Mycorrhiza (2014) 24:259–266 DOI 10.1007/s00572-013-0533-8
ORIGINAL PAPER
Simultaneous specific in planta visualization of root-colonizing fungi using fluorescence in situ hybridization (FISH) Pál Vági & Dániel G. Knapp & Annamária Kósa & Diána Seress & Áron N. Horváth & Gábor M. Kovács
Received: 29 May 2013 / Accepted: 9 October 2013 / Published online: 13 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract In planta detection of mutualistic, endophytic, and pathogenic fungi commonly colonizing roots and other plant organs is not a routine task. We aimed to use fluorescence in situ hybridization (FISH) for simultaneous specific detection of different fungi colonizing the same tissue. We have adapted ribosomal RNA (rRNA) FISH for visualization of common mycorrhizal (arbuscular- and ectomycorrhiza) and endophytic fungi within roots of different plant species. Beside general probes, we designed and used specific ones hybridizing to the large subunit of rRNA with fluorescent dyes chosen to avoid or reduce the interference with the autofluorescence of plant tissues. We report here an optimized efficient protocol of rRNA FISH and the use of both epifluorescence and confocal laser scanning microscopy for simultaneous specific differential detection of those fungi colonizing the same root. The method could be applied for the characterization of other plant–fungal interactions, too. In planta FISH with specific probes labeled with appropriate fluorescent dyes could be used not only in basic research but to detect plant colonizing pathogenic fungi in their latent life-period. Keywords Cadophora . CLSM . Dark septate . Endophyte . FISH . Glomus . Microscopy . Rhizophagus . Root . rRNA
Electronic supplementary material The online version of this article (doi:10.1007/s00572-013-0533-8) contains supplementary material, which is available to authorized users. P. Vági : D. G. Knapp : A. Kósa : D. Seress : Á. N. Horváth : G. M. Kovács (*) Department of Plant Anatomy, Institute of Biology, Eötvös Loránd University, 1117 Budapest( Pázmány Péter sétány 1/c, Hungary e-mail: [email protected] P. Vági : G. M. Kovács Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary
Introduction Fungi have a fundamental role in ecological processes as decomposers, mutualistic symbionts, and parasites/ pathogens with increasing importance as emerging infecting diseases of both plants and animals (Fisher et al. 2012). Plant tissues are fungal diversity hotspots; they are generally colonized by community of mutualistic, endophytic, and pathogenic fungi (Arnold and Lutzoni 2007; see references in Hibbett et al. 2009). The roots of the majority of the land plants are colonized by mycorrhizal and endophytic fungi playing fundamental role in ecosystem functioning (Smith and Read 2008; Schulz et al. 2006). Several plant species simultaneously form different mycorrhizal interactions and are colonized by root endophytes (e.g., Stoyke et al. 1992; Kovács and Szigetvári 2002; Girlanda et al. 2006; Grünig et al. 2008; Mandyam and Jumpponen 2008; Wagg et al. 2008; Tedersoo et al. 2009; Regvar et al. 2010; Kohout et al. 2012). Molecular diversity screening techniques, especially the nuclear ribosomal DNA (nrDNA) targeting PCR-based methods revealed an enormous diversity of fungi (Hibbett et al. 2009). Nevertheless, there is no method generally used for specific in situ visualization/localization of functioning fungi present in a certain environment, especially when specific detection is aimed, beside many other reasons, because of the emerging difficulties caused by, e.g., the autofluorescence characteristic of plant tissues. There are microscopic techniques relying on the labeling of specific fungal cell wall components, or on the autofluorescence of fungi, which have been used for in planta visualization of mycorrhizal structures, as well (e.g., Hood and Shew 1996; Séjalon-Delmas et al. 1998; Dickson and Kolesik 1999; Hoch et al. 2005; Vierheilig et al. 2005 (and references therein); Dreyer et al. 2006; Diagne et al. 2011). Fluorescent protein expressing fungal strains are used to study their interactions
260
with plants (Lorang et al. 2001; Bergero et al. 2003; Czymmek et al. 2005). Nevertheless, those methods do not make possible the selective and multiplex in situ localization of certain fungi colonizing plant tissues. Fluorescence in situ hybridization (FISH) as a powerful technique to visualize microbes and group-specific probes could help to screen microbial communities (Lundberg et al. 2012; Wagner and Haider 2012). In previous studies, FISH was used to target free-living fungi (Baschien et al. 2008; Jones et al. 2011) or, e.g., extraradical hyphae and nuclei in spores of arbuscular mycorrhizal fungi (AMF) (Trouvelot et al. 1999). We aimed to adapt FISH for simultaneous specific visualization of different root colonizing fungi within the same plant tissue. We chose common and frequent mycorrhizal and root endophytic interactions as the object of our study and decided to use rRNA FISH considering its adequate specificity and its ability to give signal in the whole cytoplasm instead of only in the nucleus. Additionally, the intensity of the visualization with rRNA FISH corresponds with the actual metabolic activity of the fungi studied. With measuring and mapping of autofluorescence of root tissues of several different plants, we aimed to select fluorophores to avoid or at least reduce the challenging problem caused by plant autofluorescence. To illustrate the potential of nrRNA FISH to visualize fungi in planta, we used and tested different oligonucleotides/probes either published in previous studies or designed in the present study.
Materials and methods Root and fungal samples To study fungal root colonization root samples of Pinus nigra, Populus alba, and Salix rosmarinifolia were collected from a semiarid sandy grassland described in detail earlier (Kovács and Szigetvári 2002; Knapp et al. 2012). Galanthus nivalis’ roots heavily colonized by AMF were collected in a private garden for initial tests of the penetration of probes into the roots and hyphae. Maize (Zea mays) was used as host plant and inoculated with two different root-colonizing fungi in in vitro synthesis system. Arbuscular mycorrhizal (AM) interaction was established with Rhizophagus intraradices (Glomus intraradices) (inoculum obtained from Dr. Yoram Kapulnik ARO Volcani Center, Israel), and root endophytic interaction was synthesized using a dark septate endophyte (DSE) Cadophora sp. strain DSE1049 (Ascomycota, Helotiales) belonging to group DSE-1 described previously (Knapp et al. 2012). For the inoculation, caryopsis of maize were surfacesterilized with 30 % H2O2 for 30 s and 70 % ethanol for
Mycorrhiza (2014) 24:259–266
60 s and washed twice in sterile tap water for 10 min. Sterilized caryopses were germinated on watered filter paper for approximately 10 days. Plant seedlings were placed into pots containing 0.1 vol. inoculum of R. intraradices and a mixture of sterilized (two times 30 min on 121 °C, 24 h between the two sterilizations) sandy soil from sampling area (see above) and zeolith (2:1). For DSE inoculation, 10–12 plugs (5 mm diameter) cored from actively growing part of fungal isolates were placed into the pots. The plants were grown in a climatized growth chamber in a 14-h light (24 °C)/10 h dark (22 °C) cycle, and the roots were harvested 7–8 weeks postinoculation. Root autofluorescence data For fluorescence spectroscopy, the roots of plant species representing a wide range of plant groups (e.g., gymnosperms and angiosperms) and living forms (annual herbaceous: Ambrosia artemisifolia , Chelidonium majus and Plantago lanceolata ; perennial herbaceous: Medicago sativa and Asclepias syriaca; bulb monocot: Allium porrum , and G. nivalis; grass: Bromus squarrosus and Z. mays; woody plant: Ailanthus altissima , Fumana procumbens , Helianthemum ovatum, S. rosmarinifolia, P. nigra, and P. alba) were collected. Three to four root pieces of 1-cm length of each plant species were washed with tap water and cleaned with paint brushes, then homogenized in 1.5 ml 87 % (v/v) glycerol (Nebotrade, Budapest, Hungary) in mortar and poured into semi-micro plastic cuvettes directly before the measurements. Five-microliter aliquots of the fluorescently labeled probes were dissolved in 87 % (v/v) glycerol and poured into similar cuvettes. The fluorescence emission spectra were measured at room temperature with a Jobin Yvon Horiba Fluoromax 3 (Paris, France) spectrofluorometer. Fluorescence emission was recorded from 420 to 660 nm using different excitation wavelengths from 380 to 580 nm with 10-nm steps. All emission scans were set to start at least 20 nm higher than the corresponding excitation wavelengths. The excitation slit was set at a bandpass width of 2 nm and the emission slit at 5 nm. The integration time was 0.1 s, the data collection density was 0.5 nm, and an average of three spectra was automatically calculated in each measurement. The spectra were analyzed with the software SPSERV V3.41 (copyright: Bagyinka, Cs., Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary); five-point linear smoothing and the correction for the wavelength-dependent sensitivity changes of the spectrofluorometer were performed. The contour maps (Supporting Information Plates S1–S4) of the fluorescence spectra were visualized with the software SURFER Version 10 (Golden Software, Inc., Colorado, USA).
