Autofluorescence Is a Common Trait in Different Oceanic Fungi

Journal of Fungi

Communication Autoﬂuorescence Is a Common Trait in Different Oceanic Fungi

Eva Breyer *, Markus Böhm, Magdalena Reitbauer, Chie Amano , Marilena Heitger and Federico Baltar *

Department of Functional and Evolutionary Ecology, University of Vienna, 1030 Vienna, Austria; [email protected] (M.B.); [email protected] (M.R.); [email protected] (C.A.); [email protected] (M.H.) * Correspondence: [email protected] (E.B.); [email protected] (F.B.)

Abstract: Natural autofluorescence is a widespread phenomenon observed in different types of tissues and organisms. Depending on the origin of the autofluorescence, its intensity can provide insights on the physiological state of an organism. Fungal autofluorescence has been reported in terrestrial and human-derived fungal samples. Yet, despite the recently reported ubiquitous presence and importance of marine fungi in the ocean, the autofluorescence of pelagic fungi has never been examined. Here, we investigated the existence and intensity of autofluorescence in five different pelagic fungal isolates. Preliminary experiments of fungal autofluorescence at different growth stages and nutrient conditions were conducted, reflecting contrasting physiological states of the fungi. In addition, we analysed the effect of natural autofluorescence on co-staining with DAPI. We found that all the marine pelagic fungi that were studied exhibited autofluorescence. The intensity of fungal autofluorescence changed depending on the species and the excitation wavelength used. Furthermore, fungal autofluorescence varied depending on the growth stage and on the concentration of available nutrients. Collectively, our results indicate that marine fungi can be auto-fluorescent, although its

intensity depends on the species and growth condition. Hence, oceanic fungal autoﬂuorescence should be considered in future studies when fungal samples are stained with ﬂuorescent probes

Citation: Breyer, E.; Böhm, M.; (i.e., fluorescence in situ hybridization) since this could lead to misinterpretation of results. Reitbauer, M.; Amano, C.; Heitger, M.; Baltar, F. Autofluorescence Is a Keywords: marine fungi; fluorescence in situ hybridisation; mycoplankton; fungal cultures; Common Trait in Different Oceanic pelagic; fluorescence Fungi. J. Fungi 2021, 7, 709. https:// doi.org/10.3390/jof7090709

Academic Editor: Wei Li 1. Introduction Natural autofluorescence is a process in which endogenous cell compounds i.e., aro- Received: 19 July 2021 matic amino acids, become fluorescent when excited with light in a specific wavelength. Accepted: 26 August 2021 Depending on the type and origin of the fluorescent compound, the intensity of the emitted Published: 29 August 2021 light can change with the morphological or physiological state of the observed cells or tissue and, hence, the organism [1]. Publisher’s Note: MDPI stays neutral Autofluorescence in fungi was first observed when human tissue sections, used to with regard to jurisdictional claims in diagnose mycotic infectious diseases, were exposed to UV-light. Diverse fungi including published maps and institutional affil- iations. Candida, Aspergillus, Blastomyces, Cryptococcus and Coccidioides, emitted fluorescent light, allowing their easy detection in infected tissue samples without prior staining [2,3]. Moreover, experiments with terrestrial-derived fungal cultures investigating autofluorescence in spores revealed differences in fluorescence intensities between fungal species, which might be related to different degrees of cellular viability [4]. In contrast, it was found Copyright: © 2021 by the authors. that arbuscular mycorrhizal fungal structures were auto-fluorescent under blue and green Licensee MDPI, Basel, Switzerland. light excitation despite their viability [5]. Likewise, fungal autofluorescence persisted after This article is an open access article cell death following sample fixation, further demonstrating its diagnostic value for direct distributed under the terms and conditions of the Creative Commons analyses of histological samples [6]. Attribution (CC BY) license (https:// The origin of fungal autofluorescence is still not clear. It has been hypothesized that creativecommons.org/licenses/by/ chitin could be the cause for fungal autofluorescence due to the similarities observed 4.0/). when fungal cells were stained with Calcofluor White (that binds to chitin in fungal cell

