1), Kanjiža (2) and Athens (3) Table 2 Number of Alternaria spores counted in selected 24h samples collected using Hirst type method in three locations in Europe. The total spore counts over the entire sample surface and the typical/atypical ratio are depicted Environmental air sampling location Sampling date Alternaria spores counts Ratio (%) Typical Atypical Athens (Greece) 7/7/2023 9250 0 0 Brussels (Belgium) 11/8/2023 5520 0 0 Kanjiža (Serbia) 13/8/2023 7346 0 0 Aerobiologia Vol.: (0123456789) from diverse analysts, a total of 18.8% intact, atypical, Alternaria conidia were present, as young ones and non-beaked conidia. The non-beaked group included non-beaked Alternaria spp., atypical conidia of usually beaked Alternaria spp. and Ulocladium spp. which has been placed in Alternaria based on molecular data (Woudenberg etal., 2013). Immature spores not detected on the examined slides are significantly smaller, almost hyaline young spores and thus difficult to differentiate from other fungi or can be obscured by particulate matter trapped on the sampling tapes. In the cultures and on natural substrates represented in the air, these spores are a small fraction and their absence in the selected dates may also be a matter of chance. The identification of Alternaria spp. based on spore morphology had always been a challenge due to the plasticity they exhibit depending on the environmental parameters. Conidia are smaller with shorter beaks and less uniform in size and morphology on artificial media than on natural substrata, but near typical conidia can be produced in vitro by choice of media and culture conditions (Misaghi etal., 1978). In order to overcome this variability, Taralova etal. (2011) suggested the use of a generalized statistical model for the three-dimensional geometric structure of the sporulation apparatus that enables automated species identification from microscopy images using statistical inference originating from all the morphologically diverse porospores produced in various conidiophores. Also, using double-immunostaining technique for detecting Alternaria gives promising results both for identification of conidia and hyphal fragments (Green etal., 2005). However, such detection approaches have limited value in high throughput real-time analysis of aerobiological samples. Aerobiological research nowadays focuses in the automatization of airborne bioparticles monitoring by training devices to identify accurately the diverse airspora. Pollen are collected directly from the anthers of flowering plants but fungi are more complex and cultures of accurately identified species should be used. In order to overcome the variations in spore morphology observed when culturing on synthetic media, standardization of growth parameters is necessary to mitigate this limitation and yield spores that are near typical as on natural substrates. 3.3 Raman spectroscopy Influence of different growth culture media on Raman spectra did not yield noticeable variations (Fig. 4). Most prominent features of presented spectra are two broad modes at 1355 and 1585 cm−1, with noticeable background signal. Obtained results are almost identical to spectra published previously by Volkov and Perry (2021) in a comprehensive study of Alternaria species with Raman and UV-Vis spectroscopy that assigned the Raman modes to characteristic molecules vibrations. According to calculated resonant Raman spectra the broad mode observed at 1585 cm−1 represents convolution of two sharp modes, originating from C-C ring stretching (Lin etal., 2020; Volkov & Perry, 2021). Almost the same Raman spectra can be obtained from different fungal species i.e. Lentinula edodes, Ganoderma lucidum, Pleurotus cornucopiae, etc. (Puliga etal., 2022), where the observed modes in 1100–1600 cm−1 range are assigned to polysaccharides. Polysaccharides play a crucial role in the structure and function of a cell wall (Gow et al., 2017; Rodrigues etal., 2011; San-Blas etal., 1994). In the context of Alternaria spores, polysaccharides are key components that provide structural integrity and protection to the spore. They form long chains of sugar molecules that are rich in C-C bonds contributing to observed vibrational modes detected in Raman spectra. The mode at 1355 cm−1 can be ascribed to Fig. 4 Comparison of averaged Raman spectra for Alternaria alternata spores grown in different culture medium. Each spectrum represents averaged data, accumulated from 10 different spores Aerobiologia Vol:. (1234567890) C-H bond vibrations (in-plane bending and wagging) (Yuen