Mycorrhiza (2014) 24:259–266
Probes tested We tested previously published fungal FISH probes used for labeling free-living fungal cells, and we also tested probes for segments of small (SSU) and large subunit (LSU) rRNA, which are the target sequences of widely used PCR primers specific for certain fungal groups like basidiomycetes or glomeromycetes (Table 1). We designed specific probes for R. intraradices based on an alignment published previously (Błaszkowski et al. 2012) of nrDNA LSU sequences of wide taxonomic range of AMF. We also used nrDNA LSU sequences of endophytic fungi published previously (Knapp et al. 2012) to design specific probes for the Cadophora sp. (Table 1). Although we aimed to design specific probes, the main goal was to find probes that can efficiently be used to illustrate the possibility of simultaneous specific visualization when those two fungi colonize the roots of a plant coinoculated with these two fungi. We applied MATHFISH (Yilmaz et al. 2011) for in silico prediction of hybridization parameters especially when the simultaneous use of two different probes was planned. Nevertheless, we always had to use higher temperature than the predicted one, especially for washing, probably because of the buffer capacity of the volume of the thick samples used in the experiments. Preparation of the samples for FISH Roots of the plants collected either from field or harvested after in vitro inoculation were extensively washed from soil particles and rinsed in distilled water. EM root tips were collected (10–15/ samples); the non-EM roots were cut into pieces (6–8 pieces/plant) of approximately 5 mm in length with fine scissors. The root pieces were transferred to CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES, 1 % Triton X100, 1 mM EGTA, in distilled water, pH=6.8) for
261
permeabilization and lipid extraction and infiltrated in vacuum for 30 min at room temperature. After extraction, samples were transferred to a fixation solution containing 4 % paraformaldehyde in phosphatebuffered saline (PBS) (0.07 M, pH=7.2) and incubated for 60 min at room temperature. We used these long permeabilization and fixation times because of the large sample volume. The fixed root pieces were washed in PBS for 3×10 min. Samples were postfixed and stored in 70 % ethanol for a maximum of 4 weeks at −20 °C. Prior to sectioning, samples were washed in PBS for 30 min and then positioned and frozen onto a Peltier-cell cooled stage covered in a drop of distilled water. Longitudinal and cross-sections of 90–120 μm thickness were cut with a Reichert microtome (Reichert, Austria) with steel microtome knife, and the sections were washed from the knife and collected in Petri dishes. The sections were infiltrated in hybridization buffer [for 20 ml: 2 ml 20× saline sodium citrate SSC (3 M NaCl, 0.3 M sodium citrate, pH=7.0), 2 g dextran sulfate (Sigma), 2 mg BSA, 8 ml distilled water, and 50 % dimetylformamide (Sigma)] for 1 h prior to the hybridization procedure. We also tested if cell wall degrading enzymatic treatment could facilitate the penetration of probes or allow us to shorten the washing steps. The enzyme solution contained 1 % (w/v) of lysing enzyme mix (lysing enzymes from Trichoderma harzianum, Sigma) in 1 ml PBS. The samples were treated in the enzyme solution for 30 min at 20 °C, then washed in hybridization buffer for 10 min prior to the hybridization procedure. Optimalization of hybridization parameters Prior to hybridization, the 400 μl hybridization buffer was supplemented with 1 μl RNase inhibitor (RiboLock RNase
Table 1 Sequences of the fluorophore-labeled probes applied in the present study Name of the probe
Sequence
Target group
Origin
EUKb310_RhodR: MY1574_Flc: AMF1-Cy3: Bas_ITS4B_af488: AMF_28G2_af488: GintrLSU01_af488: GintrLSU02_af488: GintrLSU822_af488: CadoLSU01_af546:
5′-[RhodR] TCA GGC BCC YTC TCC G -3′ 5′-[Flc] TCC TCG TTG AAG AGC -3′ 5′-[Cy3] GTT TCC CGT AAG GCG CCG AA -3′ 5′-[A488] CAG GAG ACT TGT ACA CGG TCC AG -3′ 5′-[A488] CCA TTA CGT CAA CAT CCT TAA CG -3′ 5′-[A488] CAT ACG GGC AAG TAC ACC CAA -3′ 5′-[A488] TTT CGG CAC CAG AGC AAC GAT -3′ 5′-[A488] AAC TCC TCA CGC TCC ACA GA -3′ 5′-[A546] GAG AGG AGC CAC ATT CCC AA -3′
Eukaryots Eufungi AMF Basidiomycetes AMF Rhizophagus intraradices/AMF/ R. intraradices/AMF/ R. intraradices/AMF/ Cadophora sp./DSE/
Baker et al. 2003 Baschien et al. 2008 Helgason et al. 1998 (PCR primer) Gardes and Bruns 1993. (PCR primer) da Silva et al. 2006 (PCR primer) Present study Present study Trouvelot et al. 1999 (PCR primer) Present study
FISH probes and PCR primers published previously with different selectivity were also tested RhodR Rhodamine Red-X, Flc fluorescein, A488 Alexa Fluor 488, A546 Alexa Fluor 546
262
Inhibitor 40 U/μl, Fermentas), 60 μl salmon sperm DNA (10 mg/ml in H2O, Sigma), and the probe DNA. The probe stock solution consisted of 20 μg probe DNA in 5 μl TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH=7.5) and was stored at −20 °C. These 5 μl stocks were diluted in 400 μl hybridization buffer and partitioned to eight 0.2-ml PCR tubes, each PCR tube received six root sections (thus one 5 μl stock of probe was sufficient for hybridization of 48 root sections). Hybridization conditions were optimized using pure cultures of different endophytes. We used a fixed 50 % formamide concentration and changed the hybridization and washing temperature between 32 and 72 °C in 10 °C increments. Samples were hybridized overnight at adequate temperature in a thermocycler (Hybaid PCR Sprint). Using temperature to adjust stringency in simultaneous hybridization of different probes to several samples required different conditions. Every hybridization experiment needed a separate incubator set to the desired temperature. Therefore, another protocol in which the hybridization stringency achieved by adding different concentrations of formamide to the hybridization buffer was also tested (Daims et al. 2005). In this case, a single hybridization assay was performed in two different samples: the Cadophora sp. strain containing the target sequence and the strain Periconia sp. (strain REF144, group DSE-8 sensu Knapp et al. 2012) without the target sequence. As a negative control, probe-free hybridization buffer was used on Cadophora hyphae. The samples were incubated overnight at 46 °C. Posthybridization steps After hybridization the washing steps took place in 1.5-ml Eppendorf tubes in a dry bath incubator (M.R.C. Ltd) preheated to the proper washing temperature. Root samples were washed three times in preheated washing buffer (same as the hybridization buffer, without dextran sulphate, probe DNA, RNase inhibitor, and salmon sperm DNA), three times in 2× SSC and stored in 4× SSC in dark until mounted in Fluoroshield (Sigma). The length of the washing steps in the first washing buffer was changed between 3×10 to 3×120 min. In the case of washing buffers, the stringency was also achieved by lowering the salt concentration in the corresponding washing buffer instead of adding formamide with washing steps of 3×20 min on 48 °C. Fluorescence microscopy of the root sections We used epifluorescence and confocal microscopy to analyze the root samples. With using a confocal system our aim was to gain 3D images of the fungal structures in thick root sections. Images were captured using Nikon Eclipse 80i microscope equipped with a Spot 7.4 Slider camera (Diagnostic
Mycorrhiza (2014) 24:259–266
Instruments, Inc.), differential–interference–contrast (DIC) and a filter wheel with excitation and emission filters for visualization of Alexa Fluor 488, Alexa Fluor 546, Cy3 and fluorescein-labeled probes. For 3D reconstruction stacks of 1,024×1,024 pixel 8bit grayscale images were acquired using a Zeiss laser scanning microscope (LSM) 410 confocal microscope with 488-nm argon laser line (FT 510 dichroic mirror and HQ 525/50 barrier filter for Alexa Fluor 488 and Fluorescein) and 543 nm HeNe laser line (FT 560 dichroic mirror, D 605/55 barrier filter for Alexa Fluor 546 and Cy3). Increments in the Z-plane were taken accordingly to the values suggested by the LSM software for the chosen objectives, immersion oil and mounting medium. Maximum intensity projections of the stacks were further processed using the Zeiss LSM software or Adobe Photoshop. The projections were colored in accordance with the actual probes emission wavelength. When two probes were applied simultaneously two projections were merged to show the different fungal structures in the same section.
Results Plant autoflourescence, labels, and probes tested To overcome on the challenging problem of the characteristic autofluorescence of plant tissues, we investigated the excitation/emission spectra of root homogenates of different plant species representing a wide range of plant groups (e.g., gymnosperms and angiosperms) and living forms (Supporting Information Plates S1-S4). Based on our spectroscopy results, we defined a wavelength range characteristic of most of the plant roots studied (Supporting Information Plates S1–S3) in which the background autofluorescence was weak enough not to overwhelm the fluorescence from the fluorophores (Supporting Information Plate S4). In a few special cases, the autofluorescence caused by special metabolites of the plants (like silkweed, A. syriaca, and bloodroot, C. majus) was strong enough even at the ranges with low-relative values to make the use of fluorescence probes impossible (Supporting Information Plate S3). Simultaneous detection of different fungi was based on the application of fluorescently labeled specific probes characterized by high hybridization efficiency and nonoverlapping fluorescence emission spectra with each other. Fluorescence intensity of all simultaneously used probes must be high enough to provide good contrast to the background. We tested different fluorescent labels: Alexa Fluor 488, Alexa Fluor 546, Cy3, fluorescein, and RhodamineRedX (Supporting Information Plate S4). When probes were applied in pairs, these were with a lower and a higher emission maximum (Fig. 2a, b).