J. Fungi 2021, 7, 709. https://doi.org/10.3390/jof7090709 https://www.mdpi.com/journal/jof J. Fungi 2021, 7, 709 2 of 10

walls) [5]. Recently, ergosterol, a membrane lipid found in fungal cell walls in the sub- kingdom of Dikarya used to quantify fungal biomass was suggested as another source of autoﬂuorescence [7]. Previous research only focused on terrestrial and human-associated fungi. Re- cently, the important contribution of marine fungi to oceanic microbial food webs [8–10], biomass [11,12] and functional diversity [13,14] has been highlighted. Despite the ubiquitous presence of pelagic fungi in the oceanic water column, there are no investigations studying the existence of autofluorescence of oceanic fungi, and on the factors affecting the presence and intensity of this autofluorescence. To fill this gap of knowledge, we studied the autofluorescence of five marine pelagic fungi and investigated their autofluorescence at different growth stages. Furthermore, we also investigated the effect of varying nutrient concentrations and DAPI co-staining (a common DNA-stain in microbiology) on this fungal autofluorescence. Based on previous research, we hypothesized that marine fungi would be auto-fluorescent, similarly to certain terrestrial and human-associated fungi [2,3,5]. We also hypothesized that the autofluorescence intensity changes with species and physiological state, since it is possible that the production and distribution of auto-fluorescent compounds changes in response to growth and physiological state [1].

2. Materials and Methods 2.1. Cultivation of Marine Fungal Cultures Four marine fungal cultures (Metschnikowia australis, Rhodotorula sphaerocarpa, Sak- aguchia dacryoidea, Blastobotrys parvus) obtained from the Austrian Centre of Biological Resources (ACBR), and one fungal species (Rhodotorula sp.) isolated during the ‘Poseidon’ research cruise in 2019, were used to study the existence and intensity of autofluorescence in growth experiments (Table1). All the five fungal species used (four yeasts and one hyphae-morphotype) were originally isolated from open ocean waters. The fungi were grown in the dark at room temperature on solid agar media containing (g/L): 10 g glucose, 5 g peptone, 3 g yeast extract, 3 g malt extract, 35 g artificial sea salts, 20 g agar and 0.5 g chloramphenicol.

Table 1. Marine fungi used for the autoﬂuorescence experiments.

Species Division Origin of Isolation ACBR Code Metschnikowia australis Ascomycota Antarctic Ocean HA635 Rhodotorula sphaerocarpa Basidiomycota Antarctic Ocean, Marguerite Bay HB738 Sakaguchia dacryoidea Basidiomycota Antarctic Ocean HB877 Blastobotrys parvus Ascomycota Antarctic Ocean HA1620 Rhodotorula sp. Basidiomycota Atlantic Ocean -

Aliquot samples of the yeasts were transferred and grown in liquid media (Table2) to obtain samples at speciﬁc growth stages. All yeasts were cultured in the oligotrophic medium, and S. dacryoidea was additionally cultured in the eutrophic medium. The liquid cultures were incubated on a shaker incubator (Argo Lab, Ski 4, 140 rpm, Carpi, Italy) at room temperature under normal day-night light regime. The exponential growth stage was sampled in all species according to the daily measured optical density (UV-1800 Shimadzu spectrophotometer, λ = 660 nm, Kyoto, Japan). Additionally, S. dacryoidea was sampled in the stationary growth stage. Sampling of the different growth stages were based on previous experiments and on the shape of the growth curve (Table3, Figure1). J. Fungi 2021, 7, x FOR PEER REVIEW 3 of 10 J. Fungi 2021, 7, 709 3 of 10

Table 2. Growth media used for marine yeast liquid cultures. Table 2. Growth media used for marine yeast liquid cultures. Chemicals (g/L) Eutrophic Medium Oligotrophic Medium Chemicals (g/L)Glucose Eutrophic Medium2 Oligotrophic Medium 0.2 GlucosePeptone 22 0.2 0.2 Peptoneyeast extract 22 0.2 0.2 yeast extractsea salts 235 0.2 35 sea salts 35 35 chloramphenicolchloramphenicol 0.50.5 0.5 0.5