etal., 2009), since all measured spores exhibited the same features. When the excitation laser is directed deeper into the spore there is an overall increase in Raman intensity, primarily due to a strong background signal (Fig.5). Observed behavior can be explained by the fact that the body of spore is rich in proteins, DNA and other molecules that can easily contribute to appearance of luminescence or fluorescence, which is manifesting as strong background signal (Lian etal., 2023). Recent studies already shown the potential of Raman spectroscopy in discriminating pollen species (Kendel & Zimmermann, 2020), even those that are closely related (Zimmermann, 2010). Utilization of Raman spectroscopy for process automatization in this case can introduce significant benefits. Most certainly, some of the spectral features can be used to distinguish between aerosol particles such as fungal spores or pollen. However, the efficiency of measurements in real time conditions is a challenge. Therefore, measurement parameters need to be carefully optimized. In order to collect enough signal in short time interval, laser power should be increased and grating with higher signal should be used at the cost of spectral resolution. Spectral bandwidth should be selected to a narrow range where fingerprint peaks are dominant. Another approach may involve the so-called Surface Enhanced Raman Spectroscopy (SERS) technique, where the measured Raman signal is increased by the appropriate selection of substrates (silver or gold) or with utilization of noble metal nanoparticles. This technique can offer an order of magnitude increase in Raman intensity, necessary to meet the imposed conditions of real time analysis. SERS method is also a more convenient approach to integration of Raman equipment into aerosol collectors, since the excitation setup and signal collection can be executed with optical fiber coupled to laser and monochromator. Providing sampling and measurement parameters are optimized, Raman spectroscopy can be a very powerful tool for automated aerosol monitoring and discrimination of bioparticles. And it may contribute to a variety of methods tested for specific detection of bioaerosols (Kim etal., 2016). 4 Conclusions Clear variations in both macromorphological and micromorphological features of Alternaria alternata were observed following growth and sporulation on different culture media. The macroscopic evaluation showed differences in colony morphology among the media tested, and exposure to UV light stimulated sporulation in MA without altering the overall colony morphology. Microscopic analysis revealed further insights into the micromorphological characteristics of A. alternata spores. While typical conidia with pear-shaped morphology were predominant, some atypical and young spores were also observed, particularly in cultures grown on PDA. Comparative analysis with environmental samples obtained through the Hirst method indicated differences in spore morphology between cultured and airborne spores. While cultured spores exhibited variability and atypical forms, these were not observed in the environmental samples, suggesting potential differences in spore characteristics between in vitro cultured reference material and what is detected in the outdoor air. Furthermore, analysis of the spore’s internal structure with Raman spectroscopy showed that cell wall is rich in polysaccharides, while its interior contains nucleotides (DNA), emitting a strong fluorescence signal. This implies that just alternating the focus of excitation laser could result in the variability of fluorescence measurements of single A. alternata spores. Fig. 5 Raman spectra for different depths ranging from top to bottom of Alternaria alternata spore Aerobiologia Vol.: (0123456789) In all, these findings underscore the importance of understanding the influence of culture conditions on spore characteristics, especially concerning their application in automatic detection methods such as airflow cytometry. It is crucial that the reference biological material used for training identification models by machine learning is representative of the diversity of bioaerosols found in nature, as measured by the standard Hirst method. Standardizing culture conditions and understanding the variability factors in spore morphological and fluorescence properties in order to produce spores that approach morphologically the spores from natural substrata are essential for improving the accuracy and reliability of automatic bioaerosol monitoring systems. Further