Mycorrhiza (2014) 24:259–266
263
Sample preparation and penetration of probes Although microscopy and FISH of thick plant tissues are challenging (Prieto et al. 2007), we prepared 90–120 μm thick sections of plant roots to keep the structures of colonizing fungi as intact as possible and to make specimen handling much simpler. Using thick sections allowed us to make hybridization and washing steps in PCR tubes instead of the general on-slide FISH protocol. Due to the thickness of the plant material, we increased the time of the hybridization and washing steps to several hours. This made the whole process much longer than a general FISH applied on thin sections or single cells grown on or spinned down on coverslips. The use of relatively thick sections made the 3D reconstruction of the in planta fungal structures possible with using confocal laser scanning microscopy (CLSM) (Figs. 1c and 2a–d). We tested if FISH probes could penetrate barriers of both plant root tissues and fungal cell walls. For those tests, we first Fig. 2 a–c Dual labeling of Zea mays root sections colonized with Rhizophagus intraradices and Cadophora sp. using the probes GintrLSU01_af488 and CadoLSU01_af546 (Table 1). a Intercellular R. intraradices hyphae and b vesicle and intracellular septate hyphae of Cadophora sp. visualized in green for Alexa Fluor 488 (Rhizophagus) and orange for Alexa Fluor 546 (Cadophora). Scale bars=10 μm. c R. intraradices vesicle colonized by septate hyphae of Cadophora sp. Scale bar=10 μm. d Cross-section of Salix rosmarinifolia ectomycorrhizal root tip labeled with the probe Bas_ITS4B_af488. The Hartig-net shows strong fluorescence due to its metabolic activity contrary to the fungal mantle which shows weaker signal. Scale bar=100 μm. a–d Confocal images
successfully used general eukaryotic and eufungal rRNA probes (Table 1). The treatment with cell wall degrading enzymes resulted in weaker probe signal in the cytoplasm after the washing steps on a given temperature, probably due to the higher permeability of the cell wall. Moreover, as a result of cell wall digestion the sections became soft, and this made the handling and mounting of specimens problematic. For that reason, we skipped the enzymatic treatment thereafter. Hybridization and posthybridization steps
Fig. 1 Micrographs of the longitudinal sections of Populus alba root in brightfield (a) and fluorescence mode (b). Fungal structures are invisible in DIC mode (a) and fluorescence mode shows arbuscules labeled with the probe AMF_28G2_af488 (b). Scale bars 50 μm. c Longitudinal section of Zea mays root labeled with the probe AMF_28G2_af488 shows well-developed arbuscules. Confocal image. Scale bar=10 μm
To optimize the hybridization conditions to obtain sufficient specificity, we applied a mix of two different probes: one of which was a perfect matching probe for the target and the other was a mismatch probe (e.g., CadoLSU01_af546/ GintrLSU01_af488) on pure Cadophora sp. culture. We used a fixed default formamide concentration while changing the washing temperature and the length of the washing (Table 2). Henceforth, we applied temperature/washing time combinations with which a nontarget organism did not show the probe signal, while the matching target provided sufficiently bright signal to give excellent contrast (Fig. 2a, b, Supporting
264
Mycorrhiza (2014) 24:259–266
Table 2 Washing time/temperature combinations tested on mixture of CadoLSU_AF546/GintrLSU01_af488 probes hybridized to Cadophora culture sample
Both probes gave bright signal. CadoLSU_AF546 probe gave bright signal without a signal from GintrLSU01_af488 probe. were weak in comparison to the background fluorescence
Information Figs. S1-S6). When we used GintrLSU01_af488 and CadoLSU01_af546 probes separately, the optimal washing temperature was 52–62 °C, and the minimal washing time was 3×30 min at 50 % formamide. In most cases, the specificity of the labeling increased with longer washing times, and the probe signal intensity weakened with higher temperatures (Table 2). The protocol in which the hybridization stringency was achieved by adding different concentrations of formamide to the hybridization buffer provided important information. None of the probes applied was found to bind aspecifically to nontarget samples. The CadoLSU_AF546 probe did not give any signal in the nontarget Periconia macrospinosa sample, while it gave excellent bright signal in Cadophora sp. culture samples. The fluorescent signal could only be detected in the fungal cytoplasm and not in the cell wall. The optimal formamide concentration in the hybridization buffer turned out to be between 10 and 20 %. Providing the above-defined preparation and hybridization conditions, the applied probes functioned well, and we could visualize in planta fungal structures with sufficient specificity (Figs. 1 and 2, Supporting Information Figs. S1–S6). Based on the experiences gained during the study presented here, we made a recommendation for a general protocol of in planta (nrDNA) FISH (Supporting Information Protocol S1). Visualizations of mycorrhizae and endophytes Using the optimized protocols and probes described above, mycorrhizal and endophytic fungi could be visualized to illustrate the possibilities of the nrRNA FISH for the study of fungi in planta. The general FISH probe for eukaryotic organisms (Baker et al. 2003) penetrated well into hyphae when tested on pure cultures (data not shown). The probe designed for eufungi (Baschien et al. 2008) labeled well hyphae both in pure cultures and in roots (data not shown).
Both signals
The AMF1-Cy3 probe, designed from the AM1 (Helgason et al. 1998) PCR primer specific for Glomeromycota, gave good signal when used in pilot studies to visualize AMF in roots of G. nivalis (data not shown); as described above, this probe was not used in further investigations. The probe developed from the basidiomycete specific ITS4-B (Gardes and Bruns 1993) PCR primer (Bas_ITS4B_af488) labeled well the EM structures (Hartig net, mantle) and hyphae of basidiomycetous EMF (Fig. 2d, Supporting Information Figs. S1). The probe developed from the AMF specific 28G2 (da Silva et al. 2006) PCR primer (AMF_28G2_af488) gave excellent signal in AM structures (arbuscules, vesicles, and intraradical hyphae) and made AMF visible in the roots of both woody (e.g., P. alba, Fig. 1a, b) and herbaceous plant (Z. mays, Supporting Information Figs. S2). Intraradical structures formed by R. intraradices could be visualized using the probe developed from the species-specific “8.22” (Trouvelot et al. 1999) PCR primer (GintrL SU822_af488, Supporting Information Figs. S5) and the specific probes designed in the present study for this fungus (GintrLSU01_af488, Fig. 1a–c, Supporting Information Figs. S3; GintrLSU02_af488, Supporting Information Figs. S4). The probe designed for specific labeling of the DSE Cadophora sp. visualized well the hyphae and microsclerotia of the fungus either used alone or when used simultaneously with the labeling of AMF in the same root (Fig. 2a–c, Supporting Information Figs. S6). Cadophora hyphae colonizing the vesicle of R. intraradices could be also detected (Fig. 2c) during the simultaneous specific labeling of these species.
Discussion Plant tissues are diversity hotspots of different fungi, and these symbiotic fungi colonizing both above- and belowground
Mycorrhiza (2014) 24:259–266
organs of plants play a crucial role not only in the life of their hosts but in ecosystem functioning. Localization and visualization of the fungi and/or their molecules or enzymes of special function is an important method to understand the interactions. There are several methods making in situ visualization of fungi possible. Transformed fungi expressing green fluorescent protein labeled proteins can be used to visualize certain symbionts and the use of different fluorophores makes the parallel visualization of either specific molecules or fungal strains/species possible (Lorang et al. 2001; Bergero et al. 2003; Czymmek et al. 2005). Nevertheless, these methods need the challenging transformation of fungal strains just like the maintenance of stable transformants of fungi. Moreover, the method hardly usable in environmental conditions and only cultivable fungi could be screened. Using the rRNA as target for the FISH has some important powers. As the ribosomes are dispersed in the whole hypha fluorophore-labeled oligonucleotides hybridizing to these RNAs could make the whole cytoplasm visible contrary, e.g., to DNA based nuclear probes, which could solely light the nucleus. Moreover, using rRNA the FISH could refer the metabolic activity of the fungi. There could be considerable inactive members of, e.g., ectomycorrhizal fungal communities, as it was proven when ectomycorrhizae of a Norway spruce forest were screened using viability-test dyes (Qian et al. 1998). During the study, one of the crucial tasks was to find appropriate fluorophores, which do not interfere at least not in a high extent with the autofluorescence of plants. The measure and mapping of the spectra of autofluroscence of the plant roots proved to be a useful method for finding appropriate dyes. This method was also used to study the fluorescence characteristics of fluorophores planned to be used parallel. Although the problem caused by autofluorescence could be overcome by microscopes equipped with spectral detectors, dyes chosen by this technique could make correct visualization easier and microscopes equipped with traditional barrier filters could also be used, which enables the widespread application of the method. Several probes and PCR primers generally used to amplify different groups of fungi have been applied here with different success rate especially regarding to their specificity. We designed our specific probes to hybridize to the D1–D2 region to the LSU gene, which was variable enough to find completely different regions when the ascomycete DSE Cadophora sp. and the AMF R. intraradices were studied simultaneously. Of course, this region has certain limitation and less variable as, e.g., the ITS, species-level DNA barcode region of fungi (Schoch et al. 2012). Nevertheless, because of the advantages of rRNA mentioned above, we aimed to find an rRNA segment with considerable variation, and this variable region of the LSU rRNA seemed to be appropriate. According to its
265
taxonomic resolution in, e.g., the Glomeromycota (Stockinger et al. 2010) or the ascomycete cup fungi Pezizales (Hansen et al. 2005), this region might be a proper segment to design specific probes.
Conclusion We demonstrate here the successful adaptation of rRNA FISH for simultaneous specific visualization of different fungi within the same plant root. The method might be used for aboveground tissues as well, providing that the emission of fluorophores does not overlap considerably with the autofluorescence of those tissues. We expect that in planta FISH with probes designed for specific fungi will be used not only in basic research but also might be a useful diagnostic tool of plant colonizing pathogenic fungi. Acknowledgments We thank Yoram Kapulnik (ARO Volcani Center, Israel) for kindly providing AMF inoculum. Funding was provided by the Hungarian Scientific Research Fund (OTKA, K72776, and NI72776). GMK is supported by the Bolyai János Research Fellowship (Hungarian Academy of Sciences).