Table 3. Optical density (OD) of different fungal growth stages. Table 3. Optical density (OD) of different fungal growth stages. Growth Stage OD for Eutrophic Medium OD for Oligotrophic Medium GrowthAdaptation Stage phase OD for Eutrophic0.07 Medium–0.4 OD for Oligotrophic Medium<0.15 AdaptationExponential phase phase 0.07–0.40.4–1.2 <0.15 0.15–0.27 ExponentialStationary phase phase 0.4–1.2>1.2 0.15–0.27 >0.27 Stationary phase >1.2 >0.27

Figure 1. GrowthFigure of yeast 1. Growth cultures of as yeast indicated cultures by as optical indicated density. by optical Note: S. density. dacryoidea Note: wasS. dacryoideacultured inwas two cultured different in nutrient concentrationstwo (0.2 differentand 2 g/L nutrient glucose, concentrations blue and purple (0.2 line, and respectively). 2 g/L glucose, The blue other and fungal purple species line, respectively). were grown The with 2 g/L glucose. Black otherdots indicate fungal speciesthe sampling were grown times.with 2 g/L glucose. Black dots indicate the sampling times. 2.2. Sample Preparation to Investigate Fungal Autofluorescence 2.2. Sample Preparation to Investigate Fungal Autofluorescence In the corresponding growth stages, 20 mL of the yeast cultures were sampled and In the corresponding growth stages, 20 mL of the yeast cultures were sampled and fixed with 2% final conc. of formaldehyde (Sigma-Aldrich, 37%, St. Louis, MO, USA). Mi- fixed with 2% final conc. of formaldehyde (Sigma-Aldrich, 37%, St. Louis, MO, USA). Mi- croscopic samples were prepared by filtering 250 µL of fixed culture, diluted in 5 mL MilliQ- croscopic samples were prepared by filtering 250 µL of fixed culture, diluted in 5 mL Mil- water on GTTP filters (0.22 µm, 25 mm diameter, Merck Millipore, Burlington, MA, USA). liQ-water on GTTP filters (0.22 µm, 25 mm diameter, Merck Millipore, Burlington, MA, Subsequently, filters were dried for 30 min and mounted with Vectashield (Vector Labora- tories, H-1000,USA Burlingame,). Subsequently, CA, USA) filters on were a microscopic dried for 30 slide. min and mounted with Vectashield (Vector To furtherLaboratories, investigate H the-1000 autofluorescence, Burlingame, CA, in hyphae-morphotype USA) on a microscopic fungi, slide.Blastobotrys parvus was dilutedTo infurther artificial investigate seawater the (35 autofluorescence g/L sea salts), then in hyphae filtered-morphotype and mounted fungi, with Blastobotrys Vectashieldparvus as described was diluted before. in artificial seawater (35 g/L sea salts), then filtered and mounted with To investigateVectashield fungal as described autofluorescence, before. samples were examined with a Zeiss Axio Imager 2 microscopeTo investigate (1250× magnification, fungal autofluorescence, Carl Zeiss, Jena, samples Germany) were using examined four different with a Zeiss Axio channels andImager filter 2 sets microscope provided (1250 by Zeiss:× magnification DAPI (40,6-diamidin-2-phenylindol,, Carl Zeiss, Jena, Germany) filter using set four different 49); FITC (fluorescein isothiocyanate, filter set 44); DsRed (red fluorescent protein, filter set

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43 HE); rhodamine (filter set 20 HE). For comparing potential species-specific differences in autofluorescence, each filter channel was analysed with fixed exposure times (2.6 s for DAPI, 5.2 s for FITC, 9.06 s for DsRed, 1.1 s for rhodamine) to obtain optimal results based on previous autofluorescence investigations. Here, we intentionally chose relatively long exposure times to examine autofluorescence. For usual fluorescence measurements, we used exposure times automatically calculated by the software (Axio Vision SE64-Re4.9, Carl Zeiss) of around 100–500 ms for Calcofluor-White (Sigma-Aldrich, St. Louis, MO, USA) and 150–500 ms for DAPI staining. Pictures were taken with an AxioCam MRm camera (Carl Zeiss).