research exploring additional culture parameters and their effects on spore characteristics is warranted to refine detection methods for A. alternata and other airborne fungal spores of interest. Acknowledgements COST Action ADOPT through STSMs: E-COST-GRANT-CA18226-fa273a24, E-COST-GRANTCA18226-e04047ee, E-COST-GRANT-CA18226-d7d22ed3, Ministry Science, Technological Development and Innovations of the Republic of Serbia (Grant agreement no. 200358) and the project 23NRM03 BioAirMet that has received funding from the European Partnership on Metrology, co-financed from the European Union’s Horizon Europe Research and Innovation Programme and by the Participating States. Author contributions Conceptualization: Nicolas Bruffaerts, Branko Sikoparija; Methodology: Nicolas Bruffaerts, Branko Sikoparija, Ioanna Pyrri; Formal analysis and investigation: Nicolas Bruffaerts, Branko Sikoparija, Ioanna Pyrri, Marko Radovic, Ljiljana Janjusevic; Writing—original draft preparation: Ioanna Pyrri; Writing—review and editing: Nicolas Bruffaerts, Branko Sikoparija, Elizabet D’hooge, Marko Radovic, Ljiljana Janjusevic; Funding acquisition: Nicolas Bruffaerts, Branko Sikoparija; Resources: Nicolas Bruffaerts, Branko Sikoparija, Elizabet D’hooge; Supervision: Nicolas Bruffaerts, Branko Sikoparija. Funding No funding was received for conducting this study. Data availability The data that support the findings of this study are available from the authors upon reasonable request. Declarations Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Buters, J., Clot, B., Galán, C., Gehrig, R., Gilge, S., Hentges, F., O’Connor, D., Sikoparija, B., Skjøth, C., Tummon, F., Adams-Groom, B., Antunes, C., Bruffaerts, N., Celenk, S., Crouzy, B., Guillaud, G., Hajkova, L., Seliger, A., Oliver, G., … Stjepanovic, B. (2024). Automatic detection of airborne pollen: An overview. Aerobiologia, 40, 13–37. https:// doi. org/ 10. 1007/ s1045302209750-x Erb, S., Berne, A., Burgdorfer, N., Clot, B., Graber, M.-J., Lieberherr, G., Sallin, C., Tummon, F., & Crouzy, B. (2024). Automatic real-time monitoring of fungal spores: the case of Alternaria spp. Aerobiologia, 40, 123–127. https:// doi. org/ 10. 1007/ s1045302309780-z Galán, C., Smith, M., Damialis, A., Frenguelli, G., Gehrig, R., Grinn-Gofroń, A., EAS QC Working Group. (2021). Airborne fungal spore monitoring: between analyst proficiency testing. Aerobiologia, 37, 351–361. https:// doi. org/ 10. 1007/ s1045302109698-4 Ginoya, C. M., & Gohel, N. M. (2015). Cultural and morphological variability among the isolates of Alternaria alternata (Fr.) Keissler, incitant of fruit rot of chilli. International Journal of Plant Protection, 8(1), 118–125. https:// doi. org/ 10. 15740/ HAS/ IJPP/8. 1/ 118125 Gow, N. A. R., Latge, J.-P., & Munro, C. A. (2017). The fungal cell wall: Structure, biosynthesis, and function. Microbiol Spectrum, 5(3), 10–1128. Green, B. J., Schmechel, D., & Tovey, E. R. (2005). Detection of aerosolized Alternaria alternata conidia, hyphae, and fragments by using a novel double-immunostaining technique. Clinical and Vaccine Immunology, 12(9), 1114– 1116. https:// doi. org/ 10. 1128/ cdli. 12.9. 11141116. 2005 Huffman, A. J., Perring, A. E., Savage, N. J., Clot, B., Crouzy, B., Tummon, F., etal. (2020). Realtime sensing of bioaerosols: Review and current perspectives. Aerosol Science and Technology, 54(5), 465–495. https:// doi. org/ 10. 1080/ 02786 826. 2019. 16647 24 Kendel, A., & Zimmermann, B. (2020). Chemical analysis of pollen by FT-Raman and FTIR spectroscopies. Frontiers in Plant Science, 11, 352. https:// doi. org/ 10. 3389/ fpls. 2020. 0035 Kim, J., Jin, J. H., Kim, H. S., Song, W., Shin, S. K., Yi, H., & Lee, B. Y. (2016). Fully automated field-deployable bioaerosol monitoring system using carbon nanotube-based biosensors. Environmental Science & Technology, 50(10), 5163–5171. Lian, K., Chen, G., Wang, X., Zhang, W., Hu, X., Wang, H., Li, Y., Xia, D., & Wang, Y. (2023). Fluorescent detection of brown spot of tobacco caused by Alternaria alternata based on lambda exonuclease-induced DNAzyme amplification. RSC Advances, 13, 1587. https:// doi. org/ 10. 1039/ D2RA0 5616J Lin, Y.-J., Lin, H.-K., & Lin, Y.-H. (2020). Construction of Raman spectroscopic fingerprints for the detection of Fusarium wilt of banana in Taiwan. PLoS ONE, 15(3), e0230330. https:// doi. org/ 10. 1371/ journ al. pone. 02303 30 Misaghi, I. J., Grogan, R. G., Duniway, J. M., & Kimble, K. A. (1978). Influence of environment and culture media on spore morphology of Alternaria alternata. Phytopathology, 68(1), 29–34.