References Arnold AE, Lutzoni F (2007) Diversity and host range of foliar fungal endophytes: are tropical leaves biodiversity hotspots? Ecology 88: 541–549. doi:10.1890/05-1459 Baker BJ, Hugenholtz P, Dawson SC, Banfield JF (2003) Extremely acidophilic protists host Rickettsiales-lineage endosymbionts with an intervening sequence in their 16S rRNA genes. Appl Environ Microbiol 69:5512–5518. doi:10.1128/AEM.69.9.5512-5518.2003 Baschien C, Manz W, Neu TR, Marvanová L, Szewcyk U (2008) In situ detection of freshwater fungi in an alpine stream by new taxonspecific fluorescence in situ hybridization probes. Appl Environ Microbiol 74:6427–6436. doi:10.1128/AEM.00815-08 Bergero R, Harrier LA, Franken P (2003) Reporter genes: applications to the study of arbuscular mycorrhizal (AM) fungi and their symbiotic interactions with plant roots. Plant and Soil 255:143–155. doi:10. 1104/pp. 011882 Błaszkowski J, Kovács GM, Gáspár BK, Balázs KT, Buscot F, Ryszka P (2012) The arbuscular mycorrhizal Paraglomus majewskii sp. nov. represents a distinct basal lineage in Glomeromycota. Mycologia 104:148–156. doi:10.3852/10-430 Czymmek KJ, Bourett TM, Howard RJ (2005) Fluorescent protein probes in fungi. In: Savidge T, Pothoulakis C (eds) Methods in microbiology, vol 34. Microbial Imaging Elsevier, Amsterdam, pp 27–62 da Silva GA, Lumini E, Maia LC, Bonfante P, Bianciotto V (2006) Phylogenetic analysis of Glomeromycota by partial LSU rDNA sequences. Mycorrhiza 16:183–189. doi:10.1007/s00572-0050030-9 Daims H, Stoecker K, Wagner M (2005) Fluorescence in situ hybridization for the detection of prokaryotes. In: Osborn AM, Smith C (eds) Molecular microbial ecology. New York, USA, Taylor and Francis, pp 213–239 Dickson S, Kolesik P (1999) Visualisation of mycorrhizal fungal structures and quantification of their surface area and volume using laser
266 scanning confocal microscopy. Mycorrhiza 9:205–213. doi:10. 1007/s005720050268 Diagne N, Escoute J, Lartaud M, Verdeil JL, Franche C, Kane A, Bogusz D, Diouf D, Duponnois R, Svistoonoff S (2011) Uvitex2B: a rapid and efficient stain for detection of arbuscular mycorrhizal fungi within plant roots. Mycorrhiza 21:315–321. doi:10.1007/s00572010-0357-8 Dreyer B, Morte A, Pérez-Gilabert M, Honrubia M (2006) Autofluorescence detection of arbuscular mycorrhizal fungal structures in palm roots: an underestimated experimental method. Mycol Res 110:887–897. doi:10.1016/j.mycres.2006.05.011 Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, McCraw SL, Gurr SJ (2012) Emerging fungal threats to animal, plant and ecosystem health. Nature 484:186–194. doi:10.1038/nature10947 Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118 Girlanda M, Perotto S, Luppi AM (2006) Molecular diversity and ecological roles of mycorrhiza-associated sterile fungal endophytes in Mediterranean ecosystems. In: Schulz B, Boyle C, Sieber TN (eds) Microbial root endophytes. Springer, Berlin–Heidelberg, pp 207– 226. doi:10.1007/3-540-33526-9_12 Grünig CR, Queloz V, Sieber TN, Holdenrieder O (2008) Dark septate endophytes (DSE) of the Phialocephala fortinii s.l. - Acephala applanata species complex in tree roots: classification, population biology, and ecology. Botany 86:1355–1369. doi:10.1139/B08-108 Hansen K, Lobuglio KF, Pfister DH (2005) Evolutionary relationships of the cup-fungus genus Peziza and Pezizaceae inferred multiple nuclear genes: RPB2, β-tubulin, and LSU rDNA. Mol Phylogenet Evol 36:1–23. doi:10.1016/j.ympev.2005.03.010 Helgason T, Daniell TJ, Husband R, Fitter AH, Young JPW (1998) Ploughing up the wood-wide Web? Nature 394:431. doi:10.1038/ 28764 Hibbett DS, Ohman A, Kirk PM (2009) Fungal ecology catches fire. New Phytol 184:279–282. doi:10.1111/j.1469-8137.2009.03042.x Hoch HC, Galvani CD, Szarowski DH, Turner JN (2005) Two new fluorescent dyes applicable for visualization of fungal cell walls. Mycologia 97:580–588. doi:10.3852/mycologia.97.3.580 Hood ME, Shew HD (1996) Applications of KOH-aniline blue fluorescence in the study of plant-fungal interactions. Phytopathology 86: 704–708. doi:10.1094/Phyto-86-704 Jones MD, Forn I, Gadelha C, Egan MJ, Bass D, Massana R, Richards TA (2011) Discovery of novel intermediate forms redefines the fungal tree of life. Nature 474:200–203. doi:10.1038/nature09984 Kohout P, Sýkorová Z, Čtvrtlíková M, Rydlová J, Suda J, Vohník M, Sudová R (2012) Surprising spectra of root-associated fungi in submerged aquatic plants. FEMS Microbiol Ecol 80:216–235. doi: 10.1111/j.1574-6941.2011.01291.x Knapp DG, Pintye A, Kovács GM (2012) The dark side is not fastidious—dark septate endophytic fungi of native and invasive plants of semiarid sandy areas. PLoS ONE 7:e32570. doi:10.1371/journal. pone.0032570 Kovács GM, Szigetvári C (2002) Mycorrhizae and other root-associated fungal structures of the plants of a sandy grassland on the Great Hungarian Plain. Phyton Ann Rei Bot 42:211–223 Lorang JM, Tuori RP, Martinez JP, Sawyer TL, Redman RS, Rollins JA, Wolpert TJ, Johnson KB, Rodriguez RJ, Dickman MB, Ciuffetti LM (2001) Green fluorescent protein is lighting up fungal biology. Appl Environ Microbiol 67:1987–1994. doi:10.1128/AEM.67.5.19871994.2001 Lundberg DS, Lebeis SL, Paredes SH, Yourstone S, Gehring J, Malfatti S, Tremblay J, Engelbrektson A, Kunin V, del Rio TG, Edgar RC,
Mycorrhiza (2014) 24:259–266 Eickhorst T, Ley RE, Hugenholtz P, Tringe SG, Dangl JL (2012) Defining the core Arabidopsis thaliana root microbiome. Nature 488:86–90. doi:10.1038/nature11237 Mandyam K, Jumpponen A (2008) Seasonal and temporal dynamics of arbuscular mycorrhizal and dark septate endophytic fungi in a tallgrass prairie ecosystem are minimally affected by nitrogen enrichment. Mycorrhiza 18:145–155. doi:10.1007/s00572-008-0165-6 Prieto P, Moore G, Shaw P (2007) Fluorescence in situ hybridization on vibratome sections of plant tissues. Nat Protoc 2:1831–1838. doi:10. 1038/nprot.2007.265 Qian XM, Kottke I, Oberwinkler F (1998) Activity of different ectomycorrhizal types studied by vital fluorescence. Plant and Soil 199:91–98. doi:10.1023/A:1004226220283 Regvar M, Likar M, Piltaver A, Kugonič N, Smith JE (2010) Fungal community structure under goat willows (Salix caprea L.) growing at metal polluted site: the potential of screening in a model phytostabilisation study. Plant and Soil 330:345–356. doi:10.1007/ s11104-009-0207-7 Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W, Fungal Barcoding Consortium (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci U S A 109:6241–6246. doi: 10.1073/pnas.1117018109 Schulz BJE, Boyle CJC, Sieber TN (eds) (2006) Microbial root endophytes. Soil biology, vol 9. Springer, Berlin–Heidelberg Séjalon-Delmas N, Magnier A, Douds DD, Bécard G (1998) Cytoplasmic autofluorescence of an arbuscular mycorrhizal fungus Gigaspora gigantea and nondestructive fungal observations in planta. Mycologia 90:921–926 Smith SE, Read DJ (2008) Mycorrhizal symbiosis. Academic, Cambridge Stockinger H, Krüger M, Schüßler A (2010) DNA barcoding of arbuscular mycorrhizal fungi. New Phytol 187:461–474. doi:10. 1111/j.1469-8137.2010.03262.x Stoyke G, Egger KN, Currah RS (1992) Characterization of sterile endophytic fungi from the mycorrhizae of sub-alpine plants. Can J Bot 70:2009–2016. doi:10.1139/b92-250 Tedersoo L, Pärtel K, Jairus T, Gates G, Põldmaa K, Tamm H (2009) Ascomycetes associated with ectomycorrhizas: molecular diversity and ecology with particular reference to the Helotiales. Environ Microbiol 11:3166–3178. doi:10.1111/j.1462-2920.2009.02020.x Trouvelot S, van Tuinen D, Hijri M, Gianinazzi-Perason V (1999) Visualization of ribosomal DNA loci in spore interphasic nuclei of glomalean fungi by fluorescence in situ hybridization. Mycorrhiza 8:203–206. doi:10.1007/s005720050235 Vierheilig H, Schweiger P, Brundrett M (2005) An overview of methods for the detection and observation of arbuscular mycorrhizal fungi in roots. Physiol Plant 125:393–404. doi:10.1111/j.1399-3054.2005. 00564.x Wagner M, Haider S (2012) New trends in fluorescence in situ hybridization for identification and functional analyses of microbes. Curr Opin Biotech 23:96–102. doi:10.1016/j.copbio.2011.10.010 pp. 96102 Wagg C, Pautler M, Massicote HB, Peterson RL (2008) The cooccurrence of ectomycorrhizal, arbuscular mycorrhizal, and dark septate fungi in seedlings of four members of the Pinaceae. Mycorrhiza 18:103–110. doi:10.1007/s00572-007-0157-y Yilmaz LS, Parnerkar S, Noguera DR (2011) mathFISH, a web tool that uses thermodynamics-based mathematical models for in silico evaluation of oligonucleotide probes for fluorescence in situ hybridization. Appl Environ Microbiol 77:1118–1122. doi:10.1128/AEM. 01733-10
ORIGINAL PAPER
Simultaneous specific in planta visualization of root-colonizing fungi using fluorescence in situ hybridization (FISH) Pál Vági & Dániel G. Knapp & Annamária Kósa & Diána Seress & Áron N. Horváth & Gábor M. Kovács