3. Results and Discussion Sakaguchia dacryoidea was grown in two different nutrient concentrations (0.2 and 2 g/L glucose) and sampled in the exponential and stationary growth stage (Figure1). In both nutrient concentrations, S. dacryoidea entered the exponential growth phase after 1.5 d. In the media with less nutrients, the stationary phase was reached after 2 d, with a maximum OD of about 0.5. Conversely, at high nutrient concentrations, the S. dacryoidea yield was higher, reaching OD values in the stationary phase of about 1.8. It is noteworthy that all fungal species grown with 2 g glucose/L reached similar maximum biomass in the stationary phase. When observed under the microscope, S. dacryoidea sampled in the exponential phase exhibited autofluorescence in all of the channels investigated (i.e., DAPI, FITC, DsRed and rhodamine). The strongest autofluorescence was detected in the DAPI channel as indicated by the shortest exposure time (in this case of 2.6 s) (Figure2). The autofluorescence of S. dacryoidea was weaker in the FITC, DsRed and Rhodamine than in the DAPI channel. To test whether the autofluorescence was affected by the growth stage we also examined it in the stationary phase of the same fungal culture (Figure2). We found that the autofluorescence of S.dacryoidea became weaker in all observed channels compared to the exponential phase, indicating that nutrient limitation in the stationary phase affects marine fungal autofluorescence (Figure2). To gain more insight into the potential effect of nutrient concentrations on fungal physiology and autofluorescence, the autofluorescence of S. dacryoidea was analysed after culturing in a medium with reduced (10 times less) glucose concentration (i.e., 0.2 g/L) (Figure3). In all of the analysed channels, the autofluorescence of S. dacryoidea in the exponential phase was slightly lower under lower nutrient conditions than at high nutrient concentrations (Figures2 and3). However, in the stationary phase, the autofluorescence of S. dacryoidea increased again under low nutrient conditions (Figure3). To determine whether autofluorescence is a general phenomenon in oceanic fungi or whether it is species-dependent, we compared the autofluorescence of four different marine yeasts in the DAPI channel (Figure4). We found species-specific autofluorescence intensities with autofluorescence clearly visible in S. dacryoidea, R. sphaerocarpa and Rhodotorula sp. In contrast, M. australis showed only minor autofluorescence. These results suggest that autofluorescence is not restricted to a single species, but instead might be a widely distributed feature in marine fungi, albeit species-specific. Future research including a larger number of more diverse fungi would be help in confirming whether autofluorescence is a global characteristic of marine fungi. To study the effect of additional staining with DAPI on the autofluorescence of S.dacryoidea, we stained the fungal cells sampled in both growth stages and nutrient conditions with DAPI (Figure5). J. FungiJ. Fungi 20212021, 7,,7 x, 709FOR PEER REVIEW 5 of 10 5 of 10

Figure 2.2. AutofluorescenceAutoﬂuorescence ofof S.S. dacryoideadacryoideagrown grown in in thethe eutrophiceutrophic medium medium and and sampled sampled in in the ex- theponential exponential and stationary and stationary phase. phase. (A,E (A): ,DAPIE): DAPI channel; channel; (B (,FB):,F ):FITC FITC channel; channel; (C (C,G,G):): DsRed DsRed channel; channel;(D,H): rhodamine (D,H): rhodamine channel. channel. Picture exposure Picture exposure times are times depicted are depicted in milliseconds in milliseconds (ms). (ms).Scale bar = 20 Scaleµm. bar = 20 µm.

To test whether the autofluorescence was affected by the growth stage we also examined it in the stationary phase of the same fungal culture (Figure 2). We found that the autofluorescence of S.dacryoidea became weaker in all observed channels compared to the exponential phase, indicating that nutrient limitation in the stationary phase affects marine fungal autofluorescence (Figure 2). To gain more insight into the potential effect of nutrient concentrations on fungal physiology and autofluorescence, the autofluorescence of S. dacryoidea was analysed after culturing in a medium with reduced (10 times less) glucose concentration (i.e., 0.2 g/L) (Figure 3). In all of the analysed channels, the autofluorescence of S. dacryoidea in the exponential phase was slightly lower under lower nutrient conditions than at high nutrient concentrations (Figures 2 and 3). However, in the stationary phase, the autofluorescence of S. dacryoidea increased again under low nutrient conditions (Figure 3).