Received: 29 May 2013 / Accepted: 9 October 2013 / Published online: 13 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract In planta detection of mutualistic, endophytic, and pathogenic fungi commonly colonizing roots and other plant organs is not a routine task. We aimed to use fluorescence in situ hybridization (FISH) for simultaneous specific detection of different fungi colonizing the same tissue. We have adapted ribosomal RNA (rRNA) FISH for visualization of common mycorrhizal (arbuscular- and ectomycorrhiza) and endophytic fungi within roots of different plant species. Beside general probes, we designed and used specific ones hybridizing to the large subunit of rRNA with fluorescent dyes chosen to avoid or reduce the interference with the autofluorescence of plant tissues. We report here an optimized efficient protocol of rRNA FISH and the use of both epifluorescence and confocal laser scanning microscopy for simultaneous specific differential detection of those fungi colonizing the same root. The method could be applied for the characterization of other plant–fungal interactions, too. In planta FISH with specific probes labeled with appropriate fluorescent dyes could be used not only in basic research but to detect plant colonizing pathogenic fungi in their latent life-period. Keywords Cadophora . CLSM . Dark septate . Endophyte . FISH . Glomus . Microscopy . Rhizophagus . Root . rRNA
Electronic supplementary material The online version of this article (doi:10.1007/s00572-013-0533-8) contains supplementary material, which is available to authorized users. P. Vági : D. G. Knapp : A. Kósa : D. Seress : Á. N. Horváth : G. M. Kovács (*) Department of Plant Anatomy, Institute of Biology, Eötvös Loránd University, 1117 Budapest( Pázmány Péter sétány 1/c, Hungary e-mail: [email protected] P. Vági : G. M. Kovács Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary
Introduction Fungi have a fundamental role in ecological processes as decomposers, mutualistic symbionts, and parasites/ pathogens with increasing importance as emerging infecting diseases of both plants and animals (Fisher et al. 2012). Plant tissues are fungal diversity hotspots; they are generally colonized by community of mutualistic, endophytic, and pathogenic fungi (Arnold and Lutzoni 2007; see references in Hibbett et al. 2009). The roots of the majority of the land plants are colonized by mycorrhizal and endophytic fungi playing fundamental role in ecosystem functioning (Smith and Read 2008; Schulz et al. 2006). Several plant species simultaneously form different mycorrhizal interactions and are colonized by root endophytes (e.g., Stoyke et al. 1992; Kovács and Szigetvári 2002; Girlanda et al. 2006; Grünig et al. 2008; Mandyam and Jumpponen 2008; Wagg et al. 2008; Tedersoo et al. 2009; Regvar et al. 2010; Kohout et al. 2012). Molecular diversity screening techniques, especially the nuclear ribosomal DNA (nrDNA) targeting PCR-based methods revealed an enormous diversity of fungi (Hibbett et al. 2009). Nevertheless, there is no method generally used for specific in situ visualization/localization of functioning fungi present in a certain environment, especially when specific detection is aimed, beside many other reasons, because of the emerging difficulties caused by, e.g., the autofluorescence characteristic of plant tissues. There are microscopic techniques relying on the labeling of specific fungal cell wall components, or on the autofluorescence of fungi, which have been used for in planta visualization of mycorrhizal structures, as well (e.g., Hood and Shew 1996; Séjalon-Delmas et al. 1998; Dickson and Kolesik 1999; Hoch et al. 2005; Vierheilig et al. 2005 (and references therein); Dreyer et al. 2006; Diagne et al. 2011). Fluorescent protein expressing fungal strains are used to study their interactions
260
with plants (Lorang et al. 2001; Bergero et al. 2003; Czymmek et al. 2005). Nevertheless, those methods do not make possible the selective and multiplex in situ localization of certain fungi colonizing plant tissues. Fluorescence in situ hybridization (FISH) as a powerful technique to visualize microbes and group-specific probes could help to screen microbial communities (Lundberg et al. 2012; Wagner and Haider 2012). In previous studies, FISH was used to target free-living fungi (Baschien et al. 2008; Jones et al. 2011) or, e.g., extraradical hyphae and nuclei in spores of arbuscular mycorrhizal fungi (AMF) (Trouvelot et al. 1999). We aimed to adapt FISH for simultaneous specific visualization of different root colonizing fungi within the same plant tissue. We chose common and frequent mycorrhizal and root endophytic interactions as the object of our study and decided to use rRNA FISH considering its adequate specificity and its ability to give signal in the whole cytoplasm instead of only in the nucleus. Additionally, the intensity of the visualization with rRNA FISH corresponds with the actual metabolic activity of the fungi studied. With measuring and mapping of autofluorescence of root tissues of several different plants, we aimed to select fluorophores to avoid or at least reduce the challenging problem caused by plant autofluorescence. To illustrate the potential of nrRNA FISH to visualize fungi in planta, we used and tested different oligonucleotides/probes either published in previous studies or designed in the present study.
Materials and methods Root and fungal samples To study fungal root colonization root samples of Pinus nigra, Populus alba, and Salix rosmarinifolia were collected from a semiarid sandy grassland described in detail earlier (Kovács and Szigetvári 2002; Knapp et al. 2012). Galanthus nivalis’ roots heavily colonized by AMF were collected in a private garden for initial tests of the penetration of probes into the roots and hyphae. Maize (Zea mays) was used as host plant and inoculated with two different root-colonizing fungi in in vitro synthesis system. Arbuscular mycorrhizal (AM) interaction was established with Rhizophagus intraradices (Glomus intraradices) (inoculum obtained from Dr. Yoram Kapulnik ARO Volcani Center, Israel), and root endophytic interaction was synthesized using a dark septate endophyte (DSE) Cadophora sp. strain DSE1049 (Ascomycota, Helotiales) belonging to group DSE-1 described previously (Knapp et al. 2012). For the inoculation, caryopsis of maize were surfacesterilized with 30 % H2O2 for 30 s and 70 % ethanol for
Mycorrhiza (2014) 24:259–266
60 s and washed twice in sterile tap water for 10 min. Sterilized caryopses were germinated on watered filter paper for approximately 10 days. Plant seedlings were placed into pots containing 0.1 vol. inoculum of R. intraradices and a mixture of sterilized (two times 30 min on 121 °C, 24 h between the two sterilizations) sandy soil from sampling area (see above) and zeolith (2:1). For DSE inoculation, 10–12 plugs (5 mm diameter) cored from actively growing part of fungal isolates were placed into the pots. The plants were grown in a climatized growth chamber in a 14-h light (24 °C)/10 h dark (22 °C) cycle, and the roots were harvested 7–8 weeks postinoculation. Root autofluorescence data For fluorescence spectroscopy, the roots of plant species representing a wide range of plant groups (e.g., gymnosperms and angiosperms) and living forms (annual herbaceous: Ambrosia artemisifolia , Chelidonium majus and Plantago lanceolata ; perennial herbaceous: Medicago sativa and Asclepias syriaca; bulb monocot: Allium porrum , and G. nivalis; grass: Bromus squarrosus and Z. mays; woody plant: Ailanthus altissima , Fumana procumbens , Helianthemum ovatum, S. rosmarinifolia, P. nigra, and P. alba) were collected. Three to four root pieces of 1-cm length of each plant species were washed with tap water and cleaned with paint brushes, then homogenized in 1.5 ml 87 % (v/v) glycerol (Nebotrade, Budapest, Hungary) in mortar and poured into semi-micro plastic cuvettes directly before the measurements. Five-microliter aliquots of the fluorescently labeled probes were dissolved in 87 % (v/v) glycerol and poured into similar cuvettes. The fluorescence emission spectra were measured at room temperature with a Jobin Yvon Horiba Fluoromax 3 (Paris, France) spectrofluorometer. Fluorescence emission was recorded from 420 to 660 nm using different excitation wavelengths from 380 to 580 nm with 10-nm steps. All emission scans were set to start at least 20 nm higher than the corresponding excitation wavelengths. The excitation slit was set at a bandpass width of 2 nm and the emission slit at 5 nm. The integration time was 0.1 s, the data collection density was 0.5 nm, and an average of three spectra was automatically calculated in each measurement. The spectra were analyzed with the software SPSERV V3.41 (copyright: Bagyinka, Cs., Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary); five-point linear smoothing and the correction for the wavelength-dependent sensitivity changes of the spectrofluorometer were performed. The contour maps (Supporting Information Plates S1–S4) of the fluorescence spectra were visualized with the software SURFER Version 10 (Golden Software, Inc., Colorado, USA).
Mycorrhiza (2014) 24:259–266
Probes tested We tested previously published fungal FISH probes used for labeling free-living fungal cells, and we also tested probes for segments of small (SSU) and large subunit (LSU) rRNA, which are the target sequences of widely used PCR primers specific for certain fungal groups like basidiomycetes or glomeromycetes (Table 1). We designed specific probes for R. intraradices based on an alignment published previously (Błaszkowski et al. 2012) of nrDNA LSU sequences of wide taxonomic range of AMF. We also used nrDNA LSU sequences of endophytic fungi published previously (Knapp et al. 2012) to design specific probes for the Cadophora sp. (Table 1). Although we aimed to design specific probes, the main goal was to find probes that can efficiently be used to illustrate the possibility of simultaneous specific visualization when those two fungi colonize the roots of a plant coinoculated with these two fungi. We applied MATHFISH (Yilmaz et al. 2011) for in silico prediction of hybridization parameters especially when the simultaneous use of two different probes was planned. Nevertheless, we always had to use higher temperature than the predicted one, especially for washing, probably because of the buffer capacity of the volume of the thick samples used in the experiments. Preparation of the samples for FISH Roots of the plants collected either from field or harvested after in vitro inoculation were extensively washed from soil particles and rinsed in distilled water. EM root tips were collected (10–15/ samples); the non-EM roots were cut into pieces (6–8 pieces/plant) of approximately 5 mm in length with fine scissors. The root pieces were transferred to CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES, 1 % Triton X100, 1 mM EGTA, in distilled water, pH=6.8) for
261
permeabilization and lipid extraction and infiltrated in vacuum for 30 min at room temperature. After extraction, samples were transferred to a fixation solution containing 4 % paraformaldehyde in phosphatebuffered saline (PBS) (0.07 M, pH=7.2) and incubated for 60 min at room temperature. We used these long permeabilization and fixation times because of the large sample volume. The fixed root pieces were washed in PBS for 3×10 min. Samples were postfixed and stored in 70 % ethanol for a maximum of 4 weeks at −20 °C. Prior to sectioning, samples were washed in PBS for 30 min and then positioned and frozen onto a Peltier-cell cooled stage covered in a drop of distilled water. Longitudinal and cross-sections of 90–120 μm thickness were cut with a Reichert microtome (Reichert, Austria) with steel microtome knife, and the sections were washed from the knife and collected in Petri dishes. The sections were infiltrated in hybridization buffer [for 20 ml: 2 ml 20× saline sodium citrate SSC (3 M NaCl, 0.3 M sodium citrate, pH=7.0), 2 g dextran sulfate (Sigma), 2 mg BSA, 8 ml distilled water, and 50 % dimetylformamide (Sigma)] for 1 h prior to the hybridization procedure. We also tested if cell wall degrading enzymatic treatment could facilitate the penetration of probes or allow us to shorten the washing steps. The enzyme solution contained 1 % (w/v) of lysing enzyme mix (lysing enzymes from Trichoderma harzianum, Sigma) in 1 ml PBS. The samples were treated in the enzyme solution for 30 min at 20 °C, then washed in hybridization buffer for 10 min prior to the hybridization procedure. Optimalization of hybridization parameters Prior to hybridization, the 400 μl hybridization buffer was supplemented with 1 μl RNase inhibitor (RiboLock RNase
Table 1 Sequences of the fluorophore-labeled probes applied in the present study Name of the probe
Sequence
Target group
Origin
EUKb310_RhodR: MY1574_Flc: AMF1-Cy3: Bas_ITS4B_af488: AMF_28G2_af488: GintrLSU01_af488: GintrLSU02_af488: GintrLSU822_af488: CadoLSU01_af546:
5′-[RhodR] TCA GGC BCC YTC TCC G -3′ 5′-[Flc] TCC TCG TTG AAG AGC -3′ 5′-[Cy3] GTT TCC CGT AAG GCG CCG AA -3′ 5′-[A488] CAG GAG ACT TGT ACA CGG TCC AG -3′ 5′-[A488] CCA TTA CGT CAA CAT CCT TAA CG -3′ 5′-[A488] CAT ACG GGC AAG TAC ACC CAA -3′ 5′-[A488] TTT CGG CAC CAG AGC AAC GAT -3′ 5′-[A488] AAC TCC TCA CGC TCC ACA GA -3′ 5′-[A546] GAG AGG AGC CAC ATT CCC AA -3′
Eukaryots Eufungi AMF Basidiomycetes AMF Rhizophagus intraradices/AMF/ R. intraradices/AMF/ R. intraradices/AMF/ Cadophora sp./DSE/
Baker et al. 2003 Baschien et al. 2008 Helgason et al. 1998 (PCR primer) Gardes and Bruns 1993. (PCR primer) da Silva et al. 2006 (PCR primer) Present study Present study Trouvelot et al. 1999 (PCR primer) Present study
FISH probes and PCR primers published previously with different selectivity were also tested RhodR Rhodamine Red-X, Flc fluorescein, A488 Alexa Fluor 488, A546 Alexa Fluor 546
262
Inhibitor 40 U/μl, Fermentas), 60 μl salmon sperm DNA (10 mg/ml in H2O, Sigma), and the probe DNA. The probe stock solution consisted of 20 μg probe DNA in 5 μl TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH=7.5) and was stored at −20 °C. These 5 μl stocks were diluted in 400 μl hybridization buffer and partitioned to eight 0.2-ml PCR tubes, each PCR tube received six root sections (thus one 5 μl stock of probe was sufficient for hybridization of 48 root sections). Hybridization conditions were optimized using pure cultures of different endophytes. We used a fixed 50 % formamide concentration and changed the hybridization and washing temperature between 32 and 72 °C in 10 °C increments. Samples were hybridized overnight at adequate temperature in a thermocycler (Hybaid PCR Sprint). Using temperature to adjust stringency in simultaneous hybridization of different probes to several samples required different conditions. Every hybridization experiment needed a separate incubator set to the desired temperature. Therefore, another protocol in which the hybridization stringency achieved by adding different concentrations of formamide to the hybridization buffer was also tested (Daims et al. 2005). In this case, a single hybridization assay was performed in two different samples: the Cadophora sp. strain containing the target sequence and the strain Periconia sp. (strain REF144, group DSE-8 sensu Knapp et al. 2012) without the target sequence. As a negative control, probe-free hybridization buffer was used on Cadophora hyphae. The samples were incubated overnight at 46 °C. Posthybridization steps After hybridization the washing steps took place in 1.5-ml Eppendorf tubes in a dry bath incubator (M.R.C. Ltd) preheated to the proper washing temperature. Root samples were washed three times in preheated washing buffer (same as the hybridization buffer, without dextran sulphate, probe DNA, RNase inhibitor, and salmon sperm DNA), three times in 2× SSC and stored in 4× SSC in dark until mounted in Fluoroshield (Sigma). The length of the washing steps in the first washing buffer was changed between 3×10 to 3×120 min. In the case of washing buffers, the stringency was also achieved by lowering the salt concentration in the corresponding washing buffer instead of adding formamide with washing steps of 3×20 min on 48 °C. Fluorescence microscopy of the root sections We used epifluorescence and confocal microscopy to analyze the root samples. With using a confocal system our aim was to gain 3D images of the fungal structures in thick root sections. Images were captured using Nikon Eclipse 80i microscope equipped with a Spot 7.4 Slider camera (Diagnostic
Mycorrhiza (2014) 24:259–266
Instruments, Inc.), differential–interference–contrast (DIC) and a filter wheel with excitation and emission filters for visualization of Alexa Fluor 488, Alexa Fluor 546, Cy3 and fluorescein-labeled probes. For 3D reconstruction stacks of 1,024×1,024 pixel 8bit grayscale images were acquired using a Zeiss laser scanning microscope (LSM) 410 confocal microscope with 488-nm argon laser line (FT 510 dichroic mirror and HQ 525/50 barrier filter for Alexa Fluor 488 and Fluorescein) and 543 nm HeNe laser line (FT 560 dichroic mirror, D 605/55 barrier filter for Alexa Fluor 546 and Cy3). Increments in the Z-plane were taken accordingly to the values suggested by the LSM software for the chosen objectives, immersion oil and mounting medium. Maximum intensity projections of the stacks were further processed using the Zeiss LSM software or Adobe Photoshop. The projections were colored in accordance with the actual probes emission wavelength. When two probes were applied simultaneously two projections were merged to show the different fungal structures in the same section.
Results Plant autoflourescence, labels, and probes tested To overcome on the challenging problem of the characteristic autofluorescence of plant tissues, we investigated the excitation/emission spectra of root homogenates of different plant species representing a wide range of plant groups (e.g., gymnosperms and angiosperms) and living forms (Supporting Information Plates S1-S4). Based on our spectroscopy results, we defined a wavelength range characteristic of most of the plant roots studied (Supporting Information Plates S1–S3) in which the background autofluorescence was weak enough not to overwhelm the fluorescence from the fluorophores (Supporting Information Plate S4). In a few special cases, the autofluorescence caused by special metabolites of the plants (like silkweed, A. syriaca, and bloodroot, C. majus) was strong enough even at the ranges with low-relative values to make the use of fluorescence probes impossible (Supporting Information Plate S3). Simultaneous detection of different fungi was based on the application of fluorescently labeled specific probes characterized by high hybridization efficiency and nonoverlapping fluorescence emission spectra with each other. Fluorescence intensity of all simultaneously used probes must be high enough to provide good contrast to the background. We tested different fluorescent labels: Alexa Fluor 488, Alexa Fluor 546, Cy3, fluorescein, and RhodamineRedX (Supporting Information Plate S4). When probes were applied in pairs, these were with a lower and a higher emission maximum (Fig. 2a, b).