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FigureFigure 3. AutofluorescenceAutoﬂuorescence of ofS. S. dacryoidea dacryoideagrown grown in in the the oligotrophic oligotrophic medium medium and and sampled sampled in in the exponentialthe exponential and and stationary stationary phase. phase. (A, (EA):,E DAPI): DAPI channel; channel; (B (,BF,):F ):FITC FITC channel; channel; (C (C,G,G)):: DsRed DsRed channel; (channel;D,H): rhodamine (D,H): rhodamine channel. channel. Picture exposure Picture exposure times are times depicted are depicted in milliseconds in milliseconds (ms). Scale (ms). bar = 20 µm.Scale bar = 20 µm.

To determine whether autofluorescence is a general phenomenon in oceanic fungi or whether it is species-dependent, we compared the autofluorescence of four different marine yeasts in the DAPI channel (Figure 4). We found species-specific autofluorescence intensities with autofluorescence clearly visible in S. dacryoidea, R. sphaerocarpa and Rhodotorula sp. In contrast, M. australis showed only minor autofluorescence. These results suggest that autofluorescence is not restricted to a single species, but instead might be a widely distributed feature in marine fungi, albeit species-specific. Future research including a larger number of more diverse fungi would be help in confirming whether autofluorescence is a global characteristic of marine fungi.

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Figure 4. Autofluorescence of four marine yeasts grown in eutrophic medium and sampled in the Figure 4. Autofluorescence of four marine yeasts grown in eutrophic medium and sampled in the exponential phase. All pictures were made in the DAPI channel, exposure times are depicted in exponential phase. All pictures were made in the DAPI channel, exposure times are depicted in µ millisecondsmilliseconds (ms). (ms). Scale Scale bar = 20 µm.m. DAPI-staining intensified fungal fluorescence in all conditions (note the shorter expo- sureTo time) study with the strongest effect fluorescence of additional of the staining cell nucleus with (Figure DAPI 5 on). Hence, the autofluorescence although fungal of S.dacryoideacells are auto-fluorescent,, we stained the the fungal cell nucleus cells sampled is still clearly in both visible growth and stages can be and distinguished nutrient con- ditionsfrom the with rest DAPI of the (Figure cell when 5). stained with DAPI. Finally, to study fungi with hyphae-morphotype, we investigated the autofluorescence of Blastobotrys parvus in four different channels (Figure6). When observed with the DAPI channel, B. parvus showed no autofluorescence. However, after changing to FITC, DsRed and Rhodamine channel, the autofluorescence of B. parvus was clearly visible, supporting that this phenomenon occurs in both fungal morphotypes. Collectively, this is the first time that autofluorescence is shown in marine fungal species. These results are consistent with previous studies on terrestrial and human-derived fungal species [2–6]. Our results also suggest that the intensity of fungal autofluorescence changes with the excited wavelengths. We also provide a preliminary indication that the autofluorescence of marine fungi varies between fungal species and in relation to the nutrient availability and growth stage. This is consistent with a previous study where the autofluorescence of cells/tissues was suggested to be influenced by the physiological state of the organism [1]. The existence of autofluorescence in marine fungi is important to consider, particularly when dealing with fluorescence-based techniques for analysis or identification. This autofluorescence can be used as a methodological advantage without the need of prior staining as shown for human associated fungi [2,3]. It can be misleading, however, when working with fluorescence microscopy. For instance, a very common method in microbiological studies for identification of cells and estimating their relative abundance is fluorescence in situ hybridisation (FISH). Its principle relies on staining microbial cells with, for example, DAPI, which binds to the DNA in the cells, and also with some specific probe to target a specific taxon. As a result, cell compounds that contain DNA emit a fluorescent signal when exited in a specific wavelength [15,16] and the targeted taxa with the corresponding FISH-probe will also emit fluorescence in a different wavelength to DAPI [17]. Thus, the

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existence of autofluorescence in marine fungi might lead to potential interferences, poten- J. Fungi 2021, 7, x FOR PEER REVIEWtially resulting in false “positive” results due to natural autofluorescence in marine fungal 8 of 10 cells. Therefore, natural autofluorescence in marine fungi should be investigated before applying fluorescence-dependent analytical methods.

Figure 5. Autoﬂuorescence of S. dacryoidea grown in two different nutrient concentrations and Figure 5. Autofluorescence of S. dacryoidea grown in two different nutrient concentrations and sam- sampled in the exponential and stationary phase. Right panels show additional staining with DAPI. pled in the exponential and stationary phase. Right panels show additional staining with DAPI. Exposure times are depicted in milliseconds (ms). Scale bar = 20 µm. Exposure times are depicted in milliseconds (ms). Scale bar = 20 µm.

DAPI-staining intensified fungal fluorescence in all conditions (note the shorter exposure time) with strongest fluorescence of the cell nucleus (Figure 5). Hence, although fungal cells are auto-fluorescent, the cell nucleus is still clearly visible and can be distinguished from the rest of the cell when stained with DAPI. Finally, to study fungi with hyphae-morphotype, we investigated the autofluorescence of Blastobotrys parvus in four different channels (Figure 6).

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FigureFigure 6. 6. AutofluorescenceAutofluorescence of of B.B. p parvusarvus. .((AA):): DAPI DAPI channel; channel; (B (B):): FITC FITC channel; channel; (C (C):): DsRed DsRed channel; channel; (D(D):): rhodamine rhodamine channel. PicturePicture exposureexposure times times are are depicted depicted in in milliseconds milliseconds (ms). (ms). Scale Scale bar bar = 20 =µ 20m. µm. Author Contributions: Conceptualization, F.B. and E.B.; methodology, F.B., E.B. and C.A.; investiga- tion,When M.B. and observed M.R.; resources, with the M.H.; DAPI writing—original channel, B. parvus draft showed preparation, no autofluorescence. E.B. All authors have How- read ever,and agreedafter changing to the published to FITC, version DsRed of the and manuscript. Rhodamine channel, the autofluorescence of B. parvus was clearly visible, supporting that this phenomenon occurs in both fungal mor- Funding: This research was funded by the Austrian science fond (FWF) project OCEANIDES (project photypes.number AP3430411/21) to Federico Baltar. Collectively, this is the first time that autofluorescence is shown in marine fungal species.Institutional These Review results Board are consistent Statement: withNot applicable. previous studies on terrestrial and human-de- rivedInformed fungal Consent species Statement: [2–6]. OurNot results applicable. also suggest that the intensity of fungal autofluorescence changes with the excited wavelengths. We also provide a preliminary indication Data Availability Statement: The raw data supporting the conclusions of this article will be made thatavailable the autofluorescence by the authors, without of marine undue fungi reservation varies tobetween any qualified fungal researcher. species and in relation to the nutrient availability and growth stage. This is consistent with a previous study where theAcknowledgments: autofluorescenceWe of would cells/tissues like to acknowledge was suggested Gerhard to be J. influenced Herndl, for by providing the physiological feedback on statethe drafting of the organism of the manuscript [1]. and also the Open Access Funding by the University of Vienna. ConflictsThe existence of Interest: ofThe autofluorescence authors declare noin conflictmarine of fungi interest. is important to consider, particularly when dealing with fluorescence-based techniques for analysis or identification. This References autofluorescence can be used as a methodological advantage without the need of prior 1. Monici, M. Cell and tissue autofluorescencestaining as shown research for human and diagnostic associated applications. fungi [2,3]Biotechnol.. It can be Ann. misleading, Rev. 2005, 11however,, 227–256. when 2. Mann, J.L. Autofluorescenceworking of Fungi: with An Aidfluorescence to Detection microscopy. in Tissue Sections. For instance,Am. J. Clin. a very Pathol. common1983, 79 method, 587–590. in [microbi-CrossRef] [PubMed] ological studies for identification of cells and estimating their relative abundance is fluo- Am. J. Clin. Pathol. 1983 79 3. Graham, A.R. Fungal Autofluorescencerescence in withsitu Ultraviolethybridisation Illumination. (FISH). Its principle relies on, staining, 231–234. microbial [CrossRef cells][PubMed with,] 4. Wu, C.H.; Warren, H.L. Natural Autofluorescence in Fungi, and its Correlation with Viability. Mycologia 1984, 76, 1049–1058. for example, DAPI, which binds to the DNA in the cells, and also with some specific probe [CrossRef] 5. Dreyer, B.; Morte, A.; Pérez-Gilabert,to target M.;a specific Honrubia, taxon. M. AutofluorescenceAs a result, cell detectioncompounds of arbuscular that contain mycorrhizal DNA emit fungal a fluorescent structures in palm roots: An underestimatedsignal experimental when exited method. in a specificMycol.Res. wavelength2006, 110, 887–897.[15,16] and [CrossRef the targeted][PubMed taxa] with the corre- 6. Margo, C.E.; Bombardier, T.sponding The diagnostic FISH value-probe of will fungal also autofluorescence. emit fluorescenceSurv. in Ophthalmol. a different1985 wavelengt, 29, 374–376.h to [CrossRefDAPI [17]] . 7. Mansoldo, F.R.P.; Firpo, R.;Thus, da Silva the Cardoso, existence V.; Queiroz, of autofluorescence G.N.; Cedrola, S.M.L.;in marine de Godoy, fungi M.G.;might Vermelho, lead to A.B.potential New method interfer- for rapid identification and quantificationences, potentially of fungal resulting biomass using in false ergosterol “positive” autofluorescence. results dueTalanta to natural2020, 219autofluorescence, 121238. [CrossRef in] [PubMed] marine fungal cells. Therefore, natural autofluorescence in marine fungi should be inves- 8. Gutiérrez, M.H.; Pantoja, S.;tigated Tejos, before E.; Quiñones, applying R.A. fluorescence The role of fungi-dependent in processing analytical marine methods. organic matter in the upwelling ecosystem off Chile. Mar. Biol. 2011, 158, 205–219. [CrossRef]