Mycorrhiza (2014) 24:259–266
263
Sample preparation and penetration of probes Although microscopy and FISH of thick plant tissues are challenging (Prieto et al. 2007), we prepared 90–120 μm thick sections of plant roots to keep the structures of colonizing fungi as intact as possible and to make specimen handling much simpler. Using thick sections allowed us to make hybridization and washing steps in PCR tubes instead of the general on-slide FISH protocol. Due to the thickness of the plant material, we increased the time of the hybridization and washing steps to several hours. This made the whole process much longer than a general FISH applied on thin sections or single cells grown on or spinned down on coverslips. The use of relatively thick sections made the 3D reconstruction of the in planta fungal structures possible with using confocal laser scanning microscopy (CLSM) (Figs. 1c and 2a–d). We tested if FISH probes could penetrate barriers of both plant root tissues and fungal cell walls. For those tests, we first Fig. 2 a–c Dual labeling of Zea mays root sections colonized with Rhizophagus intraradices and Cadophora sp. using the probes GintrLSU01_af488 and CadoLSU01_af546 (Table 1). a Intercellular R. intraradices hyphae and b vesicle and intracellular septate hyphae of Cadophora sp. visualized in green for Alexa Fluor 488 (Rhizophagus) and orange for Alexa Fluor 546 (Cadophora). Scale bars=10 μm. c R. intraradices vesicle colonized by septate hyphae of Cadophora sp. Scale bar=10 μm. d Cross-section of Salix rosmarinifolia ectomycorrhizal root tip labeled with the probe Bas_ITS4B_af488. The Hartig-net shows strong fluorescence due to its metabolic activity contrary to the fungal mantle which shows weaker signal. Scale bar=100 μm. a–d Confocal images
successfully used general eukaryotic and eufungal rRNA probes (Table 1). The treatment with cell wall degrading enzymes resulted in weaker probe signal in the cytoplasm after the washing steps on a given temperature, probably due to the higher permeability of the cell wall. Moreover, as a result of cell wall digestion the sections became soft, and this made the handling and mounting of specimens problematic. For that reason, we skipped the enzymatic treatment thereafter. Hybridization and posthybridization steps
Fig. 1 Micrographs of the longitudinal sections of Populus alba root in brightfield (a) and fluorescence mode (b). Fungal structures are invisible in DIC mode (a) and fluorescence mode shows arbuscules labeled with the probe AMF_28G2_af488 (b). Scale bars 50 μm. c Longitudinal section of Zea mays root labeled with the probe AMF_28G2_af488 shows well-developed arbuscules. Confocal image. Scale bar=10 μm
To optimize the hybridization conditions to obtain sufficient specificity, we applied a mix of two different probes: one of which was a perfect matching probe for the target and the other was a mismatch probe (e.g., CadoLSU01_af546/ GintrLSU01_af488) on pure Cadophora sp. culture. We used a fixed default formamide concentration while changing the washing temperature and the length of the washing (Table 2). Henceforth, we applied temperature/washing time combinations with which a nontarget organism did not show the probe signal, while the matching target provided sufficiently bright signal to give excellent contrast (Fig. 2a, b, Supporting
264
Mycorrhiza (2014) 24:259–266
Table 2 Washing time/temperature combinations tested on mixture of CadoLSU_AF546/GintrLSU01_af488 probes hybridized to Cadophora culture sample
Both probes gave bright signal. CadoLSU_AF546 probe gave bright signal without a signal from GintrLSU01_af488 probe. were weak in comparison to the background fluorescence
Information Figs. S1-S6). When we used GintrLSU01_af488 and CadoLSU01_af546 probes separately, the optimal washing temperature was 52–62 °C, and the minimal washing time was 3×30 min at 50 % formamide. In most cases, the specificity of the labeling increased with longer washing times, and the probe signal intensity weakened with higher temperatures (Table 2). The protocol in which the hybridization stringency was achieved by adding different concentrations of formamide to the hybridization buffer provided important information. None of the probes applied was found to bind aspecifically to nontarget samples. The CadoLSU_AF546 probe did not give any signal in the nontarget Periconia macrospinosa sample, while it gave excellent bright signal in Cadophora sp. culture samples. The fluorescent signal could only be detected in the fungal cytoplasm and not in the cell wall. The optimal formamide concentration in the hybridization buffer turned out to be between 10 and 20 %. Providing the above-defined preparation and hybridization conditions, the applied probes functioned well, and we could visualize in planta fungal structures with sufficient specificity (Figs. 1 and 2, Supporting Information Figs. S1–S6). Based on the experiences gained during the study presented here, we made a recommendation for a general protocol of in planta (nrDNA) FISH (Supporting Information Protocol S1). Visualizations of mycorrhizae and endophytes Using the optimized protocols and probes described above, mycorrhizal and endophytic fungi could be visualized to illustrate the possibilities of the nrRNA FISH for the study of fungi in planta. The general FISH probe for eukaryotic organisms (Baker et al. 2003) penetrated well into hyphae when tested on pure cultures (data not shown). The probe designed for eufungi (Baschien et al. 2008) labeled well hyphae both in pure cultures and in roots (data not shown).
Both signals
The AMF1-Cy3 probe, designed from the AM1 (Helgason et al. 1998) PCR primer specific for Glomeromycota, gave good signal when used in pilot studies to visualize AMF in roots of G. nivalis (data not shown); as described above, this probe was not used in further investigations. The probe developed from the basidiomycete specific ITS4-B (Gardes and Bruns 1993) PCR primer (Bas_ITS4B_af488) labeled well the EM structures (Hartig net, mantle) and hyphae of basidiomycetous EMF (Fig. 2d, Supporting Information Figs. S1). The probe developed from the AMF specific 28G2 (da Silva et al. 2006) PCR primer (AMF_28G2_af488) gave excellent signal in AM structures (arbuscules, vesicles, and intraradical hyphae) and made AMF visible in the roots of both woody (e.g., P. alba, Fig. 1a, b) and herbaceous plant (Z. mays, Supporting Information Figs. S2). Intraradical structures formed by R. intraradices could be visualized using the probe developed from the species-specific “8.22” (Trouvelot et al. 1999) PCR primer (GintrL SU822_af488, Supporting Information Figs. S5) and the specific probes designed in the present study for this fungus (GintrLSU01_af488, Fig. 1a–c, Supporting Information Figs. S3; GintrLSU02_af488, Supporting Information Figs. S4). The probe designed for specific labeling of the DSE Cadophora sp. visualized well the hyphae and microsclerotia of the fungus either used alone or when used simultaneously with the labeling of AMF in the same root (Fig. 2a–c, Supporting Information Figs. S6). Cadophora hyphae colonizing the vesicle of R. intraradices could be also detected (Fig. 2c) during the simultaneous specific labeling of these species.
Discussion Plant tissues are diversity hotspots of different fungi, and these symbiotic fungi colonizing both above- and belowground
Mycorrhiza (2014) 24:259–266
organs of plants play a crucial role not only in the life of their hosts but in ecosystem functioning. Localization and visualization of the fungi and/or their molecules or enzymes of special function is an important method to understand the interactions. There are several methods making in situ visualization of fungi possible. Transformed fungi expressing green fluorescent protein labeled proteins can be used to visualize certain symbionts and the use of different fluorophores makes the parallel visualization of either specific molecules or fungal strains/species possible (Lorang et al. 2001; Bergero et al. 2003; Czymmek et al. 2005). Nevertheless, these methods need the challenging transformation of fungal strains just like the maintenance of stable transformants of fungi. Moreover, the method hardly usable in environmental conditions and only cultivable fungi could be screened. Using the rRNA as target for the FISH has some important powers. As the ribosomes are dispersed in the whole hypha fluorophore-labeled oligonucleotides hybridizing to these RNAs could make the whole cytoplasm visible contrary, e.g., to DNA based nuclear probes, which could solely light the nucleus. Moreover, using rRNA the FISH could refer the metabolic activity of the fungi. There could be considerable inactive members of, e.g., ectomycorrhizal fungal communities, as it was proven when ectomycorrhizae of a Norway spruce forest were screened using viability-test dyes (Qian et al. 1998). During the study, one of the crucial tasks was to find appropriate fluorophores, which do not interfere at least not in a high extent with the autofluorescence of plants. The measure and mapping of the spectra of autofluroscence of the plant roots proved to be a useful method for finding appropriate dyes. This method was also used to study the fluorescence characteristics of fluorophores planned to be used parallel. Although the problem caused by autofluorescence could be overcome by microscopes equipped with spectral detectors, dyes chosen by this technique could make correct visualization easier and microscopes equipped with traditional barrier filters could also be used, which enables the widespread application of the method. Several probes and PCR primers generally used to amplify different groups of fungi have been applied here with different success rate especially regarding to their specificity. We designed our specific probes to hybridize to the D1–D2 region to the LSU gene, which was variable enough to find completely different regions when the ascomycete DSE Cadophora sp. and the AMF R. intraradices were studied simultaneously. Of course, this region has certain limitation and less variable as, e.g., the ITS, species-level DNA barcode region of fungi (Schoch et al. 2012). Nevertheless, because of the advantages of rRNA mentioned above, we aimed to find an rRNA segment with considerable variation, and this variable region of the LSU rRNA seemed to be appropriate. According to its
265
taxonomic resolution in, e.g., the Glomeromycota (Stockinger et al. 2010) or the ascomycete cup fungi Pezizales (Hansen et al. 2005), this region might be a proper segment to design specific probes.
Conclusion We demonstrate here the successful adaptation of rRNA FISH for simultaneous specific visualization of different fungi within the same plant root. The method might be used for aboveground tissues as well, providing that the emission of fluorophores does not overlap considerably with the autofluorescence of those tissues. We expect that in planta FISH with probes designed for specific fungi will be used not only in basic research but also might be a useful diagnostic tool of plant colonizing pathogenic fungi. Acknowledgments We thank Yoram Kapulnik (ARO Volcani Center, Israel) for kindly providing AMF inoculum. Funding was provided by the Hungarian Scientific Research Fund (OTKA, K72776, and NI72776). GMK is supported by the Bolyai János Research Fellowship (Hungarian Academy of Sciences).