J. Fungi 2021, 7, 709 10 of 10

9. Hassett, B.T.; Gradinger, R. Chytrids dominate arctic marine fungal communities. Environ. Microbiol. 2016, 18, 2001–2009. [CrossRef][PubMed] 10. Cleary, A.C.; Durbin, E.G.; Rynearson, T.A.; Bailey, J. Feeding by Pseudocalanus copepods in the Bering Sea: Trophic linkages and a potential mechanism of niche partitioning. Deep Sea Res. Part. II Top. Stud. Oceanogr. 2016, 134, 181–189. [CrossRef] 11. Bochdansky, A.B.; Clouse, M.A.; Herndl, G.J. Eukaryotic microbes, principally fungi and labyrinthulomycetes, dominate biomass on bathypelagic marine snow. ISME J. 2017, 11, 362–373. [CrossRef][PubMed] 12. Hassett, B.T.; Borrego, E.J.; Vonnahme, T.R.; Rämä, T.; Kolomiets, M.V.; Gradinger, R. Arctic marine fungi: Biomass, functional genes, and putative ecological roles. ISME J. 2019, 13, 1484–1496. [CrossRef][PubMed] 13. Morales, S.E.; Biswas, A.; Herndl, G.J.; Baltar, F. Global Structuring of Phylogenetic and Functional Diversity of Pelagic Fungi by Depth and Temperature. Front. Mar. Sci. 2019, 6, 131. [CrossRef] 14. Baltar, F.; Zhao, Z.; Herndl, G.J. Potential and expression of carbohydrate utilization by marine fungi in the global ocean. Microbiome 2021, 9, 106. [CrossRef][PubMed] 15. Otto, F. DAPI staining of fixed cells for high-resolution flow cytometry of nuclear DNA. In Methods in Cell Biology; Academic Press: Cambridge, MA, USA, 1990; Volume 33, pp. 105–110. 16. Hamada, S.; Fujita, S. DAPI staining improved for quantitative cytofluorometry. Histochemistry 1983, 79, 219–226. [CrossRef] [PubMed] 17. Amann, R.; Fuchs, B.M. Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Nat. Rev. Microbiol. 2008, 6, 339–348. [CrossRef][PubMed]