References Arnold AE, Lutzoni F (2007) Diversity and host range of foliar fungal endophytes: are tropical leaves biodiversity hotspots? Ecology 88: 541–549. doi:10.1890/05-1459 Baker BJ, Hugenholtz P, Dawson SC, Banfield JF (2003) Extremely acidophilic protists host Rickettsiales-lineage endosymbionts with an intervening sequence in their 16S rRNA genes. Appl Environ Microbiol 69:5512–5518. doi:10.1128/AEM.69.9.5512-5518.2003 Baschien C, Manz W, Neu TR, Marvanová L, Szewcyk U (2008) In situ detection of freshwater fungi in an alpine stream by new taxonspecific fluorescence in situ hybridization probes. Appl Environ Microbiol 74:6427–6436. doi:10.1128/AEM.00815-08 Bergero R, Harrier LA, Franken P (2003) Reporter genes: applications to the study of arbuscular mycorrhizal (AM) fungi and their symbiotic interactions with plant roots. Plant and Soil 255:143–155. doi:10. 1104/pp. 011882 Błaszkowski J, Kovács GM, Gáspár BK, Balázs KT, Buscot F, Ryszka P (2012) The arbuscular mycorrhizal Paraglomus majewskii sp. nov. represents a distinct basal lineage in Glomeromycota. Mycologia 104:148–156. doi:10.3852/10-430 Czymmek KJ, Bourett TM, Howard RJ (2005) Fluorescent protein probes in fungi. In: Savidge T, Pothoulakis C (eds) Methods in microbiology, vol 34. Microbial Imaging Elsevier, Amsterdam, pp 27–62 da Silva GA, Lumini E, Maia LC, Bonfante P, Bianciotto V (2006) Phylogenetic analysis of Glomeromycota by partial LSU rDNA sequences. Mycorrhiza 16:183–189. doi:10.1007/s00572-0050030-9 Daims H, Stoecker K, Wagner M (2005) Fluorescence in situ hybridization for the detection of prokaryotes. In: Osborn AM, Smith C (eds) Molecular microbial ecology. New York, USA, Taylor and Francis, pp 213–239 Dickson S, Kolesik P (1999) Visualisation of mycorrhizal fungal structures and quantification of their surface area and volume using laser
266 scanning confocal microscopy. Mycorrhiza 9:205–213. doi:10. 1007/s005720050268 Diagne N, Escoute J, Lartaud M, Verdeil JL, Franche C, Kane A, Bogusz D, Diouf D, Duponnois R, Svistoonoff S (2011) Uvitex2B: a rapid and efficient stain for detection of arbuscular mycorrhizal fungi within plant roots. Mycorrhiza 21:315–321. doi:10.1007/s00572010-0357-8 Dreyer B, Morte A, Pérez-Gilabert M, Honrubia M (2006) Autofluorescence detection of arbuscular mycorrhizal fungal structures in palm roots: an underestimated experimental method. Mycol Res 110:887–897. doi:10.1016/j.mycres.2006.05.011 Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, McCraw SL, Gurr SJ (2012) Emerging fungal threats to animal, plant and ecosystem health. Nature 484:186–194. doi:10.1038/nature10947 Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118 Girlanda M, Perotto S, Luppi AM (2006) Molecular diversity and ecological roles of mycorrhiza-associated sterile fungal endophytes in Mediterranean ecosystems. In: Schulz B, Boyle C, Sieber TN (eds) Microbial root endophytes. Springer, Berlin–Heidelberg, pp 207– 226. doi:10.1007/3-540-33526-9_12 Grünig CR, Queloz V, Sieber TN, Holdenrieder O (2008) Dark septate endophytes (DSE) of the Phialocephala fortinii s.l. - Acephala applanata species complex in tree roots: classification, population biology, and ecology. Botany 86:1355–1369. doi:10.1139/B08-108 Hansen K, Lobuglio KF, Pfister DH (2005) Evolutionary relationships of the cup-fungus genus Peziza and Pezizaceae inferred multiple nuclear genes: RPB2, β-tubulin, and LSU rDNA. Mol Phylogenet Evol 36:1–23. doi:10.1016/j.ympev.2005.03.010 Helgason T, Daniell TJ, Husband R, Fitter AH, Young JPW (1998) Ploughing up the wood-wide Web? Nature 394:431. doi:10.1038/ 28764 Hibbett DS, Ohman A, Kirk PM (2009) Fungal ecology catches fire. New Phytol 184:279–282. doi:10.1111/j.1469-8137.2009.03042.x Hoch HC, Galvani CD, Szarowski DH, Turner JN (2005) Two new fluorescent dyes applicable for visualization of fungal cell walls. Mycologia 97:580–588. doi:10.3852/mycologia.97.3.580 Hood ME, Shew HD (1996) Applications of KOH-aniline blue fluorescence in the study of plant-fungal interactions. Phytopathology 86: 704–708. doi:10.1094/Phyto-86-704 Jones MD, Forn I, Gadelha C, Egan MJ, Bass D, Massana R, Richards TA (2011) Discovery of novel intermediate forms redefines the fungal tree of life. Nature 474:200–203. doi:10.1038/nature09984 Kohout P, Sýkorová Z, Čtvrtlíková M, Rydlová J, Suda J, Vohník M, Sudová R (2012) Surprising spectra of root-associated fungi in submerged aquatic plants. FEMS Microbiol Ecol 80:216–235. doi: 10.1111/j.1574-6941.2011.01291.x Knapp DG, Pintye A, Kovács GM (2012) The dark side is not fastidious—dark septate endophytic fungi of native and invasive plants of semiarid sandy areas. PLoS ONE 7:e32570. doi:10.1371/journal. pone.0032570 Kovács GM, Szigetvári C (2002) Mycorrhizae and other root-associated fungal structures of the plants of a sandy grassland on the Great Hungarian Plain. Phyton Ann Rei Bot 42:211–223 Lorang JM, Tuori RP, Martinez JP, Sawyer TL, Redman RS, Rollins JA, Wolpert TJ, Johnson KB, Rodriguez RJ, Dickman MB, Ciuffetti LM (2001) Green fluorescent protein is lighting up fungal biology. Appl Environ Microbiol 67:1987–1994. doi:10.1128/AEM.67.5.19871994.2001 Lundberg DS, Lebeis SL, Paredes SH, Yourstone S, Gehring J, Malfatti S, Tremblay J, Engelbrektson A, Kunin V, del Rio TG, Edgar RC,
Mycorrhiza (2014) 24:259–266 Eickhorst T, Ley RE, Hugenholtz P, Tringe SG, Dangl JL (2012) Defining the core Arabidopsis thaliana root microbiome. Nature 488:86–90. doi:10.1038/nature11237 Mandyam K, Jumpponen A (2008) Seasonal and temporal dynamics of arbuscular mycorrhizal and dark septate endophytic fungi in a tallgrass prairie ecosystem are minimally affected by nitrogen enrichment. Mycorrhiza 18:145–155. doi:10.1007/s00572-008-0165-6 Prieto P, Moore G, Shaw P (2007) Fluorescence in situ hybridization on vibratome sections of plant tissues. Nat Protoc 2:1831–1838. doi:10. 1038/nprot.2007.265 Qian XM, Kottke I, Oberwinkler F (1998) Activity of different ectomycorrhizal types studied by vital fluorescence. Plant and Soil 199:91–98. doi:10.1023/A:1004226220283 Regvar M, Likar M, Piltaver A, Kugonič N, Smith JE (2010) Fungal community structure under goat willows (Salix caprea L.) growing at metal polluted site: the potential of screening in a model phytostabilisation study. Plant and Soil 330:345–356. doi:10.1007/ s11104-009-0207-7 Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W, Fungal Barcoding Consortium (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci U S A 109:6241–6246. doi: 10.1073/pnas.1117018109 Schulz BJE, Boyle CJC, Sieber TN (eds) (2006) Microbial root endophytes. Soil biology, vol 9. Springer, Berlin–Heidelberg Séjalon-Delmas N, Magnier A, Douds DD, Bécard G (1998) Cytoplasmic autofluorescence of an arbuscular mycorrhizal fungus Gigaspora gigantea and nondestructive fungal observations in planta. Mycologia 90:921–926 Smith SE, Read DJ (2008) Mycorrhizal symbiosis. Academic, Cambridge Stockinger H, Krüger M, Schüßler A (2010) DNA barcoding of arbuscular mycorrhizal fungi. New Phytol 187:461–474. doi:10. 1111/j.1469-8137.2010.03262.x Stoyke G, Egger KN, Currah RS (1992) Characterization of sterile endophytic fungi from the mycorrhizae of sub-alpine plants. Can J Bot 70:2009–2016. doi:10.1139/b92-250 Tedersoo L, Pärtel K, Jairus T, Gates G, Põldmaa K, Tamm H (2009) Ascomycetes associated with ectomycorrhizas: molecular diversity and ecology with particular reference to the Helotiales. Environ Microbiol 11:3166–3178. doi:10.1111/j.1462-2920.2009.02020.x Trouvelot S, van Tuinen D, Hijri M, Gianinazzi-Perason V (1999) Visualization of ribosomal DNA loci in spore interphasic nuclei of glomalean fungi by fluorescence in situ hybridization. Mycorrhiza 8:203–206. doi:10.1007/s005720050235 Vierheilig H, Schweiger P, Brundrett M (2005) An overview of methods for the detection and observation of arbuscular mycorrhizal fungi in roots. Physiol Plant 125:393–404. doi:10.1111/j.1399-3054.2005. 00564.x Wagner M, Haider S (2012) New trends in fluorescence in situ hybridization for identification and functional analyses of microbes. Curr Opin Biotech 23:96–102. doi:10.1016/j.copbio.2011.10.010 pp. 96102 Wagg C, Pautler M, Massicote HB, Peterson RL (2008) The cooccurrence of ectomycorrhizal, arbuscular mycorrhizal, and dark septate fungi in seedlings of four members of the Pinaceae. Mycorrhiza 18:103–110. doi:10.1007/s00572-007-0157-y Yilmaz LS, Parnerkar S, Noguera DR (2011) mathFISH, a web tool that uses thermodynamics-based mathematical models for in silico evaluation of oligonucleotide probes for fluorescence in situ hybridization. Appl Environ Microbiol 77:1118–1122. doi:10.1128/AEM. 01733-10
